Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity

Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity Pilar Requena, Gloria Patricia Gómez-Pérez, Matthew B. B. McCall, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6221433/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2025 Read the published version in Medical Microbiology and Immunology → Version 1 posted 9 You are reading this latest preprint version Abstract Continuous exposure to Plasmodium falciparum (Pf) has been associated with alterations in B cells. We investigated the effect of controlled human malaria infection (CHMI) on B cell phenotypes in individuals with different Pf immunity status: malaria-naïve, immunized with PfSPZ-CVac and semi-immune (lifelong-exposed) volunteers. Compared to naïve, semi-immune but not vaccinated individuals, had increased baseline frequencies of immature B cells (CD19 + CD10 + ), active naive (IgD + CD27 − CD21 − ) B cells, active atypical (IgD − CD27 − CD21 − ) memory B cells (MBCs), active classical (IgD − CD27 + CD21 − ) MBCs and CD1c + -B cells but lower frequencies of some IgG + -B cells. The frequencies of CD1c + active atypical MBCs correlated positively with anti-Pf antibodies and negatively with circulating eotaxin levels, while the opposite was observed for IgG + resting atypical MBCs. During early blood-stage infection (day 11 after CHMI), there was an expansion of resting classical (IgD − CD27 + CD21 + ) MBCs in all three groups. Vaccination, compared to placebo, altered the effect of CHMI on B cells, showing a positive association with resting classical MBCs (β = 0.190, 95%CI 0.011–0.368) and active naïve-PD1 + (β = 0.637, 95%CI 0.058–1.217) frequencies, and a negative one with CD1c + resting atypical MBCs (β=-0.328, 95%CI -0.621–-0.032). In addition, the sickle cell trait in semi-immune subjects altered the effect of CHMI on several B cells. In conclusion, lifelong but not vaccine exposure to malaria was associated with increased frequencies of multiple B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells, respectively. A single infection (CHMI) induces changes in B cell frequencies and is modulated by sickle cell trait and malaria-immunity status. Clinical Trials Registration. NCT01624961, NCT02115516, and NCT02237586. Plasmodium falciparum Controlled human malaria infection B cells cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Malaria is one of the most serious health problems worldwide, with 263 million cases and 597,000 deaths estimated in 2023 [ 1 ]. Among Plasmodium species causing human malaria, P. falciparum (Pf) is the major responsible for the high rates of mortality. Different efforts are ongoing to fight malaria disease, and one of the strategies is vaccination. Two malaria vaccines, RTS,S/AS01E (Mosquirix®) and R21/Matrix-M™, are recommended by the World Health Organization for widespread use among children living in regions with moderate to high Pf malaria transmission [ 1 ]. However, since duration and level of protection are not optimal [ 2 , 3 ], next-generation vaccines are being developed. One candidate with promising results is the Sanaria PfSPZ-CVac vaccine, which consists of intravenous (IV) injection of Pf sporozoites (PfSPZs) together with chloroquine as a chemoattenuating drug. In a clinical trial conducted in Tübingen (Germany) PfSPZ-CVac immunization resulted in full protection against controlled human malaria infection (CHMI) 8–10 weeks after the last vaccine dose [ 4 ], a finding that could be reproduced in subsequent trials [ 5 , 6 ]. CHMI, consisting of administrating drug-sensitive Pf and closely monitoring parasitemia to administer antimalarial drugs at the first sign of infection, is a powerful tool for evaluation of the efficacy of novel malaria vaccines and drugs (reviewed in [ 7 ]). CHMI provides also a unique opportunity to study in depth the early immune response to Pf infection. Immunity to clinical malaria is slowly acquired over time through repeated natural infections; however, sterile immunity to Pf infection is rarely developed. As a result, in highly endemic areas adults frequently have asymptomatic and submicroscopic infections, i.e. low parasitemias only detected by qPCR [ 8 ]. Moreover, naturally acquired immunity to clinical malaria may diminish when frequent exposure to Plasmodium spp. is discontinued, with several studies reporting declines of Pf-specific antibodies within months and even weeks after an initial potent response [ 9 – 12 ] in contrast to the antibody responses against many viruses and non-replicating antigens used in vaccines that last decades [ 9 , 13 ]. In this regard, we and others have demonstrated clear associations between high levels of exposure to Plasmodium spp. and alterations in peripheral B lymphocyte populations that are responsible for antibody production. An expansion of CD10 + immature B cells has been seen in children with acute episodes of Pf malaria, which reached up to 25% of the CD19 + B lymphocyte subset [ 14 ]. The expansion of immature B cells has also been described in HIV [ 15 ] and other human immunodeficiency states [ 16 ] implying they are associated with chronic immune stimulation. In addition, a subset of CD21 - CD27 - memory B cells (MBCs), termed as “exhausted” or "atypical" MBC are expanded in malaria, HIV and other infections [ 17 – 23 ]. It is not clear whether these malaria-related atypical MBCs are dysfunctional compared to classical MBCs. On the one hand, atypical MBCs have a transcriptional profile distinguishable from the classical ones [ 24 ], with a reduced expression of genes involved in BCR signaling [ 25 ] and antigen recognition [ 26 ], and lower surface IgG levels [ 23 , 25 , 27 ]. On the other hand, signs of proliferative activity and secretion of antibodies have been described in atypical MBCs [ 27 ]. It also remains uncertain if a single or only repeated exposure to malaria parasites is sufficient to induce these alterations in B cell subsets. Most studies have been performed in malaria endemic regions, but one study reported an expansion of atypical MBCs in malaria-naïve Dutch individuals after a single CHMI [ 28 ]. In addition, the levels of chemokine CCL11 (eotaxin) have been negatively correlated with atypical MBC [ 23 ], as well as P. vivax infection and Pf parasitemia [ 29 , 30 ]. Furthermore, frequencies of atypical MBCs had a positive correlation with serum concentrations of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-8 in a cohort with a lifelong exposure to malaria [ 23 ]. The objectives of this work were 1) to compare the B cell profiles of individuals with different Pf immunity status: malaria-naïve, PfSPZ-CVac-immunized and malaria semi-immune individuals with lifelong exposure to Pf in Africa and 2) to evaluate the effect of a single Pf infection via CHMI on these groups. MATERIALS AND METHODS Study participants This study was performed in the context of three CHMI trials (Fig.1) that differed in the level of malaria exposure of the participants: malaria lifelong exposed individuals (LACHMI-001 trial), malaria naïve individuals (TÜCHMI-001 trial) and malaria naïve PfSPZ-CVac vaccinated individuals (TÜCHMI-002 trial). The protocols have been published previously [4,31,32]. The LACHMI-001 clinical trial (NCT02237586) [31] was designed to study the effect of lifelong malaria exposure as well as sickle cell trait on healthy adults aged 18‒30 years following experimental infection with 3,200 PfSPZs (strain NF54) administered by direct venous inoculation (DVI) at CERMEL (Gabon) from July 2014 to February 2016. Volunteers with no history of antimalarial drug intake were allocated into three groups: non-immunes (N=5) minimally exposed to malaria (Europeans living temporally in Gabon), and semi-immunes with a lifelong malaria exposure (Gabonese individuals from Lambaréné), further segregated depending on the presence (hemoglobin [Hb] AS, N=9) or absence (HbAA, N=11) of sickle cell trait. Peripheral blood was collected one day before (C-1) and five (D5) and 11 (D11) days after the CHMI, and peripheral blood mononuclear cells (PBMCs) were cryopreserved. Non-immune participants were treated when parasitemia was detected, and semi-immunes when parasitemia and clinical symptoms were detected or at D28 if they were not treated before [31]. In this study eight semi-immune subjects with sufficient PBMCs were analyzed (HbAS, n=5; HbAA, n=3). The TÜCHMI-001 clinical trial (NCT01624961) [32] aimed to determine the best dose of PfSPZs administered by intravenous injection (IV) through an indwelling catheter for a CHMI regimen. The study was performed at the Eberhard Karls University of Tübingen (Germany) from June to December 2012, with a verification group with the highest dose of parasites administrated by DVI at the Hospital Clinic (Barcelona, Spain) from December 2012 to July 2013 (NCT01771848) [33]. Participants were healthy individuals (N=36) age 18-45 years with no history of malaria. Dose-escalation of PfSPZ (NF54) started with 50 and ended with a maximal dose of 3,200 PfSPZ. Peripheral blood was collected one day before the PfSPZ inoculation (C-1), 11 (D11), and 84 (D84) days after, and PBMCs were cryopreserved [32]. The number of individuals analyzed here was seven, all from the maximal dose group (3,200 PfSPZ) from TÜCHMI-001 clinical trial. The TÜCHMI-002 clinical trial (NCT02115516) [4] aimed at establishing a safe and well-tolerated vaccine regimen using three different doses by DVI (3.2x10 3 , 1.28x10 4 and 5.12x10 4 ) of purified, cryopreserved PfSPZs administered to malaria-naïve healthy adult volunteers taking chloroquine (PfSPZ-CVac vaccine). To assess vaccine efficacy, placebo and PfSPZ-CVac immunized individuals underwent CHMI 8–10 weeks later with 3200 PfSPZ (NF54) by DVI. The trial was carried out in Tübingen (Germany) from May to July of 2014. Peripheral blood was collected at C-1, D11, D84, and PBMCs were cryopreserved [4]. The number of subjects analyzed here was 33 (placebo, n=12; PfSPZ-CVac vaccinated, n=21). Ethical approval For all studies, written informed consent was obtained from all participants. The TÜCHMI-001 and TÜCHMI-002 studies were approved by the ethics committee of the University Clinic and the Medical Faculty of the University of Tübingen, and the Hospital Clinic and the Hospital de la Santa Creu i Sant Pau ethics committees in Barcelona. The LACHMI-001 study received approval by the Gabonese National Ethics Committee (Comité National d’Ethique de la Recherche) and was conducted under the USA FDA Investigational New Drug application. All these studies strictly followed the principles of the Declaration of Helsinki in its sixth revision as well as the Good Clinical Practice guidelines. Detection of Pf Pf infection was diagnosed by thick blood smear (TBS) microscopy and by 18S qPCR. From day 5 until antimalarial treatment, blood samples were withdrawn daily. Quantitative TBS were prepared as described elsewhere [34] and qPCR was performed after all the samples were collected, using published protocols [4,31,32]. Isolation of plasma and PBMCs Plasma was separated from the cellular fraction of heparinized peripheral whole blood by centrifugation at 600 g for 10 min at room temperature within 2‒4 h of collection, aliquoted and stored at –80°C. A density gradient medium (Lymphoprep 4X250mL, PALEX, Cat 1114545) was used to obtain PBMCs that were frozen in fetal bovine serum with 10% of dimethyl sulfoxide and stored in liquid nitrogen until analysis. Immunophenotyping and gating strategy PBMC samples were thawed and processed at ISGlobal (Spain) in three periods of time for logistic reasons: i) all LACHMI-001 and 11 TÜCHMI-002 samples, ii) 2 TÜCHMI-001 and 7 TÜCHMI-002 samples and iii) 5 TÜCHMI-001 and 15 TÜCHMI-002 samples. Samples were thawed by adding RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies) and 0.5µL/mL benzonase (Novagen-Merck, Cat 70664-3), and were centrifuged 500xg for 7 min at RT. After thawing, PBMC viability was measured on a Guava® Cytometer (PC550IG-C4C / 0746-2747) using ViaCount Reagent (Merck-Millipore, Cat 4000-0041). Samples with viabilitites below 70% were excluded. Between 5 × 10 5 and 1 x 10 6 PBMCs per sample were used for B cell staining. Cell suspensions were stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957), washed and blocked with bovine serum albumin 0.5% for 15 min. After washing, cells were stained with an 11-color panel. Supplementary Table 1 summarizes the antibodies used. For compensation control, BD Comp Beads (BD, Cat 552843) were used. To establish the gates for positive events, Fluorescence Minus One (FMO) controls were performed which consisted on staining samples with all the fluorophores used in the panel except one of them. The gating strategy is summarized in Figure 1. First, leukocytes were gated using a time event gate and selecting singlets. Viable B cells (VBCs) were gated by CD19-expression and by excluding CD3 + , CD14 + , CD16 + , dead and apoptotic cells. Immature VBCs were gated as CD10 + within CD19 + live cells, whereas mature VBCs were CD10 - . Mature VBC were divided into switched (IgD - ), unswitched (IgD + CD38 -/low ), plasmablast cells and germinal center cells (PCGCs) (IgD - CD38 ++ ) populations. Of note, PCGCs can also include pre-germinal cells and recent germinal cells. Switched and unswitched populations were further segregated by their expression of CD21 and CD27. Within the switched population, MBCs were classified as active classical (acMBCs) (CD27 + CD21 - ), resting classical (rcMBCs) (CD27 + CD21 + ), active atypical (aaMBCs) (CD27 - CD21 - ) and resting atypical (raMBCs) (CD27 - CD21 + ). The unswitched (IgD + ) population was classified as resting naïve (CD27 - CD21 + ) and active naïve (CD27 - CD21 - ) B cells. B cell subpopulations are reported here as percentage of total VBCs. CD1c + , IgG + and PD1 + cells were gated within each B cell subset. Cell acquisition was performed on a BD LSR II Fortessa cytometer and the data analysis was performed on FlowJo software version v10. Antibody and cytokine analyses Plasma samples were collected 1–2 days before CHMI (C-1), and on the following days after: D7, D11-13, D19 (only in the LACHMI-001 cohort), D28 (only in the TÜCHMI-001 and LACHMI-001 cohorts), and D84 (only for TÜCHMI-001 and TÜCHMI-002). Anti-IgG to 21 Pf antigens (Supplementary Table 2) were measured by quantitative suspension array technology using the xMAP™ platform (Luminex Corp., Austin, Texas) and their levels were expressed as median fluorescence intensity, as described [35,36] & [Gómez-Pérez et al, submitted]. Plasma cytokines were measured by means of the xMAP™ technology (Luminex Corp., Austin, Texas) at time points C-1, D7, D13, D19 and D28 using the Cytokine Human Magnetic 30-Plex Panel from Life Technologies™ as described before [30]. The kit included the following proteins representing major cytokine families (Th1, Th2, Th17), chemokines (proinflammatory and regulatory) and growth factors: epidermal growth factor, fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, vascular endothelial growth factor, TNF, interferon (IFN)-α, IFN-γ, IL-1 receptor agonist (RA), IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40/p70), IL-13, IL-15, IL-17, IFN-γ induced protein (IP-10), monocyte chemoattractant protein (MCP-1), monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation normal T cell expressed and secreted (RANTES) and CCL11 (eotaxin). Concentrations in pg/mL were log 10 transformed for analysis. Statistical analysis Cell population frequencies were calculated with respect to the VBCs, therefore, alterations in one population were detected as changes on the other cell subsets. To improve statistical power, all malaria-naïve individuals (TÜCHMI-001 and placebo TÜCHMI-002) were grouped and termed “malaria naïve”. The distributions of quantitative variables were analyzed using the skewness and kurtosis tests for normality. Most frequencies of B cells did not follow a Gaussian distribution (data not shown) and therefore non-parametric statistical tests were chosen for the analysis. Baseline frequencies of B cell subsets were compared between the three exposure groups (naïve, vaccinated and semi-immune) by Kruskal-Wallis followed by Dunn’s test, corrected with the Bonferroni method for multiple comparisons. The effect of the CHMI was evaluated independently in each exposure group by means of the Friedman’s test, followed by two-by-two comparisons with Bonferroni’s correction. To assess whether previous vaccination affected the B cell changes induced by CHMI, we performed generalized estimated equations and explored the interaction between time and group allocation (placebo vs. vaccine) in the TÜCHMI-2 cohort. The same analysis was performed to evaluate the interaction between time and sickle cell trait (HbAA vs HbAS) in the LACHMI-001 cohort. To analyze the association between the B cell frequencies and other population variables and infection after CHMI, individual or multivariable logistic regressions were estimated with the samples from LACHMI and the PfSPZ-CVac-vaccinated group, estimating the odds ratios (OR) and 95% confidence intervals (CI). The correlations between B cell frequencies and antibody levels or cytokine concentrations were assessed by Spearman’s test at baseline with the naïve, semi-immune and vaccinated donors data together and separately. Statistical significance was defined at p<0.05, and at p<0.1 a trend was considered. All the statistical analyses and graphs were performed using Stata v17 (Stata Corp., College Station, TX, USA, 2017), SPSS version 28.0.1.0 (IBM Corp., Armonk, N.Y., USA) and R (corrplot and tidyverse packages)[37,38] . RESULTS Population The clinical characteristics of study participants are summarized in Table 1. There were more males than females and the median body mass index was in the normal range. After CHMI, all the individuals in the TÜCHMI-001 and placebo-TÜCHMI-002 groups became parasitaemic, while only 29% of the vaccinated-TÜCHMI-002 did. In the LACHMI cohort (semi-immune), 75% of the individuals got a positive smear and 88% were positive by qPCR. In addition, all the individuals with HbAA developed microscopic parasitaemia, while among the participants with HbAS, 40% and 80% got positive smears and qPCR, respectively. Table 1. Population characteristics. LACHMI TÜCHMI-001 TÜCHMI-002 placebo TÜCHMI-002 vaccinated Total N 8 7 12 21 48 Sex (F:M) 2:6 1:6 8:4 7:14 18:30 Age mean (SD) 21.9 (2.30) 27.8 (2.27) 25.43 (4.12) 27.14 (4.86) BMI mean (SD) 21.84 (1.82) 23.89 (3.08) 21.66 (3.36) 24.99 (3.09) 23.47 (3.28) Hb status (HbAA: HbAS) 3:5 N/A N/A N/A N/A TBS test (pos:neg) 6:2 7:0 12:0 6:15 31:17 qPCR test (pos:neg) 7:1 7:0 8:0 6:15 28:16 Days to infection geometric mean (SD) 20.3 (4.4) 11.0 (0.4) 12.1 (2.3) 12.3 (1.1) 13.5 (4.1) Baseline B cell frequency differences between exposure groups Compared to naïve, semi-immune participants had higher percentages of immature B cells, aaMBC, acMBC and active naïve B cells, and lower percentages of PCGC at baseline (Table 2). However, some of these differences were not statistically significant after adjusting for multiple testing. In addition, for all B cell subsets, semi-immune donors had higher frequencies of CD1c-expressing cells than naïve individuals (Table 2). Similar results were observed when semi-immune were compared to vaccinated individuals. In contrast to CD1c-expressing cells, semi-immune individuals had lower percentages of IgG + cells for many B cell subsets compared to naïve and vaccinated donors, except aaMBCs, for which they had a higher percentage (Table 2). With regards PD1-expressing cells, only aaMBCs showed different frequencies among exposure groups, with semi-immune donors having more PD1 + aaMBC cells than naïve individuals. No differences in the frequencies of B cell subsets were observed between naïve and vaccinated individuals, with the exception of a trend towards lower IgG + aMBC (both resting and active) in vaccinated than naïve individuals (Table 2). In summary, cumulative but not a single (vaccine) malaria exposure was associated with increased frequencies of many B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells respectively. The aaMBC subset presented a different behavior, as semi-immune donors had more IgG + aaMBC and PD1 + aaMBC cells than vaccinated and naïve individuals, respectively. Table 2. Differences in frequencies of B cells between groups at baseline. Naïve (n=19) Vaccinated (n=21) Semi-immune (n=8) p-value naïve vs vacc p-value naïve vs semi p-value vacc vs semi Immature 5.5 (3.3-9.6) 7.1 (3.6-11.8) 12.1 (7.3-16.1) 0.589 0.049 0.199 PCGC 0.9 (0.4-1.6) 0.7 (0.4-1.3) 0.4 (0.3-0.6) 0.202 0.037 0.384 aaMBC 2.1 (0.7-3.7) 2.4 (1.3-3.6) 3.9 (2.9-4.4) 0.911 0.075 0.167 acMBC 2.1 (0.9-3.5) 1.8 (1.2-3.2) 3.8 (2.6-5.2) 1.000 0.079 0.085 raMBC 10.1 (7.5-16.8) 12.7 (7.1-14.4) 4.7 (5.6-9.2) 1.000 0.100 0.187 rcMBC 21.1 (11.0-28.5) 14.5 (11.7-16.4) 10.4 (7.7-16.2) 0.311 0.109 0.586 act. naive 1.48 (0.8-3.0) 2.0 (1.4-3.3) 3.4 (2.6-8.4) 0.626 0.009 0.047 naive 41.4 (35.7-51.5) 49.4 (36.7-53.2) 43.4 (37.9-50.4) 0.502 1.000 0.716 CD1c + aaMBC 13.9 (8.1-24.9) 25.0 (10.8-42.0) 50.25 (33.4-54.6) 0.251 0.002 0.037 CD1c + acMBC 33.5 (21.1-57.3) 43.0 (23.3-67.1) 70.9 (50.5-74.9) 0.629 0.038 0.149 CD1c + raMBC 22.4 (6.2-43.8) 23.2 (14.7-44.3) 60.4 (45.5-69.7) 0.724 0.002 0.009 CD1c + rcMBC 51.0 (29.1-69.9) 63.6 (36.8-73.5) 74.5 (60-78.5) 0.555 0.072 0.285 CD1c + act. naive 26.3 (16.4-42.0) 38.6 (23.6-59.3) 61.95 (45.0-71.7) 0.282 0.004 0.066 CD1c + naive 16.4 (4.6-36.1) 17.8 (9.8-50.1) 58.3 (42.5-67.5) 0.517 0.002 0.018 PD1 + aaMBC 4.49 (3.8-9.7) 5.4 (3.9-12.7) 14.7 (5.7-21.6) 0.540 0.050 0.216 PD1 + acMBC 4.4 (3.6-6.8) 5.5 (3.1-9.0) 8.0 (4.9-14.0) 0.637 0.151 0.446 PD1 + raMBC 0.3 (0.2-1.2) 0.3 (0.2-1.5) 0.9 (0.4-1.4) 0.342 0.296 1.000 PD1 + rcMBC 0.9 (0.6-1.7) 1.5 (0.9-4.0) 2.4 (1.4-3.8) 0.129 0.143 1.000 PD1 + act. naive 3.0 (1.3-6.3) 4.6 (1.8-6.5) 6.3 (3.4-10.1) 0.892 0.108 0.241 PD1 + naive 0.3 (0.2-.0.7) 0.4 (0.3-1.8) 0.9 (0.6-2.1) 0.741 0.100 0.260 IgG + aaMBC 36.3 (25.9-43.4) 26.3 (20.1-37.9) 45.35 (21.1-55.7) 0.085 0.905 0.072 IgG + acMBC 48.2 (37.7-5.9) 43.7(29.2-55.0) 25.5 (11.2-42.7) 0.426 0.030 0.183 IgG + raMBC 22.9 (12.8-39.1) 11.9 (5.3-23.4) 5.2 (3.0-9.8) 0.045 0.001 0.100 IgG + rcMBC 39.5 (23.2-54.5) 36.4 (11.6-43.4) 12.2 (5.0-15.25) 0.299 0.006 0.075 IgG + act. naive 17.5 (8.8-33.3) 10.8 (7.7-14.8) 11.7 (4.7-16.8) 0.110 0.170 1.000 IgG + naive 10.4 (6.9-43.2) 9.0 (4.5-13.6) 3.4 (1.9-5.6) 0.370 0.002 0.027 Median plus interquartile range of cellular percentages are shown in the cells. P-value corresponds to Dunn’s test corrected for multiple comparisons with the Bonferroni method. Highlighted in bold if p-value <0.1. Effect of CHMI on each malaria-exposure group and B cell subset In all the cohorts, CHMI was associated with an expansion of rcMBCs, which occurred sooner (D11) in the vaccinated and semi-immune groups (Figures 2A, 2B and 2C). This was accompanied by an expansion of PCGCs and a decrease of naïve B cells in the semi-immune cohort (Figure 2C). With regards to the expression of markers, an expansion of raMBCs-PD1 + and active naive-PD1 + B cells was observed in the naïve and vaccinated groups, respectively (Figures 2A and 2B). Finally, we observed an increase in the percentage of several MBCs producing IgG + from day 0 to day 11 in the semi-immune group (Figure 2C). Non-significant results are not shown. Next, the effect of the interaction of previous vaccination with the average B cell changes during follow-up was assessed in the TÜCHMI-002 cohort. There was a significant (or borderline significant) and positive effect of vaccination for rcMBCs (b=0.190, 95%CI 0.011–0.368, p=0.037) (Figure 3) at D11, and for active naïve-PD1 + (b=0.637, 95%CI 0.058–1.217, p=0.031) (Figure 3), acMBC-IgG + (b=0.166, 95%CI -0.025–0.358, p=0.088) and PCGCs (b=0.874, 95%CI -0.137–1.884, p=0.090) at D84. In addition, a negative effect was observed for CD1c + raMBC (b=-0.328, 95%CI -0.621–-0.032, p=0.030) (Figure 3) at D84 and CD1c + naive B cells (b=-0.201, 95%CI -0.408–0.006, p=0.058). Furthermore, the interaction of the sickle cell trait (HbAS) with the average B cell changes after the CHMI was assessed in the LACHMI cohort. During the follow-up, semi-immune individuals with sickle cell trait had more aaMBCs (b=1.244, 95%CI 0.041–2.446, p=0.043) and raMBCs (b=0.752, 95%CI 0.036–1.467, p=0.039) at D5 compared to participants with normal hemoglobin (HbAA). On the contrary, sickle cell trait had a negative effect on acMBC-PD1 + (b=-0.853, 95%CI -1.395—-0.310, p=0.002), raMBC-PD1 + (b=-0.926, 95%CI -1.445–-0.406, p<0.001) and rcMBC-PD1 + (b=-0.962, 95%CI -1.679–-0.245, p=0.009) frequencies. These results were maintained after adjusting for malaria infection post-CHMI (data not shown). Predictors of infection after CHMI This was evaluated in the vaccinated and semi-immune groups together. Semi-immune donors had 17x higher risk of having an infection after the CHMI than vaccinated donors, but none of the B cell subsets in their baseline levels were significantly associated with infection after CHMI (Table 3). At D11, aaMBC frequency had a borderline non-significant positive association with infection (Table 3). However, after adjusting for previous malaria exposure, the association was lost (OR=2.45, 95% CI 0.67–8.94, p=0.172). When we performed this analysis separately in the vaccinated and semi-immune groups, no associations of baseline or D11 B cells levels and infection were found (data not shown). Table 3. Predictors of infection Variables Baseline Day 11 OR 95% CI p-value OR 95% CI p-value Malaria exposure (semi- vs vacc(ref)) 17.50 1.76 – 174.42 0.015 - - - Sex (wom vs men (ref)) 0.50 0.10 – 2.58 0.407 - - - bmi (³25 vs <25(ref)) 0.35 0.07 – 1.61 0.176 - - - aaMBC 2.29 0.76 – 6.92 0.142 2.81 0.89 – 8.84 0.077 acMBC 1.77 0.56 – 5.59 0.330 1.45 0.55 – 3.81 0.447 rcMBC 0.48 0.10 – 2.44 0.380 0.32 0.06 – 1.68 0.178 raMBC 0.89 0.27 – 2.89 0.848 1.35 0.38 – 4.83 0.647 PCGC 0.51 0.20 – 1.29 0.156 1.62 0.61 – 4.32 0.335 Act.naive 1.46 0.61 – 3.50 0.395 1.65 0.66 – 4.15 0.287 naive 0.15 0.01 – 2.89 0.211 0.10 0.00 – 2.20 0.143 IgG + aaMBC 0.59 0.20 – 1.76 0.342 0.44 0.13 – 1.49 0.189 IgG + acMBC 0.62 0.20 – 1.93 0.406 0.49 0.14 – 1.77 0.276 IgG + raMBC 0.65 0.32 – 1.32 0.234 0.51 0.21 – 1.24 0.137 IgG + rcMBC 0.61 0.28 – 1.30 0.199 0.67 0.27 – 1.70 0.403 IgG + act. naive 0.67 0.27 – 1.63 0.376 0.78 0.39 – 1.57 0.486 IgG + naive 0.82 0.45 – 1.48 0.505 0.76 0.43 – 1.34 0.337 PD1 + aaMBC 1.27 0.63 – 2.56 0.500 0.94 0.50 – 1.76 0.843 PD1 + acMBC 0.94 0.34 – 2.59 0.902 0.76 0.29 – 2.01 0.580 PD1 + raMBC 1.01 0.61 – 1.67 0.979 0.94 0.52 – 1.71 0.850 PD1 + rcMBC 0.82 0.41 – 1.64 0.566 0.96 0.39 – 2.31 0.920 PD1 + act. naive 1.38 0.55 – 3.45 0.491 1.63 0.64 – 4.12 0.304 PD1 + naive 1.05 0.64 – 1.73 0.846 0.99 0.56 – 1.75 0.977 CD1c + aaMBC 1.45 0.55 – 3.84 0.458 2.02 0.61 – 6.72 0.252 CD1c + acMBC 1.84 0.45 – 7.48 0.397 3.12 0.64 – 15.32 0.160 CD1c + raMBC 1.77 0.63 – 4.96 0.276 1.38 0.58 – 3.30 0.471 CD1c + rcMBC 1.75 0.27 – 11.40 0.560 1.63 0.31 – 8.60 0.567 CD1c + act. naive 1.19 0.29 – 4.93 0.810 2.58 0.43 – 15.37 0.299 CD1c + naive 1.25 0.54 – 2.91 0.599 1.38 0.62 – 3.06 0.433 Relationship between B cells, Pf antibody levels, and cytokine concentrations A negative correlation was found between anti-IgG to Pf antigens that are well known markers of exposure (particularly PfMSP-1 19 , PfMSP-1 42 , PfAMA-1) and raMBC or rcMBC frequencies, in all individuals together, at baseline (rho range -0.61 ̶ -0.25, raw p<0.05, Figure S1A) and all time points together (rho range -0.44 ̶ -0.1, raw p<0.05, Figure S1B). In contrast, a positive association was observed with active naïve B cell proportions (baseline rho range 0.4 ̶ 0.24, all timepoints together rho range 0.41 ̶ 0.19, raw p<0.05). In addition, a moderate negative correlation was found between Pf antibody levels (mostly PfMSP-1s and PfAMA-1s) and IgG + B cell frequencies, while a positive moderate correlation occurred between such antibodies and CD1c + B cell frequencies (Figure 4A, Figure S1). Similar results were observed at D11 after the CHMI but not at D84 (data not shown). The antibody markers of Pf exposure PfMSP-1 19 , PfMSP-1 42 , PfAMA-1, PfEXP-1, PfEBA-175 also correlated negatively with the concentrations of eotaxin, MCP-1, IP-10, IFN-g and other cytokines when considering all groups and time points together, but mostly at baseline (Figure 4B, Figure S2). In turn, these cytokines/chemokines (mostly eotaxin) correlated moderately and negatively with the frequencies of CD1c + B cells, and positively with IgG + B cells (Figure 4C, Figure S3). Collectively, data show that higher Pf exposure, manifested by higher anti-Pf IgG levels (most prominent in the semi-immune), correlated with lower eotaxin and IFN related cytokines, as well as higher frequencies of CD1c + B cells and lower frequencies IgG + B cells. These correlations, however, were driven mostly by the semi-immune group. In LACHMI-001 semi-immune individuals alone, IgG + acMBC correlated positively with pro-inflammatory cytokines (IL-1, IL-6, IL-12, IL-15, IL-17, FGF; Figure 5A) and negatively with regulatory cytokines (IL-10, IL-13) (Figure S4), while CD1c + aaMBC correlated strongly and positively with anti-PfMSP1 19 and anti-PfMSP1 42 IgG levels (Figure 5B). PD1 + acMBC also correlated positively with several pro-inflammatory cytokines including IFN-a, IL-1b, IL-6, IL-12, IL-17 and IL-2 in semi-immune participants (Figure 5C). In those individuals, the frequency of aaMBCs correlated negatively with eotaxin levels, but this result did not reach statistical significance (Figure S4). DISCUSSION Before the CHMI and compared to naïve individuals, semi-immune but not vaccinated individuals had increased frequencies of immature and active naïve B cells, as well as active classical and atypical MBCs. First acute malaria episodes have been associated with an expansion of plasmablast cells, atypical MBCs and/or naïve B cells [28,39], however the frequencies may decline to baseline levels 35 days after a single malaria infection. This suggests that multiple rather than single malaria exposures are necessary for durable changes in B cell profiles, consistent with our findings. Interestingly, baseline frequencies of CD1c + cells within each B cell subset, were higher in the semi-immune compared to naïve individuals, correlated negatively with eotaxin and positively with anti-Pf antibodies, the latter well-known markers of malaria exposure (reviewed in [40]). CD1c expression in dendritic cells has been positively associated to Pf-malaria exposure and protection [41,42]. However, to the best of our knowledge, the expression of this marker on B cells in relation to malaria has not been previously explored and more studies are necesary to confirm whether it is a marker of exposure. On the other hand, the percentages of IgG + cells were lower in semi-immune than naive donors, for most B cell subsets and, accordingly, a negative correlation of IgG + cells with anti-Pf antibody levels was found. Of note, the decreased frequency of IgG + was not detected for the expanded aaMBC population. This negative association between IgG + cell frequencies and malaria exposure is in contrast with previous results observed in a cohort of pregnant and non-pregnant individuals from Papua New Guinea [23,43], where no association was mostly observed. But pregnancy itself, exposure to both Pf and P. vivax malaria or other population factors rather than malaria exposure may be responsible for that finding. CHMI resulted in an expansion of rcMBCs in all groups, and of IgG + MBCs at D11 in the semi-immune group, as expected after an infection (reviewed in [44]). Besides, an expansion of certain PD1 + B cell populations occurred after the CHMI. We and others have previously demonstrated that malaria exposure/infection is associated with elevated frequencies of PD1 + B cells compared to healthy individuals [43,45]. However, in this study no correlation was found between PD1 + MBCs and anti-Pf antibody levels. The function of PD1 is not yet much recognized on B cells, whilst it is a well-known inhibitory marker in T cells, associated largely to malaria infection/exposure [46–48], also in NK cells [49]. Although it is classically considered that PD1 + T cells are exhausted and associated with impaired parasite control and infection chronicity [50], some studies have reported an active role for leukocytes expressing this marker [49,51]. Indeed, PD1 is expressed in activated cells and is involved in immune-homeostatic mechanisms. In consonance with all these results, previous vaccination with PfSPZ-CVac was associated with increased percentages of rcMBCs and PD1 + active naïve B cells over post-CHMI follow-up, compared to placebo treatment. Furthermore, PD1+ acMBC were positively correlated with inflammatory cytokines in semi-immune individuals suggesting PD1 as a marker of immune activation. One-third of the population estimated to have the sickle cell trait lives in sub-Saharan Africa, and children with HbAS have decreased susceptibility to clinical malaria [52]. Among the potential explanations for this protection are: i) enhanced naturally-acquired immunity to malaria, ii) impaired growth of Pf on HbAS-erythrocytes or increased splenic removal, and iii) altered surface expression of cytoadherence proteins on infected HbAS-erythrocytes [53]. In our CHMI study, an increase of atypical MBC frequencies was observed at D5 on individuals with the HbAS, and a decrease of PD1 + MBCs, even after adjusting for current infection status. Antibody analysis including all semi-immune participants (N=20) from the same study (LACHMI-001) showed that sickle cell trait individuals compared with the semi-immune with normal hemoglobin had significantly lower IgM and IgG4 levels, and a trend of higher IgG1 and IgG3 response against certain pre-erythrocytic and blood stage antigens [Gómez-Pérez et al, submitted]. To the best of our knowledge, the B cell phenotype change after malaria (or any other) infection has not been described in carriers of the sickle cell trait, and more studies are necessary to confirm these findings and the associated antibody response, especially considering our restricted sample size. The negative correlation of Pf antibodies with eotaxin is in line with the significantly lower eotaxin levels found in lifelong malaria exposed semi-immune individuals compared to the vaccinated or naïve groups (Moncunill et al, in preparation). Other cytokines related to IFN responses, were also negatively correlated with exposure, suggesting a broad impact of cumulative malaria episodes on the immune profile of individuals towards a tolerogenic profile. Consistently these cytokines were negatively associated with B cell subsets expressing CD1c, which increased frequencies were linked to malaria exposure. Ex vivo studies show that human B cells-activation via BCR crosslinking, but not by CD40L alone (T-cell help), upregulate CD1c in naïve, memory and marginal zone-like B cells, and that elevated CD1c enhanced their function as antigen presenting cells [54]. Hence, CD1c expression might be a marker of B cell activation via BCR crosslinking by Pf antigens, and could be related to a regulatory and tolerogenic role given the observed negative correlation with IFN related cytokines. Accordingly, the semi-immune, especially individuals with sickle cell trait, were asymptomatic or had less malaria symptoms than the malaria-naïve [31]. However, we did not find the previously observed negative correlation between eotaxin and atypical MBCs in endemic populations [23]. This may be explained because in this study individuals with long and short exposure to malaria were included, and the semi-immune cohort was actually too small (n=8) to observe significant differences. Nevertheless, in those individuals, frequencies of MBC expressing IgG, diminished by exposure with the exception of aaMBC, were positively correlated with cytokines related to inflammation, Th1 and Th17 responses, suggesting a more tolerogenic and blunt immune status at baseline related with the decreased frequencies of these B cell subsets. This study is limited by the small sample size of the cohorts and the need to treat individuals at first detection of parasites, which might have influenced the natural immune response. Another limitation is having analyzed the cellular samples in three different periods of time, which may have hampered comparability. Nevertheless, important strengths of our study include its novelty, being the first to analyze the B cell phenotype dynamics as a response to CHMI in individuals with different immunity and hemoglobin status, as well as the analysis of B cell surface markers not previously studied in the context of malaria such as CD1c. In summary, cumulative but not a single (vaccine) malaria exposure was associated with increased frequencies of many B lymphocyte subsets, with higher and lower percentages of CD1c and IgG expressing cells, respectively, and a decrease of circulating cytokines previously linked to altered MBC phenotypes, mostly eotaxin, overall suggesting a tolerogenic profile. The CHMI was associated with an early expansion of rcMBC in all three cohorts, and this was increased in the vaccine- compared to the placebo-recipients, suggesting boosting of B cell memory in the vaccinated. The effect of CHMI on B cell phenotypes in semi-immune individuals was also modified by the sickle cell trait. The correlation of these B cell subsets with anti-Pf antibodies suggests that this effect is truly driven by the intensity of exposure to Pf parasites and supports further studies to assess the impact of those changes on the humoral response and naturally acquired immunity against malaria. Declarations Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. The data will be shared after a consideration of the request, ensuring that the purpose aligns with the ethical guidelines and the informed consent obtained from study participants. Data will be provided in a de-identified format to ensure participant confidentiality. Author contributions Conceptualization: B. Kim Lee Sim, Stephen L. Hoffman, Peter Kremsner, Maria Yazdabankhsh, Benjamin Mordmüller, Carlota Dobaño, Gemma Moncunill; Methodology: Gloria Patricia Gómez-Pérez, Diana Barrios, Marta Vidal, Joseph J. Campo, B. Kim Lee Sim, Stephen L. Hoffman, Benjamin Mordmüller, Gemma Moncunill; Formal analysis and investigation: Pilar Requena, Gloria Patricia Gómez-Pérez, Matthew B. McCall, Diana Barrios, Ruth Aguilar, Julia Fernandez-Morata, Marta Vidal, Carla Sanchez, Gemma Moncunill; Writing - original draft preparation: Pilar Requena, Carlota Dobaño, Gemma Moncunill; Writing - review and editing: Gloria Patricia Gómez-Pérez, Matthew B. McCall, Diana Barrios, Ruth Aguilar, Julia Fernandez-Morata, Marta Vidal, Joseph J. Campo, Carla Sanchez, Peter Kremsner, B. Kim Lee Sim, Stephen L. Hoffman, Maria Yazdabankhsh, Benjamin Mordmüller, Benjamin Mordmüller; Funding acquisition: Stephen L. Hoffman, Peter Kremsner, Maria Yazdabankhsh, Benjamin Mordmüller, Carlota Dobaño; Resources: B. Kim Lee Sim, Stephen L. Hoffman, Maria Yazdabankhsh, Carlota Dobaño; Supervision: Carlota Dobaño, Gemma Moncunill. Statements and Declarations E.R.J., B.K.L.S., and S.L.H. work for Sanaria Inc., a company that produces the Plasmodium falciparum sporozoite products used in the studies described in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. Acknowledgments We would like to thank the volunteers participating in these clinical studies, as well as the clinical and lab teams at CERMEL and Tübingen University and Laura Puyol from ISGlobal. The study was supported by funds from NIH-NIAID (R01AI095789, U01AI165745, R01GM9987654), German Center for Infection Research (Grant number TTU 03.702, TTU 03.903) and EDCTP strategic primer grant SP.2011.41304.062. G.P.G-P held a European Community- Erasmus Mundus Joint Doctorate Programme scholarship. G.M. was supported by RYC 2020–029886-I/AEI/10.13039/501100011033, co-funded by European Social Fund (ESF). This research is part of the ISGlobal Program on the Molecular Mechanisms of Malaria, which is partially supported by the Fundación Ramón Areces. Grant CEX2023-0001290-S funded by MCIN/AEI/10.13039/501100011033, and support from the Generalitat de Catalunya through the CERCA Program. 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J Immunol [Internet]. 2011 [cited 2012 Mar 23];186:5261–72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21451111 Additional Declarations Competing interest reported. E.R.J., B.K.L.S., and S.L.H. work for Sanaria Inc., a company that produces the Plasmodium falciparum sporozoite products used in the studies described in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. Supplementary Files manuscriptSupplTUTULABcells20250310.docx Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2025 Read the published version in Medical Microbiology and Immunology → Version 1 posted Editorial decision: Revision requested 07 Jun, 2025 Reviews received at journal 29 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviews received at journal 07 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 23 Mar, 2025 Submission checks completed at journal 15 Mar, 2025 First submitted to journal 13 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6221433","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440963185,"identity":"b451441b-69fd-432b-9634-2cf3916465bb","order_by":0,"name":"Pilar Requena","email":"","orcid":"","institution":"Universidad de Granada","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Requena","suffix":""},{"id":440963186,"identity":"63e7074b-89fa-472d-9e02-61faf237b173","order_by":1,"name":"Gloria Patricia Gómez-Pérez","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Gloria","middleName":"Patricia","lastName":"Gómez-Pérez","suffix":""},{"id":440963187,"identity":"eb6f1e6e-a00e-479a-bc17-0d59cd79b763","order_by":2,"name":"Matthew B. B. McCall","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"B. B.","lastName":"McCall","suffix":""},{"id":440963188,"identity":"d008f1be-e164-4d53-8e91-ea764196eb58","order_by":3,"name":"Diana Barrios","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"","lastName":"Barrios","suffix":""},{"id":440963189,"identity":"b2d50598-9c39-4486-a784-b094f4463bd7","order_by":4,"name":"Ruth Aguilar","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Ruth","middleName":"","lastName":"Aguilar","suffix":""},{"id":440963190,"identity":"c8f50284-63f0-475a-9edb-5f0a475ea6cb","order_by":5,"name":"Julia Fernández-Morata","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Fernández-Morata","suffix":""},{"id":440963191,"identity":"a19e3306-a0e0-4175-abef-8525cfb6132b","order_by":6,"name":"Marta Vidal","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Vidal","suffix":""},{"id":440963192,"identity":"4393642d-3a9d-46e6-8e0e-c6c4a2a9e74b","order_by":7,"name":"Joseph J. Campo","email":"","orcid":"","institution":"Antigen Discovery (United States)","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"J.","lastName":"Campo","suffix":""},{"id":440963196,"identity":"c86db31f-9044-492f-9e1a-9e80bb0ddaaf","order_by":8,"name":"Carla Sanchez","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"","lastName":"Sanchez","suffix":""},{"id":440963198,"identity":"8702f0e1-0d0b-46bb-aebb-5471ad333cd9","order_by":9,"name":"Maria Yazdabankhsh","email":"","orcid":"","institution":"Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Yazdabankhsh","suffix":""},{"id":440963200,"identity":"54253bf9-0db5-4fc4-bca9-e854b448a8dd","order_by":10,"name":"B. Kim Lee Sim","email":"","orcid":"","institution":"Sanaria Inc","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"Kim Lee","lastName":"Sim","suffix":""},{"id":440963202,"identity":"80610667-53f1-493d-beca-c81113ca11a8","order_by":11,"name":"Stephen L. Hoffman","email":"","orcid":"","institution":"Sanaria Inc","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"L.","lastName":"Hoffman","suffix":""},{"id":440963205,"identity":"fec20f59-ff3c-42dd-945d-8409cedf353b","order_by":12,"name":"Peter Kremsner","email":"","orcid":"","institution":"Centre de Recherches Médicales de Lambaréné (CERMEL)","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Kremsner","suffix":""},{"id":440963207,"identity":"37947853-1fa3-4515-9156-0d9eca177aac","order_by":13,"name":"Bertrand Lell","email":"","orcid":"","institution":"Centre de Recherches Médicales de Lambaréné (CERMEL)","correspondingAuthor":false,"prefix":"","firstName":"Bertrand","middleName":"","lastName":"Lell","suffix":""},{"id":440963209,"identity":"b1496e29-c626-4b3f-94e5-ee8ff6504edc","order_by":14,"name":"Benjamin Mordmüller","email":"","orcid":"","institution":"Radboud University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Mordmüller","suffix":""},{"id":440963214,"identity":"61ac205b-4cd6-4c8e-8982-6d453471c506","order_by":15,"name":"Carlota Dobaño","email":"","orcid":"","institution":"ISGlobal","correspondingAuthor":false,"prefix":"","firstName":"Carlota","middleName":"","lastName":"Dobaño","suffix":""},{"id":440963216,"identity":"09cd8ff7-f081-4bef-8836-86bc7cc748a4","order_by":16,"name":"Gemma Moncunill","email":"data:image/png;base64,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","orcid":"","institution":"ISGlobal","correspondingAuthor":true,"prefix":"","firstName":"Gemma","middleName":"","lastName":"Moncunill","suffix":""}],"badges":[],"createdAt":"2025-03-13 15:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6221433/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6221433/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00430-025-00847-x","type":"published","date":"2025-09-27T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80941057,"identity":"4b334b4a-4133-4cd4-97ff-9e6495352e1e","added_by":"auto","created_at":"2025-04-19 10:50:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10296,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of clinical trials. A) Life-long exposed individuals trial (LACHMI-001). B) Malaria naïve individuals clinical trial (TÜCHMI-001). C) Malaria naïve PfSPZ-CVac vaccinated individuals trial (TÜCHMI-002). Days of blood sample collection for PBMC cryopreservation and subsequent B cell analysis are indicated.\u003c/p\u003e","description":"","filename":"Scheme.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/fb1fa54dea7257027f487d45.jpg"},{"id":80940568,"identity":"b0087042-02a6-4a10-a8b2-58afd26d48eb","added_by":"auto","created_at":"2025-04-19 10:34:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGating strategy.\u003c/strong\u003e Excluded “DUMP” population (CD3\u003csup\u003e+\u003c/sup\u003eCD14\u003csup\u003e+\u003c/sup\u003eCD16\u003csup\u003e+\u003c/sup\u003e); viable B cells (VBC) (CD19\u003csup\u003e+\u003c/sup\u003e and not excluded); immature B cells (VBC and CD10\u003csup\u003e+\u003c/sup\u003e); plasmablasts and germinal center cells (VBC, but not Immature VBCs, IgD\u003csup\u003e-\u003c/sup\u003e and CD38\u003csup\u003e++\u003c/sup\u003e); switched (VBC, but not immature VBCs and CD38\u003csup\u003e+\u003c/sup\u003e (not high) and IgD\u003csup\u003e-\u003c/sup\u003e); acMBCs abbreviates active classical memory B cells (MBCs) (Switched: CD21\u003csup\u003e-\u003c/sup\u003e CD27\u003csup\u003e+\u003c/sup\u003e); rcMBCs, resting classical MBCs (switched: CD21\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e); aaMBC, active atypical MBCs (switched: CD21\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e); raMBCs, resting atypical MBCs (switched: CD21\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e); unswitched (VBC not Immature VBCs, not CD38\u003csup\u003e++\u003c/sup\u003e and IgD\u003csup\u003e+\u003c/sup\u003e); resting naïve (unswitched: CD21\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e); active naïve (unswitched: CD21\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/999e82c0498e2636de7c3ec3.jpg"},{"id":80940955,"identity":"832af216-9cf6-409d-b4a8-a8ed1310aa85","added_by":"auto","created_at":"2025-04-19 10:42:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CHMI on B cell subsets distribution.\u003c/strong\u003e Boxplots show the percentages of selected B cell populations before (Day 0) and at different timepoints after the CHMI, in the categorized groups: A) naive, B) vaccinated, and C) semi-immune individuals. Median, and 25th and 75th percentiles (lower and upper hinge respectively) are represented as boxes. Outside values are not displayed in the graphs. Differences between timepoints were assessed by Friedman’s test, followed by two-by-two comparisons corrected with Bonferroni’s test for multiple comparisons.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/d8cd4bde33cfd2581fa0d024.jpg"},{"id":80940571,"identity":"3d742bf7-90a3-4165-9693-6367f540ab1f","added_by":"auto","created_at":"2025-04-19 10:34:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of vaccination status in the B cell distribution at different follow-up periods after CHMI\u003c/strong\u003e. Mean cell subset frequencies plus standard error of the mean are represented for the different arms of the TÜCHMI-2 cohort over time. Only B cell populations with statistically significant interactions with vaccination are shown.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/e9d98bb82e358eaf11d90348.jpg"},{"id":80941058,"identity":"cc658a43-ca7c-411d-8ce1-06bbc446e0b5","added_by":"auto","created_at":"2025-04-19 10:50:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant correlations of B cell frequencies, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e IgG levels (surrogates of Pf exposure) and cytokine concentrations at baseline\u003c/strong\u003e. Scatter plots of immune responses measured in semi-immune (LACHMI-001, red), vaccinated (TÜCHMI-002, blue dots) and naïve (TÜHMI-001, green) individuals together, with\u003cstrong\u003e \u003c/strong\u003eSpearman’s coefficients and raw p-values.\u003cstrong\u003e A)\u003c/strong\u003e IgG to AMA-1 correlated negatively with IgG\u003csup\u003e+\u003c/sup\u003e MBC and positively with CD1c\u003csup\u003e+\u003c/sup\u003e MBC. \u003cstrong\u003eB) \u003c/strong\u003eNegative correlation of IgG to MSP1\u003csub\u003e42\u003c/sub\u003e vs. eotaxin, IFN-g or MCP-1. \u003cstrong\u003eC)\u003c/strong\u003e Eotaxin correlated negatively with CD1c\u003csup\u003e+\u003c/sup\u003e MBC and positively with IgG\u003csup\u003e+ \u003c/sup\u003eMBC.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/121a87e964944add99cc9221.jpg"},{"id":80940576,"identity":"f524d438-20ed-471f-9cd8-63deb35149f1","added_by":"auto","created_at":"2025-04-19 10:34:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant correlations of B cell frequencies, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e IgG levels (surrogates of Pf exposure) and cytokine concentrations at baseline in semi-immune individuals\u003c/strong\u003e. \u003cstrong\u003eA)\u003c/strong\u003e Positive correlations of IgG\u003csup\u003e+\u003c/sup\u003e MBC vs. pro-inflammatory cytokines in semi-immune volunteers. B) CD1c\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eaaMBC correlated positively with anti-PfMSP1\u003csub\u003e19\u003c/sub\u003e and anti-PfMSP1\u003csub\u003e42\u003c/sub\u003e IgG levels C) PD1\u003csup\u003e+\u003c/sup\u003e acMBC correlated positively with pro-inflammatory cytokines. Full correlograms of all immune markers are shown in supplementary materials.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/a67cd56acda0bbdaa1bc3476.jpg"},{"id":92430776,"identity":"96576234-dc7b-4e77-aabc-292def06d02a","added_by":"auto","created_at":"2025-09-29 16:07:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1768336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/0032507b-5c1a-43b0-9445-9b3e8d94dfb4.pdf"},{"id":80940572,"identity":"acb4eaf9-6760-4299-9d3f-3912a8542959","added_by":"auto","created_at":"2025-04-19 10:34:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1570642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptSupplTUTULABcells20250310.docx","url":"https://assets-eu.researchsquare.com/files/rs-6221433/v1/d674c6a3b57596863a66b305.docx"}],"financialInterests":"Competing interest reported. E.R.J., B.K.L.S., and S.L.H. work for Sanaria Inc., a company that produces the Plasmodium falciparum sporozoite products used in the studies described in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.","formattedTitle":"Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMalaria is one of the most serious health problems worldwide, with 263\u0026nbsp;million cases and 597,000 deaths estimated in 2023 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among \u003cem\u003ePlasmodium\u003c/em\u003e species causing human malaria, \u003cem\u003eP. falciparum\u003c/em\u003e (Pf) is the major responsible for the high rates of mortality. Different efforts are ongoing to fight malaria disease, and one of the strategies is vaccination. Two malaria vaccines, RTS,S/AS01E (Mosquirix\u0026reg;) and R21/Matrix-M\u0026trade;, are recommended by the World Health Organization for widespread use among children living in regions with moderate to high Pf malaria transmission [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, since duration and level of protection are not optimal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], next-generation vaccines are being developed. One candidate with promising results is the Sanaria PfSPZ-CVac vaccine, which consists of intravenous (IV) injection of Pf sporozoites (PfSPZs) together with chloroquine as a chemoattenuating drug. In a clinical trial conducted in T\u0026uuml;bingen (Germany) PfSPZ-CVac immunization resulted in full protection against controlled human malaria infection (CHMI) 8\u0026ndash;10 weeks after the last vaccine dose [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], a finding that could be reproduced in subsequent trials [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCHMI, consisting of administrating drug-sensitive Pf and closely monitoring parasitemia to administer antimalarial drugs at the first sign of infection, is a powerful tool for evaluation of the efficacy of novel malaria vaccines and drugs (reviewed in [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]). CHMI provides also a unique opportunity to study in depth the early immune response to Pf infection.\u003c/p\u003e \u003cp\u003eImmunity to clinical malaria is slowly acquired over time through repeated natural infections; however, sterile immunity to Pf infection is rarely developed. As a result, in highly endemic areas adults frequently have asymptomatic and submicroscopic infections, i.e. low parasitemias only detected by qPCR [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, naturally acquired immunity to clinical malaria may diminish when frequent exposure to \u003cem\u003ePlasmodium spp.\u003c/em\u003e is discontinued, with several studies reporting declines of Pf-specific antibodies within months and even weeks after an initial potent response [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] in contrast to the antibody responses against many viruses and non-replicating antigens used in vaccines that last decades [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this regard, we and others have demonstrated clear associations between high levels of exposure to \u003cem\u003ePlasmodium spp.\u003c/em\u003e and alterations in peripheral B lymphocyte populations that are responsible for antibody production. An expansion of CD10\u003csup\u003e+\u003c/sup\u003e immature B cells has been seen in children with acute episodes of Pf malaria, which reached up to 25% of the CD19\u003csup\u003e+\u003c/sup\u003e B lymphocyte subset [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The expansion of immature B cells has also been described in HIV [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and other human immunodeficiency states [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] implying they are associated with chronic immune stimulation. In addition, a subset of CD21\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e-\u003c/sup\u003e memory B cells (MBCs), termed as \u0026ldquo;exhausted\u0026rdquo; or \"atypical\" MBC are expanded in malaria, HIV and other infections [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is not clear whether these malaria-related atypical MBCs are dysfunctional compared to classical MBCs. On the one hand, atypical MBCs have a transcriptional profile distinguishable from the classical ones [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], with a reduced expression of genes involved in BCR signaling [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and antigen recognition [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and lower surface IgG levels [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On the other hand, signs of proliferative activity and secretion of antibodies have been described in atypical MBCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It also remains uncertain if a single or only repeated exposure to malaria parasites is sufficient to induce these alterations in B cell subsets. Most studies have been performed in malaria endemic regions, but one study reported an expansion of atypical MBCs in malaria-na\u0026iuml;ve Dutch individuals after a single CHMI [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, the levels of chemokine CCL11 (eotaxin) have been negatively correlated with atypical MBC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], as well as \u003cem\u003eP. vivax\u003c/em\u003e infection and Pf parasitemia [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, frequencies of atypical MBCs had a positive correlation with serum concentrations of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-8 in a cohort with a lifelong exposure to malaria [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe objectives of this work were 1) to compare the B cell profiles of individuals with different Pf immunity status: malaria-na\u0026iuml;ve, PfSPZ-CVac-immunized and malaria semi-immune individuals with lifelong exposure to Pf in Africa and 2) to evaluate the effect of a single Pf infection via CHMI on these groups.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003ch3\u003eStudy\u0026nbsp;participants\u003c/h3\u003e\n\u003cp\u003eThis study was performed in the context of three CHMI trials (Fig.1) that differed in the level of\u0026nbsp;malaria exposure of the participants: malaria lifelong exposed individuals (LACHMI-001 trial), malaria na\u0026iuml;ve individuals (T\u0026Uuml;CHMI-001 trial) and malaria na\u0026iuml;ve PfSPZ-CVac vaccinated\u0026nbsp;individuals (T\u0026Uuml;CHMI-002\u0026nbsp;trial). The protocols have been published previously [4,31,32].\u003c/p\u003e\n\u003cp\u003eThe LACHMI-001 clinical trial (NCT02237586) [31] was designed to study the effect of lifelong malaria exposure as well as sickle cell trait on healthy adults aged 18‒30 years following experimental infection with 3,200 PfSPZs (strain NF54) administered by direct venous inoculation (DVI) at CERMEL (Gabon) from July 2014 to February 2016. Volunteers with no history of antimalarial drug intake were allocated into three groups: non-immunes (N=5) minimally exposed to malaria (Europeans living temporally in Gabon), and semi-immunes with a lifelong malaria exposure (Gabonese individuals from Lambar\u0026eacute;n\u0026eacute;), further segregated depending on the presence (hemoglobin [Hb] AS, N=9) or absence (HbAA, N=11) of sickle cell trait. Peripheral blood was collected one day before (C-1) and five (D5) and 11 (D11) days after the CHMI, and peripheral blood mononuclear cells (PBMCs) were cryopreserved. Non-immune participants were treated when parasitemia was detected, and semi-immunes when parasitemia and clinical symptoms were detected or at D28 if they were not treated before [31]. In this study eight semi-immune subjects with sufficient PBMCs were analyzed (HbAS, n=5; HbAA, n=3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe T\u0026Uuml;CHMI-001 clinical trial (NCT01624961) [32] aimed to\u0026nbsp;determine the best dose of PfSPZs administered by intravenous injection (IV) through an indwelling catheter for a CHMI regimen.\u0026nbsp;The\u0026nbsp;study\u0026nbsp;was\u0026nbsp;performed\u0026nbsp;at\u0026nbsp;the\u0026nbsp;Eberhard Karls University of T\u0026uuml;bingen\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Germany) from June to December 2012, with a verification group with the highest dose of parasites administrated by DVI at the Hospital Clinic (Barcelona, Spain) from December 2012 to July 2013 (NCT01771848) [33]. Participants were healthy individuals (N=36) age 18-45 years with no history of malaria. Dose-escalation of PfSPZ (NF54) started with 50 and ended with a maximal dose of 3,200 PfSPZ. Peripheral blood was collected one day before the PfSPZ inoculation (C-1), 11 (D11), and 84 (D84) \u0026nbsp; days after, and PBMCs were cryopreserved [32]. The number of individuals analyzed here was seven, all from the maximal dose group (3,200 PfSPZ) from T\u0026Uuml;CHMI-001 clinical trial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe T\u0026Uuml;CHMI-002 clinical trial (NCT02115516) [4]\u0026nbsp;aimed at establishing a safe and\u0026nbsp;well-tolerated\u0026nbsp;vaccine\u0026nbsp;regimen\u0026nbsp;using\u0026nbsp;three\u0026nbsp;different\u0026nbsp;doses by DVI\u0026nbsp;(3.2x10\u003csup\u003e3\u003c/sup\u003e,\u0026nbsp;1.28x10\u003csup\u003e4\u003c/sup\u003e and 5.12x10\u003csup\u003e4\u003c/sup\u003e) of purified, cryopreserved PfSPZs administered to malaria-na\u0026iuml;ve healthy adult volunteers taking chloroquine (PfSPZ-CVac vaccine). To assess vaccine efficacy, placebo and PfSPZ-CVac immunized individuals underwent CHMI 8\u0026ndash;10 weeks later with 3200 PfSPZ (NF54) by DVI. The trial was carried out in T\u0026uuml;bingen (Germany) from May to July of 2014. Peripheral blood was collected at C-1, D11, D84, and PBMCs were cryopreserved [4]. The number of subjects analyzed here was 33 (placebo, n=12; PfSPZ-CVac vaccinated, n=21).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eFor\u0026nbsp;all\u0026nbsp;studies,\u0026nbsp;written\u0026nbsp;informed\u0026nbsp;consent\u0026nbsp;was\u0026nbsp;obtained\u0026nbsp;from\u0026nbsp;all\u0026nbsp;participants. The T\u0026Uuml;CHMI-001 and T\u0026Uuml;CHMI-002\u0026nbsp;studies\u0026nbsp;were\u0026nbsp;approved\u0026nbsp;by\u0026nbsp;the\u0026nbsp;ethics\u0026nbsp;committee\u0026nbsp;of\u0026nbsp;the\u0026nbsp;University\u0026nbsp;Clinic\u0026nbsp;and\u0026nbsp;the\u0026nbsp;Medical Faculty of the University of T\u0026uuml;bingen, and the Hospital Clinic and\u0026nbsp;the Hospital de la\u0026nbsp;Santa\u0026nbsp;Creu\u0026nbsp;i Sant Pau\u003cem\u003e\u0026nbsp;\u003c/em\u003eethics\u0026nbsp;committees in Barcelona. The\u0026nbsp;LACHMI-001\u0026nbsp;study\u0026nbsp;received\u0026nbsp;approval\u0026nbsp;by\u0026nbsp;the\u0026nbsp;Gabonese\u0026nbsp;National\u0026nbsp;Ethics\u0026nbsp;Committee (Comit\u0026eacute; National d\u0026rsquo;Ethique de la Recherche) and was conducted under the USA\u0026nbsp;FDA\u0026nbsp;Investigational\u0026nbsp;New\u0026nbsp;Drug\u0026nbsp;application.\u0026nbsp;All\u0026nbsp;these\u0026nbsp;studies\u0026nbsp;strictly\u0026nbsp;followed\u0026nbsp;the\u0026nbsp;principles of\u0026nbsp;the\u0026nbsp;Declaration\u0026nbsp;of\u0026nbsp;Helsinki\u0026nbsp;in\u0026nbsp;its\u0026nbsp;sixth\u0026nbsp;revision\u0026nbsp;as\u0026nbsp;well\u0026nbsp;as\u0026nbsp;the\u0026nbsp;Good\u0026nbsp;Clinical\u0026nbsp;Practice\u0026nbsp;guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Pf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePf infection was diagnosed by thick blood smear (TBS) microscopy and by 18S qPCR. From day 5 until antimalarial treatment, blood samples were withdrawn daily. Quantitative TBS were prepared as described elsewhere [34] and qPCR was performed after all the samples were collected, using published protocols [4,31,32].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation\u0026nbsp;of\u0026nbsp;plasma\u0026nbsp;and\u0026nbsp;PBMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma was separated from the cellular fraction of heparinized peripheral whole blood by centrifugation at 600 g for 10 min at room temperature within 2‒4 h of collection, aliquoted and stored at \u0026ndash;80\u0026deg;C. A density gradient medium (Lymphoprep 4X250mL, PALEX, Cat 1114545) was used to obtain PBMCs that were frozen in fetal bovine serum with 10% of dimethyl sulfoxide and stored in liquid nitrogen until analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eImmunophenotyping\u0026nbsp;and\u0026nbsp;gating\u0026nbsp;strategy\u003c/h3\u003e\n\u003cp\u003ePBMC samples were thawed and processed at ISGlobal (Spain) in three periods of time for logistic reasons: i) all LACHMI-001 and 11 T\u0026Uuml;CHMI-002\u0026nbsp;samples, ii) 2 T\u0026Uuml;CHMI-001 and 7 T\u0026Uuml;CHMI-002\u0026nbsp;samples\u0026nbsp;and iii) 5 T\u0026Uuml;CHMI-001 and 15 T\u0026Uuml;CHMI-002 samples. Samples were thawed by adding RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies)\u0026nbsp;and 0.5\u0026micro;L/mL benzonase (Novagen-Merck, Cat 70664-3), and were centrifuged\u0026nbsp;500xg for 7 min at RT. After thawing, PBMC\u0026nbsp;viability was measured on a Guava\u0026reg;\u0026nbsp;Cytometer (PC550IG-C4C /\u0026nbsp;0746-2747) using ViaCount Reagent (Merck-Millipore, Cat 4000-0041). Samples with viabilitites below 70% were excluded.\u003c/p\u003e\n\u003cp\u003eBetween 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e and 1 x 10\u003csup\u003e6\u003c/sup\u003e PBMCs per sample were used for B cell staining. Cell suspensions were stained with LIVE/DEAD\u0026reg; Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957), washed and blocked with bovine serum albumin 0.5% for 15 min. After washing, cells were stained with an 11-color panel. Supplementary Table 1 summarizes the antibodies used. For compensation control, BD Comp Beads (BD, Cat 552843) were used. To establish the gates for positive events, Fluorescence Minus One (FMO) controls\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere\u0026nbsp;performed\u0026nbsp;which\u0026nbsp;consisted\u0026nbsp;on\u0026nbsp;staining\u0026nbsp;samples\u0026nbsp;with\u0026nbsp;all\u0026nbsp;the\u0026nbsp;fluorophores\u0026nbsp;used\u0026nbsp;in\u0026nbsp;the\u0026nbsp;panel\u0026nbsp;except one of them. The\u0026nbsp;gating\u0026nbsp;strategy is\u0026nbsp;summarized\u0026nbsp;in\u0026nbsp;Figure\u0026nbsp;1.\u003c/p\u003e\n\u003cp\u003eFirst, leukocytes were gated using a time event gate and selecting singlets. Viable B cells (VBCs) were gated by CD19-expression and by excluding CD3\u003csup\u003e+\u003c/sup\u003e, CD14\u003csup\u003e+\u003c/sup\u003e, CD16\u003csup\u003e+\u003c/sup\u003e, dead and apoptotic cells. Immature VBCs were gated as CD10\u003csup\u003e+\u003c/sup\u003e within CD19\u003csup\u003e+\u003c/sup\u003e live cells, whereas mature VBCs were CD10\u003csup\u003e-\u003c/sup\u003e. Mature VBC were divided into switched (IgD\u003csup\u003e-\u003c/sup\u003e), unswitched (IgD\u003csup\u003e+\u003c/sup\u003e CD38\u003csup\u003e-/low\u003c/sup\u003e), plasmablast cells and germinal center cells (PCGCs) (IgD\u003csup\u003e-\u003c/sup\u003e CD38\u003csup\u003e++\u003c/sup\u003e) populations. Of note, PCGCs can also include pre-germinal cells and recent germinal cells. Switched and unswitched populations were further segregated by their expression of CD21 and CD27. Within the switched population, MBCs were classified as active classical (acMBCs) (CD27\u003csup\u003e+\u003c/sup\u003e CD21\u003csup\u003e-\u003c/sup\u003e), resting classical (rcMBCs) (CD27\u003csup\u003e+\u003c/sup\u003e CD21\u003csup\u003e+\u003c/sup\u003e), active atypical (aaMBCs) (CD27\u003csup\u003e-\u003c/sup\u003e CD21\u003csup\u003e-\u003c/sup\u003e) and resting atypical (raMBCs) (CD27\u003csup\u003e-\u003c/sup\u003e CD21\u003csup\u003e+\u003c/sup\u003e). The unswitched (IgD\u003csup\u003e+\u003c/sup\u003e) population was classified as resting na\u0026iuml;ve (CD27\u003csup\u003e-\u003c/sup\u003e CD21\u003csup\u003e+\u003c/sup\u003e) and active na\u0026iuml;ve (CD27\u003csup\u003e-\u003c/sup\u003e CD21\u003csup\u003e-\u003c/sup\u003e) B cells. B cell subpopulations are reported here as percentage of total VBCs. CD1c\u003csup\u003e+\u003c/sup\u003e, IgG\u003csup\u003e+\u003c/sup\u003e and PD1\u003csup\u003e+\u003c/sup\u003e cells were gated within each B cell subset. Cell acquisition was performed on a BD LSR II Fortessa cytometer and the data analysis was performed on FlowJo software version v10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody and cytokine analyses\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasma samples were collected 1\u0026ndash;2 days before CHMI (C-1), and on the following days after: D7, D11-13, D19 (only in the LACHMI-001 cohort), D28 (only in the T\u0026Uuml;CHMI-001 and LACHMI-001 cohorts), and D84 (only for T\u0026Uuml;CHMI-001 and T\u0026Uuml;CHMI-002). Anti-IgG to 21 Pf antigens (Supplementary Table 2) were measured by quantitative suspension array technology using the xMAP\u0026trade; platform (Luminex Corp., Austin, Texas) and their levels were expressed as median fluorescence intensity, as described [35,36] \u0026amp; [G\u0026oacute;mez-P\u0026eacute;rez et al, submitted]. Plasma cytokines were measured by means of the xMAP\u0026trade; technology (Luminex Corp., Austin, Texas) at time points C-1, D7, D13, D19 and D28 using the Cytokine Human Magnetic 30-Plex Panel from Life Technologies\u0026trade; as described before [30]. The kit included the following proteins representing major cytokine families (Th1, Th2, Th17), chemokines (proinflammatory and regulatory) and growth factors: epidermal growth factor, fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, vascular endothelial growth factor, TNF, interferon (IFN)-\u0026alpha;, IFN-\u0026gamma;, IL-1 receptor agonist (RA), IL-1\u0026beta;, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40/p70), IL-13, IL-15, IL-17, IFN-\u0026gamma; induced protein (IP-10), monocyte chemoattractant protein (MCP-1), monokine induced by IFN-\u0026gamma; (MIG), macrophage inflammatory protein (MIP)-1\u0026alpha;, MIP-1\u0026beta; and regulated on activation normal T cell expressed and secreted (RANTES) and CCL11 (eotaxin). Concentrations in pg/mL were log\u003csub\u003e10\u003c/sub\u003e transformed for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell population frequencies were calculated with respect to the VBCs, therefore, alterations in one population were detected as changes on the other cell subsets. To improve statistical power, all malaria-na\u0026iuml;ve individuals (T\u0026Uuml;CHMI-001 and placebo T\u0026Uuml;CHMI-002) were grouped and termed \u0026ldquo;malaria na\u0026iuml;ve\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eThe distributions of quantitative variables were analyzed using the skewness and kurtosis tests for normality. Most frequencies of B cells did not follow a Gaussian distribution (data not shown) and therefore non-parametric statistical tests were chosen for the analysis. Baseline frequencies of B cell subsets were compared between the three exposure groups (na\u0026iuml;ve, vaccinated and semi-immune) by Kruskal-Wallis followed by Dunn\u0026rsquo;s test, corrected with the Bonferroni method for multiple comparisons. The effect of the CHMI was evaluated independently in each exposure group by means of the Friedman\u0026rsquo;s test, followed by two-by-two comparisons with Bonferroni\u0026rsquo;s correction. To assess whether previous vaccination affected the B cell changes induced by CHMI, we performed generalized estimated equations and explored the interaction between time and group allocation (placebo vs. vaccine) in the T\u0026Uuml;CHMI-2 cohort. The same analysis was performed to evaluate the interaction between time and sickle cell trait (HbAA vs HbAS) in the LACHMI-001 cohort. To analyze the association between the B cell frequencies and other population variables and infection after CHMI, individual or multivariable logistic regressions were estimated with the samples from LACHMI and the PfSPZ-CVac-vaccinated group, estimating the odds ratios (OR) and 95% confidence intervals (CI).\u003c/p\u003e\n\u003cp\u003eThe correlations between B cell frequencies and antibody levels or cytokine concentrations were assessed by Spearman\u0026rsquo;s test at baseline with the na\u0026iuml;ve, semi-immune and vaccinated donors data together and separately.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical significance was defined at p\u0026lt;0.05, and at p\u0026lt;0.1 a trend was considered. All the statistical analyses and graphs were performed using Stata v17 (Stata Corp., College Station, TX, USA, 2017), SPSS version 28.0.1.0 (IBM Corp., Armonk, N.Y., USA) and R (corrplot and tidyverse packages)[37,38] .\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePopulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical characteristics of study participants are summarized in Table 1. There were more males than females and the median body mass index was in the normal range. After CHMI, all the individuals in the T\u0026Uuml;CHMI-001 and placebo-T\u0026Uuml;CHMI-002 groups became parasitaemic, while only 29% of the vaccinated-T\u0026Uuml;CHMI-002 did. In the LACHMI cohort (semi-immune), 75% of the individuals got a positive smear and 88% were positive by qPCR. In addition, all the individuals with HbAA developed microscopic parasitaemia, while among the participants with HbAS, 40% and 80% got positive smears and qPCR, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Population characteristics.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"640\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eLACHMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eT\u0026Uuml;CHMI-001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eT\u0026Uuml;CHMI-002 placebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eT\u0026Uuml;CHMI-002 vaccinated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eSex (F:M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2:6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1:6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e8:4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e7:14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e18:30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eAge mean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e21.9 (2.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e27.8 (2.27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e25.43 (4.12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e27.14 (4.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eBMI mean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e21.84 (1.82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e23.89 (3.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e21.66 (3.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e24.99 (3.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e23.47 (3.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eHb status (HbAA: HbAS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3:5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eTBS test (pos:neg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6:2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e12:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e6:15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e31:17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eqPCR test (pos:neg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e7:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e8:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e6:15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e28:16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eDays to infection geometric mean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e20.3 (4.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e11.0 (0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e12.1 (2.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e12.3 (1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e13.5 (4.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eBaseline B cell frequency differences between exposure groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to na\u0026iuml;ve, semi-immune participants had higher percentages of immature B cells, aaMBC, acMBC and active na\u0026iuml;ve B cells, and lower percentages of PCGC at baseline (Table 2). However, some of these differences were not statistically significant after adjusting for multiple testing. In addition, for all B cell subsets, semi-immune donors had higher frequencies of CD1c-expressing\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells than na\u0026iuml;ve individuals (Table 2). Similar results were observed when semi-immune were compared to vaccinated individuals. In contrast to CD1c-expressing cells, semi-immune individuals had lower percentages of IgG\u003csup\u003e+\u003c/sup\u003e cells for many B cell subsets compared to na\u0026iuml;ve and vaccinated donors, except aaMBCs, for which they had a higher percentage (Table 2). With regards PD1-expressing cells, only aaMBCs showed different frequencies among exposure groups, with semi-immune donors having more PD1\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eaaMBC cells than na\u0026iuml;ve individuals. No differences in the frequencies of B cell subsets were observed between na\u0026iuml;ve and vaccinated individuals, with the exception of a trend towards lower IgG\u003csup\u003e+\u003c/sup\u003e aMBC (both resting and active) in vaccinated than na\u0026iuml;ve individuals (Table 2).\u003c/p\u003e\n\u003cp\u003eIn summary, cumulative but not a single (vaccine) malaria exposure was associated with increased frequencies of many B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells respectively. The aaMBC subset presented a different behavior, as semi-immune donors had more IgG\u003csup\u003e+\u003c/sup\u003e aaMBC and PD1\u003csup\u003e+\u003c/sup\u003eaaMBC cells than vaccinated and na\u0026iuml;ve individuals, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Differences in frequencies of B cells between groups at baseline.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNa\u0026iuml;ve (n=19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eVaccinated (n=21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eSemi-immune (n=8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003ep-value \u0026nbsp;\u003cbr\u003e\u0026nbsp;na\u0026iuml;ve vs vacc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep-value \u0026nbsp;\u003cbr\u003e\u0026nbsp;na\u0026iuml;ve vs semi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003ep-value \u0026nbsp;\u003cbr\u003e\u0026nbsp;vacc vs semi\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eImmature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e5.5 (3.3-9.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e7.1 (3.6-11.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e12.1 (7.3-16.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.049\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.199\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ePCGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9 (0.4-1.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.7 (0.4-1.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.4 (0.3-0.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.037\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.384\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.1 (0.7-3.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.4 (1.3-3.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e3.9 (2.9-4.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.911\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.075\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.167\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.1 (0.9-3.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.8 (1.2-3.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e3.8 (2.6-5.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.079\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.085\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e10.1 (7.5-16.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e12.7 (7.1-14.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e4.7 (5.6-9.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.187\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ercMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e21.1 (11.0-28.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e14.5 (11.7-16.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e10.4 (7.7-16.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.48 (0.8-3.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.0 (1.4-3.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e3.4 (2.6-8.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.009\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.047\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e41.4 (35.7-51.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e49.4 (36.7-53.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e43.4 (37.9-50.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e13.9 (8.1-24.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e25.0 (10.8-42.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e50.25 (33.4-54.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.037\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e33.5 (21.1-57.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e43.0 (23.3-67.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e70.9 (50.5-74.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.629\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n 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style=\"width: 132px;\"\u003e\n \u003cp\u003e26.3 (16.4-42.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e38.6 (23.6-59.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e61.95 (45.0-71.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.066\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e16.4 (4.6-36.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e17.8 (9.8-50.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e58.3 (42.5-67.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e4.49 (3.8-9.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e5.4 (3.9-12.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e14.7 (5.7-21.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.050\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.216\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e4.4 (3.6-6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e5.5 (3.1-9.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e8.0 (4.9-14.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n 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132px;\"\u003e\n \u003cp\u003e0.9 (0.6-1.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.5 (0.9-4.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e2.4 (1.4-3.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e3.0 (1.3-6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e4.6 (1.8-6.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e6.3 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style=\"width: 86px;\"\u003e\n \u003cp\u003e0.260\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e36.3 (25.9-43.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e26.3 (20.1-37.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e45.35 (21.1-55.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.085\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.072\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e48.2 (37.7-5.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e43.7(29.2-55.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e25.5 (11.2-42.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.030\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.183\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e22.9 (12.8-39.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e11.9 (5.3-23.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e5.2 (3.0-9.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.045\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003ercMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e39.5 (23.2-54.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e36.4 (11.6-43.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e12.2 (5.0-15.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.006\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.075\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e17.5 (8.8-33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e10.8 (7.7-14.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e11.7 (4.7-16.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e10.4 (6.9-43.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e9.0 (4.5-13.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e3.4 (1.9-5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.027\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Median plus interquartile range of cellular percentages are shown in the cells. P-value corresponds to Dunn\u0026rsquo;s test corrected for multiple comparisons with the Bonferroni method. Highlighted in bold if p-value \u0026lt;0.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of CHMI on each malaria-exposure group and B cell subset\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all the cohorts, CHMI was associated with an expansion of rcMBCs, which occurred sooner (D11) in the vaccinated and semi-immune groups (Figures 2A, 2B and 2C). This was accompanied by an expansion of PCGCs and a decrease of na\u0026iuml;ve B cells in the semi-immune cohort (Figure 2C). With regards to the expression of markers, an expansion of raMBCs-PD1\u003csup\u003e+\u003c/sup\u003e and active naive-PD1\u003csup\u003e+\u003c/sup\u003e B cells was observed in the na\u0026iuml;ve and vaccinated groups, respectively (Figures 2A and 2B). Finally, we observed an increase in the percentage of several MBCs producing IgG\u003csup\u003e+\u003c/sup\u003e from day 0 to day 11 in the semi-immune group (Figure 2C). Non-significant results are not shown.\u003c/p\u003e\n\u003cp\u003eNext, the effect of the interaction of previous vaccination with the average B cell changes during follow-up was assessed in the T\u0026Uuml;CHMI-002 cohort. There was a significant (or borderline significant) and positive effect of vaccination for rcMBCs (b=0.190, 95%CI 0.011\u0026ndash;0.368, p=0.037) (Figure 3) at D11, and for active na\u0026iuml;ve-PD1\u003csup\u003e+\u003c/sup\u003e (b=0.637, 95%CI 0.058\u0026ndash;1.217, p=0.031) (Figure 3), acMBC-IgG\u003csup\u003e+\u003c/sup\u003e (b=0.166, 95%CI -0.025\u0026ndash;0.358, p=0.088) and PCGCs (b=0.874, 95%CI -0.137\u0026ndash;1.884, p=0.090) at D84. In addition, a negative effect was observed for CD1c\u003csup\u003e+\u003c/sup\u003eraMBC (b=-0.328, 95%CI -0.621\u0026ndash;-0.032, p=0.030) (Figure 3) at D84 and CD1c\u003csup\u003e+\u003c/sup\u003e naive B cells (b=-0.201, 95%CI -0.408\u0026ndash;0.006, p=0.058).\u003c/p\u003e\n\u003cp\u003eFurthermore, the interaction of the sickle cell trait (HbAS) with the average B cell changes after the CHMI was assessed in the LACHMI cohort. During the follow-up, semi-immune individuals with sickle cell trait had more aaMBCs (b=1.244, 95%CI 0.041\u0026ndash;2.446, p=0.043) and raMBCs (b=0.752, 95%CI 0.036\u0026ndash;1.467, p=0.039) at D5 compared to participants with normal hemoglobin (HbAA). On the contrary, sickle cell trait had a negative effect on acMBC-PD1\u003csup\u003e+\u003c/sup\u003e (b=-0.853, 95%CI -1.395\u0026mdash;-0.310, p=0.002), raMBC-PD1\u003csup\u003e+\u003c/sup\u003e (b=-0.926, 95%CI -1.445\u0026ndash;-0.406, p\u0026lt;0.001) and rcMBC-PD1\u003csup\u003e+\u003c/sup\u003e (b=-0.962, 95%CI -1.679\u0026ndash;-0.245, p=0.009) frequencies. These results were maintained after adjusting for malaria infection post-CHMI (data not shown).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePredictors of infection after CHMI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was evaluated in the vaccinated and semi-immune groups together. Semi-immune donors had 17x higher risk of having an infection after the CHMI than vaccinated donors, but none of the B cell subsets in their baseline levels were significantly associated with infection after CHMI (Table 3). At D11, aaMBC frequency had a borderline non-significant positive association with infection (Table 3). However, after adjusting for previous malaria exposure, the association was lost (OR=2.45, 95% CI 0.67\u0026ndash;8.94, p=0.172). When we performed this analysis separately in the vaccinated and semi-immune groups, no associations of baseline or D11 B cells levels and infection were found (data not shown).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Predictors of infection\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 233px;\"\u003e\n \u003cp\u003eBaseline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 225px;\"\u003e\n \u003cp\u003eDay 11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003eOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003eOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eMalaria exposure\u003c/p\u003e\n \u003cp\u003e(semi- vs vacc(ref))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e17.50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.76\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e174.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.015\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eSex (wom vs men (ref))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.407\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ebmi\u0026nbsp;(\u0026sup3;25 vs \u0026lt;25(ref))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.176\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e6.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.81\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.84\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.077\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e5.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.447\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ercMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.178\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.848\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e4.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.647\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ePCGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e4.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eAct.naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.395\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.287\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.189\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.276\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003ercMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.403\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.376\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.486\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIgG\u003csup\u003e+\u003c/sup\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.337\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n 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valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.580\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n 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style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.920\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e4.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.304\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003ePD1\u003csup\u003e+\u003c/sup\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.977\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eaaMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e6.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eacMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e7.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.397\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e15.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eraMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e4.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.471\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003ercMBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e11.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e8.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003eact. naive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e4.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.810\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e15.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.299\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eCD1c\u003csup\u003e+\u003c/sup\u003enaive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0.599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.433\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eRelationship between B cells, Pf antibody levels, and cytokine concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA negative correlation was found between anti-IgG to Pf antigens that are well known markers of exposure\u0026nbsp;(particularly PfMSP-1\u003csub\u003e19\u003c/sub\u003e, PfMSP-1\u003csub\u003e42\u003c/sub\u003e, PfAMA-1)\u0026nbsp;and raMBC or rcMBC frequencies, in all individuals together, at baseline (rho range -0.61 ̶ -0.25, raw p\u0026lt;0.05, Figure S1A) and all time points together (rho range -0.44 ̶ -0.1, raw p\u0026lt;0.05, Figure S1B). In contrast, a positive association was observed with active na\u0026iuml;ve B cell proportions (baseline rho range 0.4 ̶ 0.24, all timepoints together rho range 0.41 ̶ 0.19, raw p\u0026lt;0.05). In addition, a moderate negative correlation was found between Pf antibody levels (mostly PfMSP-1s and PfAMA-1s)\u0026nbsp;and IgG\u003csup\u003e+\u003c/sup\u003e B cell frequencies, while a positive moderate correlation occurred between such antibodies and CD1c\u003csup\u003e+\u003c/sup\u003e B cell frequencies (Figure 4A, Figure S1). Similar results were observed at D11 after the CHMI but not at D84 (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe antibody markers of Pf exposure PfMSP-1\u003csub\u003e19\u003c/sub\u003e, PfMSP-1\u003csub\u003e42\u003c/sub\u003e, PfAMA-1, PfEXP-1, PfEBA-175 also correlated negatively with the concentrations of eotaxin, MCP-1, IP-10, IFN-g\u0026nbsp;and other cytokines when considering all groups and time points together, but mostly at baseline\u0026nbsp;(Figure 4B,\u0026nbsp;Figure S2). In turn, these cytokines/chemokines (mostly eotaxin) correlated moderately and negatively with the frequencies of CD1c\u003csup\u003e+\u003c/sup\u003e B cells, and positively with IgG\u003csup\u003e+\u003c/sup\u003e B cells (Figure 4C, Figure S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, data show that\u0026nbsp;higher Pf exposure, manifested by higher anti-Pf IgG levels (most prominent in the semi-immune), correlated with lower eotaxin and IFN related cytokines, as well as higher frequencies of CD1c\u003csup\u003e+\u003c/sup\u003e B cells and lower frequencies IgG\u003csup\u003e+\u003c/sup\u003e B cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese correlations, however, were driven mostly by the semi-immune group.\u0026nbsp;In LACHMI-001 semi-immune individuals alone, IgG\u003csup\u003e+\u003c/sup\u003e acMBC correlated positively with pro-inflammatory cytokines (IL-1, IL-6, IL-12, IL-15, IL-17, FGF; Figure 5A) and negatively with regulatory cytokines (IL-10, IL-13) (Figure S4), while CD1c\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eaaMBC correlated strongly\u003cem\u003e\u0026nbsp;\u003c/em\u003eand positively with anti-PfMSP1\u003csub\u003e19\u003c/sub\u003e and anti-PfMSP1\u003csub\u003e42\u003c/sub\u003e IgG levels (Figure 5B). PD1\u003csup\u003e+\u003c/sup\u003e acMBC also correlated positively with several pro-inflammatory cytokines including IFN-a, IL-1b, IL-6, IL-12, IL-17 and IL-2 in semi-immune participants (Figure 5C). In those individuals, the frequency of aaMBCs correlated negatively with eotaxin levels, but this result did not reach statistical significance (Figure S4).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBefore the CHMI and compared to naïve individuals, semi-immune but not vaccinated individuals had increased frequencies of immature and active naïve B cells, as well as active classical and atypical MBCs. First acute malaria episodes have been associated with an expansion of plasmablast cells, atypical MBCs and/or naïve B cells [28,39], however the frequencies may decline to baseline levels 35 days after a single malaria infection. This suggests that multiple rather than single malaria exposures are necessary for durable changes in B cell profiles, consistent with our findings. Interestingly, baseline frequencies of CD1c\u003csup\u003e+\u003c/sup\u003e cells within each B cell subset, were higher in the semi-immune compared to naïve individuals, correlated negatively with eotaxin and positively with anti-Pf antibodies, the latter well-known markers of malaria exposure (reviewed in [40]). CD1c expression in dendritic cells has been positively associated to Pf-malaria exposure and protection [41,42]. However, to the best of our knowledge, the expression of this marker on B cells in relation to malaria has not been previously explored and more studies are necesary to confirm whether it is a marker of exposure. On the other hand, the percentages of IgG\u003csup\u003e+\u003c/sup\u003e cells were lower in semi-immune than naive donors, for most B cell subsets and, accordingly, a negative correlation of IgG\u003csup\u003e+\u003c/sup\u003e cells with anti-Pf antibody levels was found. Of note, the decreased frequency of IgG\u003csup\u003e+\u003c/sup\u003e was not detected for the expanded aaMBC population. This negative association between IgG\u003csup\u003e+\u003c/sup\u003e cell frequencies and malaria exposure is in contrast with previous results observed in a cohort of pregnant and non-pregnant individuals \u0026nbsp;from Papua New Guinea [23,43], where no association was mostly observed.\u0026nbsp;But pregnancy itself, exposure to both Pf and \u003cem\u003eP. vivax\u003c/em\u003e malaria or other population factors rather than malaria exposure may be responsible for that finding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCHMI resulted in an expansion of rcMBCs in all groups, and of IgG\u003csup\u003e+\u003c/sup\u003e MBCs at D11 in the semi-immune group, as expected after an infection (reviewed in [44]). Besides, an expansion of certain PD1\u003csup\u003e+\u003c/sup\u003e B cell populations occurred after the CHMI. We and others have previously demonstrated that malaria exposure/infection is associated with elevated frequencies of PD1\u003csup\u003e+\u003c/sup\u003e B cells compared to healthy individuals [43,45]. However, in this study no correlation was found between PD1\u003csup\u003e+\u003c/sup\u003e MBCs and anti-Pf antibody levels. The function of PD1 is not yet much recognized on B cells, whilst it is a well-known inhibitory marker in T cells, associated largely to malaria infection/exposure [46–48], also in NK cells [49]. Although it is classically considered that PD1\u003csup\u003e+\u003c/sup\u003e T cells are exhausted and associated with impaired parasite control and infection chronicity [50], some studies have reported an active role for leukocytes expressing this marker [49,51]. Indeed, PD1 is expressed in activated cells and is involved in immune-homeostatic mechanisms. In consonance with all these results, previous vaccination with PfSPZ-CVac was associated with increased percentages of rcMBCs and PD1\u003csup\u003e+\u003c/sup\u003e active naïve B cells over post-CHMI follow-up, compared to placebo treatment. Furthermore, PD1+ acMBC were positively correlated with inflammatory cytokines in semi-immune individuals suggesting PD1 as a marker of immune activation.\u003c/p\u003e\n\u003cp\u003eOne-third of the population estimated to have the sickle cell trait lives in sub-Saharan Africa, and children with HbAS have decreased susceptibility to clinical malaria [52]. Among the potential explanations for this protection are: i) enhanced naturally-acquired immunity to malaria, ii) impaired growth of Pf on HbAS-erythrocytes or increased splenic removal, and iii) altered surface expression of cytoadherence proteins on infected HbAS-erythrocytes [53]. In our CHMI study, an increase of atypical MBC frequencies was observed at D5 on individuals with the HbAS, and a decrease of PD1\u003csup\u003e+\u003c/sup\u003e MBCs, even after adjusting for current infection status. Antibody analysis including all semi-immune participants (N=20) from the same study (LACHMI-001) showed that sickle cell trait individuals compared with the semi-immune with normal hemoglobin had significantly lower IgM and IgG4 levels, and a trend of higher IgG1 and IgG3 response against certain pre-erythrocytic and blood stage antigens [Gómez-Pérez et al, submitted]. To the best of our knowledge, the B cell phenotype change after malaria (or any other) infection has not been described in carriers of the sickle cell trait, and more studies are necessary to confirm these findings and the associated antibody response, especially considering our restricted sample size.\u003c/p\u003e\n\u003cp\u003eThe negative correlation of Pf antibodies with eotaxin is in line with the significantly lower eotaxin levels found in lifelong malaria exposed semi-immune individuals compared to the vaccinated or naïve groups\u0026nbsp;(Moncunill\u0026nbsp;et al, in preparation). Other cytokines related to IFN responses, were also negatively correlated with exposure, suggesting a broad impact of cumulative malaria episodes on the immune profile of individuals towards a tolerogenic profile. Consistently these cytokines were negatively associated with B cell subsets expressing CD1c, which increased frequencies were linked to malaria exposure. Ex vivo studies show that human B cells-activation via BCR crosslinking, but not by CD40L alone (T-cell help), upregulate CD1c in naïve, memory and marginal zone-like B cells, and that elevated CD1c enhanced their function as antigen presenting cells [54]. Hence, CD1c expression might be a marker of B cell activation via BCR crosslinking by Pf antigens, and could be related to a regulatory and tolerogenic role given the observed negative correlation with IFN related cytokines. Accordingly, the semi-immune, especially individuals with sickle cell trait, were asymptomatic or had less malaria symptoms than the malaria-naïve [31]. However, we did not find the previously observed negative correlation between eotaxin and atypical MBCs in endemic populations [23]. This may be explained because in this study individuals with long and short exposure to malaria were included, and the semi-immune cohort was actually too small (n=8) to observe significant differences. Nevertheless, in those individuals, frequencies of MBC expressing IgG, diminished by exposure with the exception of aaMBC, were positively correlated with cytokines related to inflammation, Th1 and Th17 responses, suggesting a more tolerogenic and blunt immune status at baseline related with the decreased frequencies of these B cell subsets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study is limited by the small sample size of the cohorts and the need to treat individuals at first detection of parasites, which might have influenced the natural immune response. Another limitation is having analyzed the cellular samples in three different periods of time, which may have hampered comparability. Nevertheless, important strengths of our study include its novelty, being the first to analyze the B cell phenotype dynamics as a response to CHMI in individuals with different immunity and hemoglobin status, as well as the analysis of B cell surface markers not previously studied in the context of malaria such as CD1c.\u003c/p\u003e\n\u003cp\u003eIn summary, cumulative but not a single (vaccine) malaria exposure was associated with increased frequencies of many B lymphocyte subsets, with higher and lower percentages of CD1c and IgG expressing cells, respectively, and a decrease of circulating cytokines previously linked to altered MBC phenotypes, mostly eotaxin, overall suggesting a tolerogenic profile. The CHMI was associated with an early expansion of rcMBC in all three cohorts, and this was increased in the vaccine- compared to the placebo-recipients, suggesting boosting of B cell memory in the vaccinated. The effect of CHMI on B cell phenotypes in semi-immune individuals was also modified by the sickle cell trait. The correlation of these B cell subsets with anti-Pf antibodies suggests that this effect is truly driven by the intensity of exposure to Pf parasites and supports further studies to assess the impact of those changes on the humoral response and naturally acquired immunity against malaria.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. The data will be shared after a consideration of the request, ensuring that the purpose aligns with the ethical guidelines and the informed consent obtained from study participants. Data will be provided in a de-identified format to ensure participant confidentiality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: B. Kim Lee Sim, Stephen L. Hoffman, Peter Kremsner, Maria Yazdabankhsh, Benjamin Mordmüller, Carlota Dobaño, Gemma Moncunill; Methodology: Gloria Patricia Gómez-Pérez, Diana Barrios, Marta Vidal, Joseph J. Campo, B. Kim Lee Sim, Stephen L. Hoffman, Benjamin Mordmüller, Gemma Moncunill; Formal analysis and investigation: Pilar Requena, Gloria Patricia Gómez-Pérez, Matthew B. McCall, Diana Barrios, Ruth Aguilar, Julia Fernandez-Morata, Marta Vidal, Carla Sanchez, Gemma Moncunill; Writing - original draft preparation: Pilar Requena, Carlota Dobaño, Gemma Moncunill; Writing - review and editing: Gloria Patricia Gómez-Pérez, Matthew B. McCall, Diana Barrios, Ruth Aguilar, Julia Fernandez-Morata, Marta Vidal, Joseph J. Campo, Carla Sanchez, Peter Kremsner, B. Kim Lee Sim, Stephen L. Hoffman, Maria Yazdabankhsh, Benjamin Mordmüller, Benjamin Mordmüller; Funding acquisition: Stephen L. Hoffman, Peter Kremsner, Maria Yazdabankhsh, Benjamin Mordmüller, Carlota Dobaño; Resources: B. Kim Lee Sim, Stephen L. Hoffman, Maria Yazdabankhsh, Carlota Dobaño; Supervision: Carlota Dobaño, Gemma Moncunill.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.R.J., B.K.L.S., and S.L.H. work for Sanaria Inc., a company that produces the \u003cem\u003ePlasmodium falciparum\u003c/em\u003e sporozoite products used in the studies described in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the volunteers participating in these clinical studies, as well as the clinical and lab teams at CERMEL and Tübingen University and Laura Puyol from ISGlobal. The study was supported by funds from NIH-NIAID (R01AI095789, U01AI165745, R01GM9987654), German Center for Infection Research (Grant number TTU 03.702, TTU 03.903) and EDCTP strategic primer grant SP.2011.41304.062. G.P.G-P held a European Community- Erasmus Mundus Joint Doctorate Programme scholarship. G.M. was supported by RYC 2020–029886-I/AEI/10.13039/501100011033, co-funded by European Social Fund (ESF). This research is part of the ISGlobal Program on the Molecular Mechanisms of Malaria, which is partially supported by the Fundación Ramón Areces. Grant CEX2023-0001290-S funded by MCIN/AEI/10.13039/501100011033, and support from the Generalitat de Catalunya through the CERCA Program.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGeneva: World Health Organization. World malaria report 2024: addressing inequity in the global malaria response. [Internet]. 2024. Available from: https://www.wipo.int/amc/en/mediation/%0Ahttps://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023\u003c/li\u003e\n\u003cli\u003eRTS, S Clinical Trials Partnership*. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: Final results of a phase 3, individually randomised, controlled trial. The Lancet. 2015;386:31\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eDatoo MS, Natama HM, Som\u0026eacute; A, Bellamy D, Traor\u0026eacute; O, Rouamba T, et al. Efficacy and immunogenicity of R21/Matrix-M vaccine against clinical malaria after 2 years\u0026rsquo; follow-up in children in Burkina Faso: a phase 1/2b randomised controlled trial. Lancet Infect Dis. 2022;22:1728\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eMordm\u0026uuml;ller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 2017 542:7642. 2017;542:445\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eMwakingwe-Omari A, Healy SA, Lane J, Cook DM, Kalhori S, Wyatt C, et al. Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity. Nature [Internet]. 2021 [cited 2025 Feb 25];595:289\u0026ndash;94. Available from: https://pubmed.ncbi.nlm.nih.gov/34194041/\u003c/li\u003e\n\u003cli\u003eSulyok Z, Fendel R, Eder B, Lorenz FR, Kc N, Karnahl M, et al. Heterologous protection against malaria by a simple chemoattenuated PfSPZ vaccine regimen in a randomized trial. Nat Commun [Internet]. 2021 [cited 2025 Feb 25];12. Available from: https://pubmed.ncbi.nlm.nih.gov/33947856/\u003c/li\u003e\n\u003cli\u003eStanisic DI, McCarthy JS, Good MF. Controlled Human Malaria Infection: Applications, Advances, and Challenges. Infect Immun. 2017;86:479\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003ePierce SK, Miller LH. World Malaria Day 2009: what malaria knows about the immune system that immunologists still do not. J Immunol. 2009;182:5171\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eLy A, Hansen DS. Development of B cell memory in malaria. Front Immunol. 2019;10:435267.\u003c/li\u003e\n\u003cli\u003eCrompton PD, Kayala MA, Traore B, Kayentao K, Ongoiba A, Weiss GE, et al. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A. 2010;107:6958\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eAkpogheneta OJ, Duah NO, Tetteh KKA, Dunyo S, Lanar DE, Pinder M, et al. Duration of naturally acquired antibody responses to blood-stage Plasmodium falciparum is age dependent and antigen specific. Infect Immun. 2008;76:1748\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eWeiss GE, Traore B, Kayentao K, Ongoiba A, Doumbo S, Doumtabe D, et al. The plasmodium falciparum-specific human memory b cell compartment expands gradually with repeated malaria infections. PLoS Pathog. 2010;6:1\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eAmanna IJ, Carlson NE, Slifka MK. Duration of Humoral Immunity to Common Viral and Vaccine Antigens. New England Journal of Medicine. 2007;357:1903\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eAsito AS, Moormann AM, Kiprotich C, Ng\u0026rsquo;ang\u0026rsquo;a ZW, Ploutz-Snyder R, Rochford R. Alterations on peripheral B cell subsets following an acute uncomplicated clinical malaria infection in children. Malar J. 2008;7:238.\u003c/li\u003e\n\u003cli\u003eMalaspina A, Moir S, Ho J, Wang W, Howell ML, O\u0026rsquo;Shea MA, et al. 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Available from: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-015-0628-0\u003c/li\u003e\n\u003cli\u003eG\u0026oacute;mez-P\u0026eacute;rez GP, Legarda A, Mu\u0026ntilde;oz J, Sim BKL, Ballester MR, Doba\u0026ntilde;o C, et al. Controlled human malaria infection by intramuscular and direct venous inoculation of cryopreserved Plasmodium falciparum sporozoites in malaria-na\u0026iuml;ve volunteers: effect of injection volume and dose on infectivity rates. Malar J [Internet]. 2015 [cited 2025 Feb 25];14:306. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4527105/\u003c/li\u003e\n\u003cli\u003ePlanche T, Krishna S, Kombila M, Engel K, Faucher JF, Ngou-Milama E, et al. Comparison of methods for the rapid laboratory assessment of children with malaria. Am J Trop Med Hyg. 2001;65:599\u0026ndash;602.\u003c/li\u003e\n\u003cli\u003eUbillos I, Jim\u0026eacute;nez A, Vidal M, Bowyer PW, Gaur D, Dutta S, et al. 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Available from: https://pubmed.ncbi.nlm.nih.gov/13115700/\u003c/li\u003e\n\u003cli\u003eBailey RD, Lawton JG, Niangaly A, Stucke EM, Bailey JA, Berry AA, et al. Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens. Journal of Infection. 2024;89:106257.\u003c/li\u003e\n\u003cli\u003eAllan LL, Stax AM, Zheng D-J, Chung BK, Kozak FK, Tan R, et al. CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling. J Immunol [Internet]. 2011 [cited 2012 Mar 23];186:5261\u0026ndash;72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21451111\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-microbiology-and-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mmim","sideBox":"Learn more about [Medical Microbiology and Immunology](http://link.springer.com/journal/430)","snPcode":"430","submissionUrl":"https://submission.nature.com/new-submission/430/3","title":"Medical Microbiology and Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plasmodium falciparum, Controlled human malaria infection, B cells, cytokines","lastPublishedDoi":"10.21203/rs.3.rs-6221433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6221433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eContinuous exposure to \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (Pf) has been associated with alterations in B cells. We investigated the effect of controlled human malaria infection (CHMI) on B cell phenotypes in individuals with different Pf immunity status: malaria-na\u0026iuml;ve, immunized with PfSPZ-CVac and semi-immune (lifelong-exposed) volunteers. Compared to na\u0026iuml;ve, semi-immune but not vaccinated individuals, had increased baseline frequencies of immature B cells (CD19\u003csup\u003e+\u003c/sup\u003eCD10\u003csup\u003e+\u003c/sup\u003e), active naive (IgD\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD21\u003csup\u003e\u0026minus;\u003c/sup\u003e) B cells, active atypical (IgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD21\u003csup\u003e\u0026minus;\u003c/sup\u003e) memory B cells (MBCs), active classical (IgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD21\u003csup\u003e\u0026minus;\u003c/sup\u003e) MBCs and CD1c\u003csup\u003e+\u003c/sup\u003e-B cells but lower frequencies of some IgG\u003csup\u003e+\u003c/sup\u003e-B cells. The frequencies of CD1c\u003csup\u003e+\u003c/sup\u003e active atypical MBCs correlated positively with anti-Pf antibodies and negatively with circulating eotaxin levels, while the opposite was observed for IgG\u003csup\u003e+\u003c/sup\u003e resting atypical MBCs. During early blood-stage infection (day 11 after CHMI), there was an expansion of resting classical (IgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD21\u003csup\u003e+\u003c/sup\u003e) MBCs in all three groups. Vaccination, compared to placebo, altered the effect of CHMI on B cells, showing a positive association with resting classical MBCs (β\u0026thinsp;=\u0026thinsp;0.190, 95%CI 0.011\u0026ndash;0.368) and active na\u0026iuml;ve-PD1\u003csup\u003e+\u003c/sup\u003e (β\u0026thinsp;=\u0026thinsp;0.637, 95%CI 0.058\u0026ndash;1.217) frequencies, and a negative one with CD1c\u003csup\u003e+\u003c/sup\u003e resting atypical MBCs (β=-0.328, 95%CI -0.621\u0026ndash;-0.032). In addition, the sickle cell trait in semi-immune subjects altered the effect of CHMI on several B cells. In conclusion, lifelong but not vaccine exposure to malaria was associated with increased frequencies of multiple B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells, respectively. A single infection (CHMI) induces changes in B cell frequencies and is modulated by sickle cell trait and malaria-immunity status.\u003c/p\u003e \u003cp\u003e \u003cb\u003eClinical Trials Registration.\u003c/b\u003e NCT01624961, NCT02115516, and NCT02237586.\u003c/p\u003e","manuscriptTitle":"Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-19 10:34:41","doi":"10.21203/rs.3.rs-6221433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-07T10:20:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-29T14:39:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308673371269793392606451924031503707610","date":"2025-05-20T18:32:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T06:58:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173191744343429230973437593335048144793","date":"2025-04-01T01:39:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T06:06:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-23T14:41:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-15T07:13:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Microbiology and Immunology","date":"2025-03-13T15:29:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-microbiology-and-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mmim","sideBox":"Learn more about [Medical Microbiology and Immunology](http://link.springer.com/journal/430)","snPcode":"430","submissionUrl":"https://submission.nature.com/new-submission/430/3","title":"Medical Microbiology and Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3fd4dc98-ec3d-4991-b216-7dc70e106fa3","owner":[],"postedDate":"April 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:06:17+00:00","versionOfRecord":{"articleIdentity":"rs-6221433","link":"https://doi.org/10.1007/s00430-025-00847-x","journal":{"identity":"medical-microbiology-and-immunology","isVorOnly":false,"title":"Medical Microbiology and Immunology"},"publishedOn":"2025-09-27 15:58:17","publishedOnDateReadable":"September 27th, 2025"},"versionCreatedAt":"2025-04-19 10:34:41","video":"","vorDoi":"10.1007/s00430-025-00847-x","vorDoiUrl":"https://doi.org/10.1007/s00430-025-00847-x","workflowStages":[]},"version":"v1","identity":"rs-6221433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6221433","identity":"rs-6221433","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}