A monoclonal antibody selectively recognizing PfEMP1 proteins associated with cerebral malaria

A monoclonal antibody selectively recognizing PfEMP1 proteins associated with cerebral malaria | 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 Article A monoclonal antibody selectively recognizing PfEMP1 proteins associated with cerebral malaria Nanna Dalgaard, Rebecca W Olsen, Yvonne Adams, Blanca L Mendez, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6252569/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 21 You are reading this latest preprint version Abstract The frequently fatal outcome of cerebral malaria has been linked to the adhesion and accumulation in the cerebral microvasculature of infected erythrocytes (IEs), which express a particular type of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This type, found in the A and B/A subsets of PfEMP1, contains a particular structural motif (DBLβ motif ) and has dual affinity for the host vascular receptors ICAM-1 and EPCR. Here, we report the functional characterization of a mouse monoclonal antibody, mAb02, raised against eight different DBLβ motif domains. The antibody selectively recognizes DBLβ motif -positive PfEMP1 proteins and inhibits their binding to ICAM-1. It also recognizes IEs expressing DBLβ motif -positive PfEMP1 proteins on their surface and inhibits their adhesion to ICAM-1. The mAb02 epitope is located in a disordered region of the ICAM-1-binding site of DBLβ motif and includes residues directly involved in the interaction between DBLβ motif and ICAM-1, as well as residues that are important for the positioning of the interacting residues. Our study shows that mAb02 targets a broadly conserved epitope that is only found in PfEMP1 proteins binding to ICAM-1 and EPCR and implicated in the pathogenesis of cerebral malaria (CM). This suggests the potential of mAb02 in the development of monoclonal antibody-based intervention against CM and for identification of IEs with capacity to causing CM. Biological sciences/Immunology Health sciences/Molecular medicine Health sciences/Pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plasmodium falciparum parasites are a major cause of morbidity and mortality among children in sub-Saharan African [ 1 ]. The virulence of these parasites has been linked to the infected erythrocyte (IE) surface expression of members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants. PfEMP1 mediates sequestration of IEs by binding to vascular host receptors leading to tissue-specific inflammation, circulatory obstruction and organ dysfunction [ 2 ]. The PfEMP1 proteins are encoded by the var multi-gene family with approximately 60 members per haploid parasite genome. Despite their extensive inter- and intra-clonal diversity, the var genes can be classified into three major groups (A, B, and C) based on their sequence, chromosomal location and transcriptional direction [3; 4]. Transcription of group A var genes, which are less diverse than the other groups, has repeatedly been linked to the development of severe malaria (SM) [5; 6; 7; 8]. This is consistent with the restricted serological diversity of P. falciparum parasites from SM patients and with the relatively rapid acquisition of immunity to severe disease in individuals living in areas with high transmission of P. falciparum [9; 10; 11; 12]. PfEMP1 proteins consist of two to seven highly polymorphic Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR) extracellular domains, a conserved transmembrane domain, and an intracellular acidic terminal segment (ATS) [3; 13; 14]. DBL and CIDR domains can be divided into different types and subtypes (α, β, γ, δ, ε, ζ, х and α, β, γ, respectively) based on sequence similarity. Furthermore, tandem domain cassettes (DCs) of specific DBL and CIDR domains are shared by different parasite clones [ 13 ]. Examples include DC4 (DBLα1.1/1.4-CIDRα1.6-DBLβ3) and DC13 (DBLα1.7-CIDRα1.4) in group A, and DC8 (DBLα2-CIDRα1.1-DBLβ12-DBLγ4/6) in group B/A [13; 15]. CIDRα1 domains in DC8 and DC13 bind endothelial protein receptor C (EPCR) [16; 17], whereas DBLβ domains in DC4 bind intercellular adhesion molecule 1 (ICAM-1) [15; 18]. These adhesion phenotypes have both been linked to SM [5; 15; 19; 20], and parasites expressing PfEMP1 proteins containing both EPCR- and ICAM-1-binding domains (dual binders) have been specifically implicated in the pathogenesis of cerebral malaria (CM) [21; 22]. These proteins contain an EPCR-binding CIDRα1 domain followed immediately by a highly conserved ICAM-1-binding DBLβ domain [ 22 ]. The latter share a DBLβ sequence motif (DBLβ motif : I[V/L]X 3 N[E]GG[P/A]XYX 27 GPPX 3 H) that is specific for dual binders and contains the amino acid residues needed for their ICAM-1 binding. DBLβ motif is restricted to group A and a few ICAM-1-binding group B/A PfEMP1 proteins, and is thus absent from ICAM-1-binding DBLβ domains in the group B and group C PfEMP1 proteins that have been linked to uncomplicated malaria [22; 23; 24; 25]. The above studies have thus established associations between specific PfEMP1 subgroups and specific clinical presentation of P. falciparum malaria. In addition, the pathogenesis of CM appears to involve the presence of dual-binding IEs within brain microvasculature endothelial cells [ 26 ]. Broadly cross-reactive monoclonal antibodies targeting DBLβ motif of most, if not all, group A and B/A DBLβ ICAM-1-binding domains would be a powerful tool in elucidating the molecular basis of CM pathogenesis and might have potential in anti-CM therapy. IgG that targets DBLβ motif domains and inhibits their binding to ICAM-1 is detectable in the plasma of naturally P. falciparum -exposed individuals, and can be induced by immunization of rats with recombinant DBLβ motif protein [ 27 ]. Importantly, however, the antibodies do not target all variants. We have previously described a mouse monoclonal antibody (24E9) that binds the convex surface of DC4DBLβ and interferes with adhesion of DC4-positive IEs to ICAM-1 [ 28 ], but does not interfere with the adhesion of IEs positive for other (non-DC4) CM-associated group A or B/A PfEMP1 proteins (unpublished data). The current study was therefore designed to investigate whether broadly cross-reactive and ICAM-1 adhesion inhibitory monoclonal antibodies could be induced by immunization with combinations of multiple DBLβ motif proteins. Results Identification of additional DBLβ motif domains We have previously defined a 53-amino acid sequence motif (DBLβ motif ) that contains the determinants for ICAM-1 binding of group A and B/A PfEMP1 proteins [ 22 ] (Fig. 1 A). To further examine the diversity of this motif, we extracted 62,000 DBLβ sequences from the Pf3K database of P. falciparum genomes collected worldwide [ 29 ]. We identified 3,042 DBLβ motif domains, of which 578 were unique, but similar, to the 15 previously published DBLβ motif variants [ 27 ]. The sequences could be grouped into three major groups (I-III) containing a total of 18 clusters (Fig. 1 B). To test their capacity for binding to ICAM-1, we produced nine different recombinant proteins (underlined in Fig. 1 A, Table S1 ). [ 22 ]Their sequences were obtained from different clusters (Fig. 1 B) and selected to be distant from previously identified ICAM-1 binding sequences (Table S1 ) [ 22 ], and to be common in the Pf3K dataset. All nine proteins bound ICAM-1 (Supplementary Figure S1 ). Position sequence similarity matrix (PSSM) analysis showed that the nine new and the 15 previously published [15; 22; 25] proteins showed 77% sequence similarity within the motif region and 58% sequence similarity if the sequences up- and down-stream of the motif were included (Fig. 1 C) mAb02 is cross-reactive against motif-containing PfEMP1 Parasites that express DBLβ motif -containing PfEMP1 proteins on the IE surface adhere to both ICAM-1 and EPCR (dual binders) and have been linked to sequestration of IEs in the microvasculature of the brain [22; 26; 30]. In an attempt to generate a monoclonal antibody selectively recognizing all or most DBLβ motif , we immunized with a total of eight different DBLβ motif domains (MA01, MA05, MA07, MA16, MA17, MA20, MA22, MA23, Table S1 ), two at a time. Of several monoclonal antibodies generated from these mice, mAb02 (IgG1, κ light chains) reacted at high titres with all 24 DBLβ motif domains tested (Fig. 2 A, left) and not with any of 13 ICAM-1-binding DBLβ domains from Group B or C DBLβ and not containing DBLβ motif (Fig. 2 A, right). mAb02 also reduced the binding of all 24 DBLβ motif domains to ICAM-1 in a concentration-dependent manner but had no effect on the binding of two non-motif ICAM-1-binding DBLβ domains (Fig. 2 B). Of note, mAb02 reduced ICAM-1 binding of both DC4 and non-DC4 group A and B/A DBLβ motif domains, unlike the previously described monoclonal antibody 24E9 [ 28 ], which only binds DBLβ domains in DC4. The two monoclonal antibodies bound with similar picomolar range affinity to a typical DC4 DBLβ motif domain (MA06, PFD1235W, Fig. 2 C; Table 1 ). Table 1 Kinetic parameters derived from surface plasmon resonance (SPR) experiments on mAbs interaction with PFD1235W DBLβ motif Interaction [nM] 1 k a [M − 1 s − 1 ] k d [s − 1 ] K D [pM] Model mAb02::MA06 3.7 1.96 (± 0.02) x 10 7 2.13 (± 0.01) x 10 − 4 10.80 1–1 interaction model 24E9mAb::MA06 3.7 1.33 (± 0.01) x 10 8 1.84 (± 0.01) x 10 − 4 1.39 1–1 interaction model Binding kinetics of immobilized mAb02 and 24E9 monoclonal antibodies binding to MA06 (PFD1235W DBLβ D4). 1 Maximum concentration tested mAb02 recognises a linear epitope mAb02 recognised both reduced and non-reduced DBLβ motif proteins (MA06, MA10, MA17) in Western blot and showed no reactivity with the control protein NB35 (Fig. 3 A). These results were corroborated by dot blots employing different reduced and non-reduced DBLβ-derived peptides (Fig. 3 B, table S2 ). The dot blot data further indicated that the DBLβ motif epitope targeted by mAb02 was the highly conserved sequence GGPGYYNTEVQKKDR (shaded grey in Fig. 3 B). This inference was verified by peptide ELISA, as mAb02 efficiently recognized Peptide 10 (Fig. 3 C), representing the ICAM-1-binding epitope of Group A and B/A DBLβ domains (Table S2 ). In contrast, 24E9 did not recognize Peptide 10, in accordance with our earlier data that 24E9 recognizes a conformational epitope that involves the Peptide 10 sequence, but also additional amino acids outside it [ 28 ]. The Peptide 10 sequence maps to the disordered regions of the ICAM-1 binding site in the crystal structure (5MZA) of DBLβ motif (MA01, PF11_0521) bound to ICAM-1 [ 22 ] (Fig. 3 D), and is highly conserved among our panel of verified DBLβ motif ICAM-1 binders (Fig. 1 A) and the 3,042 DBLβ motif domains in the Pf3K database (Fig. 3 E). mAb02 reacts with native PfEMP1 variants and inhibits IE adhesion to ICAM-1 Both mAb02 and 24E9 bound to BM057- and 3D7-IEs s expressing DC4-containing PfEMP1 with DBLβ motif (Fig. 4 A). In contrast, only mAb02 bound to HB3-IEs expressing the non-DC4, but DBLβ motif -containing PfEMP1 protein HB3VAR03 (Fig. 4 A). IEs expressing DBLβ motif adhere to ICAM-1, a phenotype linked to sequestration of IEs in the microvasculature of the brain [31; 32]. The adhesion of BM057-IEs to ICAM-1 under physiological flow conditions (wall shear stress of 1 dyn/cm 2 ) was inhibited by mAb02 in a concentration-dependent manner, with efficiency similar to that previously reported for 24E9 [ 28 ] (Fig. 4 B). Cross-reactive DBLβ motif antibodies induced during P. falciparum infections The plasma of adults and children living in areas with stable P. falciparum transmission contains DBLβ motif -reactive IgG [ 33 ]. We measured levels of IgG specific for a typical DBLβ motif (Peptide 10) and the entire DBLβ domain containing it (MA06) in 94 plasma samples from Beninese children with CM (N = 21), non-CM severe malaria (nonCM-SM; N = 33) and uncomplicated malaria (UM; N = 40) (Table 2 , Fig. 5 ). A high proportion of the samples reacted with Peptide 10 (42/94; 45%) and/or with MA06 (57/94, 61%). Overall, the Peptide 10 and MA06 responses were weakly correlated (r = 0.22; P = 0.03). This was almost entirely due to the CM samples (r = 0.45; P = 0.04), as these responses were not significantly correlated when considering either the SM samples (r = 0.02; P = 0.91) or the UM samples (r = 0.21; P = 0.19) separately. The IgG reactivity to Peptide 10 and MA06 did not depend significantly on age (Supplementary Fig. 2). Table 2 Characteristics of study participants Characteristic CM (n = 21) nCSM (n = 33) CM + SM (n = 54) UM (n = 40) Age (months) 42 (30; 48) 36 (18; 54) 37.5 (21.75; 49.5) 36 (16.25; 48) Males/Females (%) 57.1/42.9 54.5/45.5 55.6/44.4 62.5/37.5 Haemoglobin (g/dl) 4.3 (3.3; 6.95) 4.6 (3.4; 5.9) 4.6 (3.4; 6.2) 8.6 (7.15; 10.35) Blantyre score (0–5) 2 (2; 2) 5 (5; 5) 4 (2; 5) NA Parasite density (p/µl) 3,840 (942.5; 976,00) 39,112 (3,280; 240,000.5) 16,727.5 (1,900; 185,333.5) 60,933.5 (6,582; 158,000) Mortality rate (%) 14 (n = 3) 0 6 (n = 3) 0 Values are medians (25th; 75th percentile). CM: Cerebral malaria, nCSM: non-cerebral severe malaria, SM: severe malaria, UM: Uncomplicated malaria. NA: Not applicable (no coma). Discussion CM is caused by cerebral sequestration of IEs [ 34 ]. These IEs often express structurally and serologically related PfEMP1 proteins that can bind both ICAM-1 and EPCR (dual binders) [22; 30; 35]. The ICAM-1 binding site of these PfEMP1 resides within a particular sequence motif (DBLβ motif ) that can be found among PfEMP1 proteins belonging to group A and B/A. Using blood-brain barrier (BBB) organoids, we recently showed that DBLβ motif -positive IEs are selectively taken up by brain endothelial cells in vitro [ 26 ]. The uptake depends on ICAM-1 and results in breakdown of the BBB and swelling of the BBB organoids [ 26 ]. This suggests that internalization of DBLβ motif -positive IEs is involved in the pathogenesis of CM [ 36 ]. In this study, we describe a monoclonal antibody, mAb02, which selectively recognizes group A and B/A PfEMP1 proteins carrying DBLβ motif . Importantly, the antibody recognizes an epitope in DBLβ motif that is conserved among such domains both within and outside DC4 (Fig. 2 and Fig. 3 ). This sets it apart from a previously described monoclonal antibody, 24E9, which only binds to DBLβ motif -containing domains within DC4 [ 28 ]. 24E9 was derived from mice immunized with a single DC4 DBLβ domain (MA06, PFD1235w; DBLβ3_D4), whereas mAb02 was obtained after sequential immunization with eight different DBLβ motif -containing DBLβ domains. The latter domains were chosen after analysis of 62,000 DBLβ domains in the Pf3K database, and selected to capture the global diversity of the 3,042 DBLβ motif -containing domains identified there, within as well as outside DC4 (Fig. 1 ). Together, these data indicate that the immunization strategy used here can focus the immune response on broadly conserved and conformational epitope(s) shared by the immunogens. Of biological significance, mAb02 recognized native PfEMP1 proteins on the surface of erythrocytes infected by three different parasite lines (3D7, BM057, HB3) that had been selected for expression of DBLβ motif PfEMP1s including one (HB3), which was not recognized by the 24E9 monoclonal antibody (Fig. 4 A). Furthermore, sub-nanomolar concentrations of mAb02 significantly inhibited the adhesion of BM057-IEs to ICAM-1 binding under physiologically realistic flow conditions (Fig. 4 B). The target epitope of mAb02 mapped to a continuous linker region between two α-helices (Fig. 3 ), which contain amino acid residues strictly conserved among DBLβ motif domains recognised by mAb02 and known to be critical for the ICAM-1 interaction and the architecture of the ICAM-1-binding site [22; 28]. This indicates that mAb02 functions by steric hindrance and masks the ICAM-1 binding site of DBLβ motif domains. The clinical significance of the DBLβ motif epitope recognized by mAb02 is supported by the observation that levels of IgG specific for a representative DBLβ motif epitope (Peptide 10) and the DBLβ within which it resides (MA06) correlated significantly among Beninese children with CM, but not among sympatric children with either non-CM severe malaria or uncomplicated malaria (Fig. 5 ). It suggests that the CM children were infected by parasites expressing PfEMP1 variants containing a DBLβ motif epitope. In conclusion, we have identified a monoclonal antibody (mAb02) that targets a functionally important epitope defining DBLβ domains of dual-binding PfEMP1 proteins implicated in the pathogenesis of CM [22; 26; 30; 31]. This epitope is therefore a promising candidate for inclusion in a vaccine designed to protect against CM. In addition, mAb02 itself may be exploited to develop biological therapeutics for cerebral malaria, an approach that is currently receiving a lot of attention in the fight against malaria [ 37 ]. Methods Ethics statement All methods were carried out in accordance with relevant guidelines and regulations. All animal procedures were approved by The Danish Animal Procedures Committee (Dyreforsøgstilsynet) as described in permit no. 2013-15-2934-00920, and all experiments were done according to the guidelines described in Danish act LBK 1306 (23/11/2007) and BEK 1273 (12/12/2005). The mice immunizations were conducted in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines and reported according to ARRIVE guidelines. The studies involving collection of human plasma approved by the DRFMT Comité National d’Ethique pour la Recherche en Sante, No. 87/MS/DC/SGM/DRFMT/CNERS/SA Cotonou, Benin. Recombinant proteins and peptides DNA sequences encoding DBLβ domains MA01 to MA24 (group A, DBLβ motif ), NB31 to NB36 and NC37 (ICAM-1 binding group B and C, non-DBLβ motif ), and NA24, NA28 and NA30-33 (non-ICAM-1 binding group A, non-DBLβ motif ) were PCR-amplified from P. falciparum genomic DNA or produced as synthetic constructs (Eurofins). Amplicons were cloned into a modified pET15b vector and expressed as HIS-tagged proteins (Table S1 ) in E. coli Shuffle C3029 (New England Biolabs) as described [15; 22; 25; 27]. All proteins were purified by immobilized metal ion affinity chromatography using HisTrap HP 1 mL columns (GE Healthcare). DBLβ motif domains used for immunization of mice (MA1, MA5, MA7, MA16, MA19, MA21, MA22, MA24; Table S1 ) were further purified using Fc-tagged ICAM-1 coupled to a HiTrap NHS-activated HP column as described [ 28 ]. The DBLβ proteins were eluted using 0.1 M Glycine-HCl buffer (pH 2.75) and neutralized in 1 M Tris-HCl buffer (pH 10). Proteins used for surface plasmon resonance (SPR) analysis (see below) were purified using size-exclusion gel filtration purification column HiPrep 16/60 Sephacryl S-300 HR (Cytiva). Recombinant Fc-tagged ICAM-1 was expressed in HEK293 cells and purified on a HiTrap Protein G HP (GE Healthcare) as described [ 38 ]. Peptides representing the ICAM-1 binding region, or smaller sites within of DBLβ domains found in MA01, MA06, MA09 or MA10 (PF11_0521, PFD1235W D4, DD2VAR32 or KM364031 respectively, table S2 ) were obtained from Schafer-N (Denmark). Immunization, hybridomas and monoclonal antibody production The study involved 6-week-old female Balb/c ByJR mice purchased from Janvier Labs that were immunized with two different group A ICAM-1 binding DBLβ domains (20 µg of each domain in Addavax 1:1/mouse) at each of four time points, i.e., with a total of eight different domains. The following DBLβ domains was used: MA07 and MA16 (intramuscularly (i.m.), day 0), MA21 and MA22 (i.m., day 13), MA19 and MA05 (i.m., day 31), and MA24 and MA01 (intraperitoneally, Day 52). The mice were euthanized using isoflurane and cervical dislocation and bled out on day 55, and the spleen were taken out. Single splenocytes were fused to immortalized myeloma cells (SP2/0-Ag14) cells using polyethylene glycol (PEG) and selected using semisolid medium HAT-containing medium according to manufacturer’s protocol (ClonaCell-HY hybridoma cloning kit, StemCell Technologies). Hybridomas were picked after two weeks and moved to individual wells of flatbottomed 96-well plates (Nunc) and after further culture for one-week, undiluted cell supernatants were tested for DBLβ-reactive antibodies by ELISA (see below). Cells from positive wells were single cell-sorted using a BD FACS Melody Cell Sorter and cultured as above. Positive clones were adapted to DMEM medium containing 1% L-glutamine (Sigma), 1% Pen/Strep (Sigma) and 10% heat-inactivated HY-clone FBS ultra-low fetal calf serum (GE Healthcare) and subsequently cultured in one-litre Cell-Line bioreactor flask (Sigma-Aldrich) following manufacturer’s instructions. Monoclonal antibodies were purified from filtered (0.22 µm) supernatants on a Protein G HP column (GE Healthcare). Bound IgG was eluted in low-pH buffer 0.1 M Glycine-HCl buffer (pH 2.75) and directly neutralized using 1 M Tris-HCl (pH 9.0) to neutral pH, and subsequently buffer-exchanged to PBS using centrifugal VivaSpin columns (Sigma Aldrich). Human plasma samples Plasma samples from children with CM (n = 21), severe non-CM malaria (SM; n = 33), or uncomplicated P. falciparum malaria (n = 40), as defined by the World Health Organization [ 39 ] were collected in 2019 at three hospital centers in Cotonou, Benin. Plasma samples were collected at two hospitals in Cotonou, Southern Benin. The samples were collected during malaria transmission season from June-September 2019 [ 33 ]. Blood samples were collected at day of hospitalization and children below 5 years of age were subjected to clinical investigation and screened by rapid diagnostic test for malaria (Malaria Pf/Pan or DiaQuick). Clinical categorizations were determined according to WHO definitions 1 . Patients categorized with cerebral malaria presented with unarousable coma (Blantyre coma score ≤ 2) an no other cause of coma. Non-cerebral severe malaria patients presented with hyper parasitemia (> 50,000), and/or severe anemia (hemoglobin < 5 g/dl) and no coma. Uncomplicated malaria patients presented with fever, headache or myalgia without signs of severe disease (eg organ dysfunction). Parasitaemia was quantified by microscopy. All samples were collected upon informed consent obtained from parent or legal guardian Hybridoma screening ELISA MaxiSorp plates (Sigma-Aldrich) were coated with recombinant DBLβ proteins (50 µl; 2 µg/ml) in 0.1 M Glycine-HCl buffer (pH 2.75) overnight at 4°C, washed and blocked as described [ 28 ]. Two screenings of hybridoma supernatants were done, the first using a pool of the DBLβ domains used for immunization and the second using individual MA01-M24 pool as coating antigen (Table S1 ). 40 µl hybridoma supernatant (containing mAbs) were incubated in wells 1 h while shaking at room temperature. After wash, mAbs were bound with HRP-conjugated anti-mouse IgG (1:3000 in blocking buffer (1% Triton X-100, 10% BSA, 0.01% phenol red in PBS); 50 µl/well for 1 h), followed by wash with an additional washing step, using 200 µl PBS. And the bound antibodies were detected using 3,3',5,5'-Tetramethylbenzidine (TMB) PLUS2 substrate (50 µl/well). The enzymatic reaction was stopped with 0.2 M H 2 SO 4 (50 µl/well), and the OD values read at 450 nm using a HiPo Microplate Photometer MPP-96 (Biosan). Monoclonal and human antibody peptide ELISA MaxiSorp plates were coated overnight at 4°C with Peptide 7 or Peptide 10 (10 µM, 50 µl/well) dissolved in 50 mM sodium bicarbonate buffer (pH 9.5), or MA06 (18 nM, 50 µl/well, dissolved in 0.1 M Glycine-HCl buffer pH 2.75). mAb02 (323 nM, 50 µl/well) or human plasma (1:100, 50 µl/well) were added following a washing and blocking step and incubated for 1 h. Bound antibody was detected using HRP-conjugated anti-mouse or anti-human IgG (1:3,000 in blocking buffer; 50 µl/well) and developed as described above. Competition ELISA The ability of mAb02 to inhibit binding of DBLβ to ICAM-1 was tested using Maxisorp plates coated overnight (2 µg/ml, 50 µl/well, 4°C) with recombinant ICAM-1 [ 38 ]. Following blocking and washing the DBLβ domains were diluted in blocking buffer according to their ICAM-1-binding affinity tested in prior titration experiments (final concentrations; MA10: 5nM; MA01, MA11, MA23: 9nM; MA02, MA04, MA05, MA06, MA14, MA17, MA22: 18nM; MA03, MA07, MA09, MA16, NB33 and ND34: 36 nM; MA12, MA18: 73 nM; MA15, MA19, MA21, MA24: 145 nM; MA08, MA13, MA20: 582 nM). The diluted DBLβ domains were mixed 1:1 with mAb02 in 2-fold increasing concentrations (final mAb02 concentration 0.8 to 51.6 nM) and immediately added to the ICAM-1-coated wells and incubated while shaking for 1 h. Following a washing step, bound DBLβ domains were detected with HRP-conjugated anti-PentaHIS antibody (1:3000 in blocking buffer; 1 h, Qiagen) as described above. The inhibitory capacity of mAb02 was determined relative to control wells with DBLβ, but without mAb02. Peptide dot blotting Peptides (Table S2 ) were prepared in PBS with or without 50 mM DTT (heated 5 min at 95°C) and dotted (5 µg/peptide) onto 0.45 µM nitrocellulose membranes. The membranes were dried, blocked in TBS-T blocking buffer (TBS + 0.05% Tween20, 5% skimmed-milk) for 1 h and washed three times 5 min in TBS-T. mAb02 (10 µg/ml) in TBS-T plus 5% BSA was added to the membrane and incubated for 1 h. Following a washing step in TBS-T, HRP-conjugated anti-mouse IgG (1:3000 in TBS-T + 5% BSA) (Agilent) was added and incubated for 1 h. A final TBS-T wash was performed before the membrane was developed using KPL LumiGLO Reserve Chemiluminescent Substrates (Sera Care) and pictures captured using a BioRad ChemiDoc system. Western blotting Purified DBLβ proteins treated with or without DTT (95°C; 5 min) were run in a 4–12% BisTris gel (NuPAGE, Novex) using MOPS SDS running buffer (Invitrogen). Western blots were prepared on nitrocellulose membranes (0.45 µm) by wet transfer using standard methods. Binding sites were blocked in TBS-T containing 5% skimmed milk for 1 h before three times 5 min washes in TBS-T. Blot were probed with mAb02 (5 µg/ml in TBS-T, 5% BSA) for 1 h and subsequently washed. Bound antibody was detected using HRP-conjugated anti-mouse IgG (1:3000 in TBS-T + 5% skimmed milk) (Agilent). Three times 5 min washes between steps were in TBS-T. The membrane was developed using KPL LumiGLO Reserve Chemiluminescent substrate as described above. Bioinformatics analysis The DBLβ motif sequence of the ICAM-1-binding 3D7 PfEMP1 protein PFD1235w (also known as PF3D7_0425800) was used to BLASTp-extract 578 similar sequences from assemblies of Illumina whole genome sequencing data, as described previously [ 16 ]. This analysis included sequence data from worldwide P. falciparum genomes made available by MalariaGen [22; 29], supplemented by sequence data from PlasmoDB, NCBI, and the Broad Institute, but no new sequences were generated in this study. The average amino acid sequence identity of DBLβ domains was calculated using the Praline multiple sequence alignment tool using default settings [40; 41; 42; 43]. Frequencies and distribution of DBLβ motif -containing DBLβ domains for each geographical region was estimated from counting motif occurrence in the var sequence contigs assembled from P. falciparum genomes. Relatedness of the motifs was inferred by using the maximum likelihood method and the IQ-tree web server software algorithm [44; 45; 46]. The tree was visualized using the FigTree software [ 47 ]. A WebLogo3 sequence logo [ 48 ] was generated based on alignment of 3,042 protein sequences with the ICAM-1-binding motif (I[V/L]x3N[E]GG[P/A]xYx27GPPx3H) [ 22 ]. Protein structure visualization of sequence similarity was done using the ConSurf server using standard settings [49; 50; 51; 52], with the 5mza PDB file used as input [ 22 ]. All protein structure visualization was done using PymMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). Surface plasmon resonance (SPR) All SPR experiments were performed at 25°C on a Biacore T200 instrument equipped with a Series S CM5 sensor chip (Cytiva, Uppsala, Sweden). SPR running buffers and amine-coupling reagents (N-ethly-N’-(3-dimethlyaminoproply) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanol-amine HCl) were purchased from Cytiva. PBS-P with NaH 2 PO 4 -Na 2 HPO 4 (11.9 mM; pH 7.4), NaCl (137 mM), KCl (2.7 mM), and surfactant P20 (0.05% v/v) was used as running buffer. Anti-mouse antibody (Mouse Antibody Capture Kit, BR-1008-38, Cytiva, Uppsala, Sweden; 30 µg/ml) was immobilized at 11,000 relative units (RU) on flow cells 1 to 4 of the CM5 chip using standard EDC/NHS coupling chemistry and following the manufacturer’s protocol. For kinetic experiments, mAb02 and 24E9 [ 28 ] at 5 µg/ml (flow rate 10 µl/min, contact time 15 s) were captured at low-density (60–80 RU) in flow cells 2 (Fc2) and 3 (Fc3) of the anti-mouse antibody-coated CM5 chip. Flow cell 1 (Fc1) was used as a reference cell for subtraction of systematic instrumental drift. Serial threefold dilutions starting at 3.7 nM of MA06 (PFD1235W DBLβ D4) in SPR running buffer were injected sequentially in all flow cells at a flow rate of 60 µl/min and 600 s dissociation time. Regeneration solution (10 mM Glycine-HCl pH 1.7) was injected for 12 s at a flow rate of 20 µl/min after each injection to remove both captured antibodies and bound DBLβ proteins. Data processing and kinetic fitting were done using the BiaEvaluation software (v. 3.2.1, Cytiva, Uppsala, Sweden). The raw sensorgrams were double referenced (referring to the subtraction of the data over the reference surface and the average of the buffer injections from the binding responses), and the association and dissociation phases were fit using a 1:1 interaction model yielding single values for k a , k d , and the equilibrium dissociation constant KD (k d /k a ). Malaria parasites, antibody selection of IEs, and detection of native PfEMP1 P. falciparum parasite lines 3D7, BM057, HB3 were cultured in vitro and antibody-selected for IE surface expression of specific PfEMP1 proteins as described [15; 18]. We used the human mAb AB01 [ 53 ] to select 3D7 for PFD1235w-positive IEs, 24E9 [ 28 ] to select BM057 for JN037695-positive IEs, and a rat antiserum against HB3VAR03-DBLβ [ 22 ] to obtain HB3VAR03-positive IEs. Native PfEMP1 on the IE surface was detected by flow cytometry, essentially as described [18; 54]. In brief, late-stage IEs were purified on a magnet-activated cell sorting column (MACS) and incubated with mAb02, 24E9, an HB3VAR21-specific monoclonal antibody (150 µg/ml; negative mAb), negative mouse IgG (100 µg/ml) or rat serum (2.5 µL) depleted of human IE reactivity. IE-bound antibody was detected with goat-anti-mouse IgG PE (Abcam), fluorescein-conjugated goat-anti-mouse IgG (1∶100 in PBS + 2% BSA; Vectorlabs), or fluorescein-conjugated goat-anti-rat IgG (1∶100 in PBS + 2% BSA; Vectorlabs). Samples were DNA-stained using ethidium bromide (20 µg/ml) or Hoechst 33342 (10 µg/ml, Thermo Fischer) and run on a CytoFlex S4 flow cytometer (Beckman Coulter). Data were analysed using FlowJo software (Becton Dickinson). IEs were gated based on DNA stain, forward, and side scatter to exclude cell debris and uninfected erythrocytes. The genotypic identity of all parasite lines was routinely verified by genotyping [ 55 ] and mycoplasma infection was excluded using the MycoAlert mycoplasma detection kit (Lonza) according to manufacturer´s instructions. Assay for adhesion of IEs to ICAM-1 under flow Microslides VI 0.1 (ibidi) were coated overnight at 4°C with recombinant ICAM-1-Fc (50 µg/ml), then blocked (1% BSA in PBS) for one hour at 4°C. The microslides were connected to a syringe pump (Alladin, WPI) to generate a wall shear stress of 1 dyn/cm 2 mimicking physiological flow conditions found in the microvasculature. Infected erythrocytes (3–5% parasitemia or 1% haematocrit) were resolved in RPMI-1640 supplemented with 2% normal human serum and flowed over the microslide for 5 mins. The number of bound IEs per square millimetre for five separate fields was counted at 20 x magnification for a minimum of three independent experiments in triplicate were done. To inhibit ICAM-1 binding, IEs were pre-incubated with mAb02 (0.065-65 nM) for 15 minutes prior to the assay. Mouse IgG (65 nM) was included as a negative IgG control. The ICAM-1-specific antibody 15.2 (258 nM) and the DC4-DBLβ motif -specific monoclonal antibody 24E9 (65 nM) were included as additional controls. Declarations Availability of Data and Materials All data generated or analyzed during this study are included in this article and the supplementary information. Acknowledgements We thank Mette Ulla Madsen for excellent technical assistance and Maria Rosaria Bassi for her excellent help with the mouse work. Funding This work was supported by the Lundbeck Foundation (R313-2019-322), the Novo Nordisk Foundation (NNF170C0026778), and the Faculty of Health and Medical Sciences, University of Copenhagen. The funder had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions ATRJ, ND, and LB designed the study and the included experiments. ND, LB, RWO, JJR, BLM, MRW, and YA conducted the experiments. AM, NTN and RT collected the human samples. ND, LH and ATRJ wrote the manuscript. All authors reviewed the manuscript and approved the submitted version. Competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References W. World Health Organization, World Malaria Report 2022. (2022). Hviid, L. & Jensen, A. T. PfEMP1 - A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis. Adv. Parasitol. 88 , 51–84 (2015). Kraemer, S. M. & Smith, J. D. Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family. Mol. Microbiol. 50 , 1527–1538 (2003). Lavstsen, T., Salanti, A., Jensen, A. 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11:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6252569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6252569/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18465-1","type":"published","date":"2025-10-06T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82000430,"identity":"b707ad8c-eb67-4089-b963-4c9c608819fd","added_by":"auto","created_at":"2025-05-05 19:28:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1261470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogeny and conservation of the ICAM‑1 binding motif of DBLβ (DBLβ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emotif\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) ClustalW alignment of the ICAM‑1 binding motifs of 24 DBLβ\u003csub\u003emotif \u003c/sub\u003edomains (MA01-MA24, Table S1). Fully conserved residues are indicated by vertical shading. The DBLβ\u003csub\u003e \u003c/sub\u003edomains that were used for mouse immunizations are indicated by arrows. Underlined domains were produced specifically for this study. *The DBLb\u003csub\u003emotif\u003c/sub\u003e sequences of MA05 and MA06 are identical, but the flanking sequences are not. Triangles above the diagram indicate residues that interact directly with ICAM‑1: red triangles, amino acid residues critical for the ICAM‑1 interaction: orange triangles, and residues important for the structure of the ICAM‑1 binding site: blue triangles, as defined in [22]. Numbers above panel label residues according to MA01 (PF11_0521) (\u003cstrong\u003eb\u003c/strong\u003e) Maximum likelihood phylogram of 578 variants of DBLβ\u003csub\u003emotif\u003c/sub\u003e. The shaded areas indicate three major groups I-III, and the colored nodes represent 18 different clusters (C1-C18). (\u003cstrong\u003ec\u003c/strong\u003e) Ribbon representation of the ConSurf server generated model illustrating the more conserved motif (space filled spheres) compared to the surrounding sequence (ribbon structure) of the 24 DBLβ\u003csub\u003emotif\u003c/sub\u003e sequences. The structure was modelled on MA01 crystal structure (PF11_051 [22], PDB: 5mza). Residues are coloured according to their conservation score from high (purple) to low (blue). Βeta sheets and linker regions of ICAM-1 are shown in grey.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/645b031d9f66c18dd4d1f48c.jpeg"},{"id":82000423,"identity":"77735458-2f52-44f3-b640-b77dbd543824","added_by":"auto","created_at":"2025-05-05 19:28:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":605476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emAb02 reactivity against group A and group B PfEMP1 DBLβ domains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eHeatmap of the mAb02 reactivity (two-fold titrations 0-13 nM) with 24 DBLb proteins containing the ICAM-1 motif (MA01-MA24; left) or with ICAM-1-binding group B DBLβ domains (NB31-36 and NC37, right top) or non-ICAM-1-binding group A domains (NA24, NA28 and NA30-NA33, right bottom) without the motif (two-fold titrations 0-52 nM). Colors indicates ELISA optical density (OD) values as indicated by the bar. Domains used for immunization are indicated by triangles. (\u003cstrong\u003eb\u003c/strong\u003e) The ability of mAb02 (two-fold titrations 0-52 nM) to inhibit ICAM-1-binding of the 24 DBLβ\u003csub\u003emotif\u003c/sub\u003e domains or ICAM-1 binding group B NB33 and NB34 not containing the motif. Each DBLβ domain was tested by competition ELISA at a single concentration between 5\u0026nbsp;nM and 582\u0026nbsp;nM (for details, please refer to the Materials and Methods section). (\u003cstrong\u003ec\u003c/strong\u003e) SPR sensorgrams of the binding kinetics of MA06 to immobilized mAb02 (top) and 24E9 (bottom). The kinetic parameters for the interaction are determined from the fitting to a simple 1:1 interaction model and the standard errors of the fit are reported in Table\u0026nbsp;1. The experimental data are shown in black, and the kinetic fit in red.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/0a114944e2c370b941874d26.jpeg"},{"id":82000422,"identity":"9d3412c5-de26-4ed5-a285-23ad9d9faed0","added_by":"auto","created_at":"2025-05-05 19:28:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMapping of the mAb02 binding site on DBLβ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emotif\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Western blotting of selected DBLβ domains containing (MA06, MA10, MA17) or not containing (NB35) DBLβmotif under DTT-reducing (+) or non-reducing (–) conditions. (\u003cstrong\u003eb\u003c/strong\u003e) Dot blots showing mAb02 recognition of selected DTT-reduced (+) and non-reduced (–) peptides containing (Peptides 17-19, 24-26) or not containing (Peptide\u0026nbsp;7 and Peptide\u0026nbsp;11) DBLbmotif. Number above peptide sequences indicate residue position in MA01 (PF11_0521). Red triangles: residues with direct ICAM‑1 interaction; orange triangles: amino acid residues critical for the ICAM‑1 interaction; blue triangles: residues important for the structural architecture of the ICAM‑1 binding site (Fig.\u0026nbsp;1B and [22]). Grey shading indicate sequence of Peptide 10 used as minimal binding site for mAb02 in Figure 3C. (\u003cstrong\u003ec\u003c/strong\u003e) mAb02 and 24E9 reactivity with Peptide 10 (DBLbmotif, including three residues important for the architecture of the ICAM‑1 binding site and one residue with direct interaction with ICAM‑1) and with Peptide 7 (outside DBLbmotif). Mean OD values and their SD of three independent experiments are shown. (\u003cstrong\u003ed\u003c/strong\u003e) Close-up view of MA01 (PF11_0521) DBLβ green, bound to ICAM‑1D1D2 (grey PDB 5MZA). The amino acids corresponding to Peptide 10 and recognized by mAb02 are in a linker region (red loop) between two a-helices. (\u003cstrong\u003ee\u003c/strong\u003e) Sequence logo showing conservation of the 3,042 Peptide\u0026nbsp;10-like DBLbmotif sequences extracted from the Pf3K dataset.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/f600d7c8dea7e03c2857f90d.jpeg"},{"id":82000687,"identity":"6753d5cb-9e7f-47c3-b914-a6664b2d9926","added_by":"auto","created_at":"2025-05-05 19:36:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":518070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIgG recognition of native PfEMP1 proteins containing DBLβ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emotif\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Recognition of ICAM‑1-adhering IEs expressing native PfEMP1 proteins containing (BM057, 3D7VAR04) or not containing (HB3VAR03) DC4 by mAb02 (blue), 24E9 (grey), and either a control monoclonal antibody specific for a DBLb domain not containing DBLb\u003csub\u003emotif\u003c/sub\u003e (HB3VAR21_DBLβ) or control mouse IgG (purple). Lastly unstained or blank IEs were also controlled (black). Inserts in top right corner show IE recognition by positive control MA06- or MA08-specific rat antisera (green) and control rat IgG (dark green). (\u003cstrong\u003eb\u003c/strong\u003e) Inhibition (mean and SD) of BM057-IE adhesion under flowing conditions by mAb02 (blue), 24E9 (grey), 15.2 (a neutralizing ICAM‑1-specific monoclonal antibody), and control mouse IgG (black). Each condition was run in triplicate for a minimum of three independent experiments and expressed as average number bound per squared mm compared with untreated control. Statistical analysis was done using unpaired t-test with Welch’ correction (* P \u0026lt; 0.005; ** P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/2677deb11af009397b840f4f.jpeg"},{"id":82000691,"identity":"4dfd6923-66e9-4ccd-ab0d-f714df334d80","added_by":"auto","created_at":"2025-05-05 19:36:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":330038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune plasma IgG recognition of DBLβ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emotif\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eLevels of IgG specific for a representative DBLb\u003csub\u003emotif\u003c/sub\u003e (Peptide\u0026nbsp;10) and the DBLb domain it resides in (MA06) in immune plasma from Beninese children. Cerebral malaria, CM (red triangles; N=21); uncomplicated malaria, UM (blue squares; N=40); non-cerebral malaria, non-CM severe malaria (light blue circles; N=33). Individual data points, the linear regression line for all the data points (solid) calculated as Pearson correlation coefficient and its 95% confidence lines (dashed) are shown. Negative cut-offs are indicated by grey shading.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/e3edbd34397ac7abbb703d56.jpeg"},{"id":93419755,"identity":"7b69ee75-1411-4f94-913c-11d4e29a9476","added_by":"auto","created_at":"2025-10-13 16:07:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4393358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/6ab4d25a-8085-42a3-8990-f68129fdb55a.pdf"},{"id":82000421,"identity":"64b7b2f6-659e-43ca-b007-0ac337e8c4ba","added_by":"auto","created_at":"2025-05-05 19:28:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":95930,"visible":true,"origin":"","legend":"","description":"","filename":"Suppfigure1fixedforpublishing.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/e2b3709822a748465d61dadd.pdf"},{"id":82000684,"identity":"a7191e2a-8fba-4d9c-8c62-c1862f09cad4","added_by":"auto","created_at":"2025-05-05 19:36:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":98727,"visible":true,"origin":"","legend":"","description":"","filename":"Suppfigure2fixedforpublishing.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6252569/v1/f8935b9d529d433e26f32af9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A monoclonal antibody selectively recognizing PfEMP1 proteins associated with cerebral malaria","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e parasites are a major cause of morbidity and mortality among children in sub-Saharan African [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The virulence of these parasites has been linked to the infected erythrocyte (IE) surface expression of members of the \u003cem\u003ePlasmodium falciparum\u003c/em\u003e erythrocyte membrane protein 1 (PfEMP1) variants. PfEMP1 mediates sequestration of IEs by binding to vascular host receptors leading to tissue-specific inflammation, circulatory obstruction and organ dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The PfEMP1 proteins are encoded by the \u003cem\u003evar\u003c/em\u003e multi-gene family with approximately 60 members per haploid parasite genome. Despite their extensive inter- and intra-clonal diversity, the \u003cem\u003evar\u003c/em\u003e genes can be classified into three major groups (A, B, and C) based on their sequence, chromosomal location and transcriptional direction [3; 4]. Transcription of group A \u003cem\u003evar\u003c/em\u003e genes, which are less diverse than the other groups, has repeatedly been linked to the development of severe malaria (SM) [5; 6; 7; 8]. This is consistent with the restricted serological diversity of \u003cem\u003eP. falciparum\u003c/em\u003e parasites from SM patients and with the relatively rapid acquisition of immunity to severe disease in individuals living in areas with high transmission of \u003cem\u003eP. falciparum\u003c/em\u003e [9; 10; 11; 12]. PfEMP1 proteins consist of two to seven highly polymorphic Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR) extracellular domains, a conserved transmembrane domain, and an intracellular acidic terminal segment (ATS) [3; 13; 14]. DBL and CIDR domains can be divided into different types and subtypes (α, β, γ, δ, ε, ζ, х and α, β, γ, respectively) based on sequence similarity. Furthermore, tandem domain cassettes (DCs) of specific DBL and CIDR domains are shared by different parasite clones [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Examples include DC4 (DBLα1.1/1.4-CIDRα1.6-DBLβ3) and DC13 (DBLα1.7-CIDRα1.4) in group A, and DC8 (DBLα2-CIDRα1.1-DBLβ12-DBLγ4/6) in group B/A [13; 15]. CIDRα1 domains in DC8 and DC13 bind endothelial protein receptor C (EPCR) [16; 17], whereas DBLβ domains in DC4 bind intercellular adhesion molecule 1 (ICAM-1) [15; 18]. These adhesion phenotypes have both been linked to SM [5; 15; 19; 20], and parasites expressing PfEMP1 proteins containing both EPCR- and ICAM-1-binding domains (dual binders) have been specifically implicated in the pathogenesis of cerebral malaria (CM) [21; 22]. These proteins contain an EPCR-binding CIDRα1 domain followed immediately by a highly conserved ICAM-1-binding DBLβ domain [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The latter share a DBLβ sequence motif (DBLβ\u003csub\u003emotif\u003c/sub\u003e: I[V/L]X\u003csub\u003e3\u003c/sub\u003eN[E]GG[P/A]XYX\u003csub\u003e27\u003c/sub\u003eGPPX\u003csub\u003e3\u003c/sub\u003eH) that is specific for dual binders and contains the amino acid residues needed for their ICAM-1 binding. DBLβ\u003csub\u003emotif\u003c/sub\u003e is restricted to group A and a few ICAM-1-binding group B/A PfEMP1 proteins, and is thus absent from ICAM-1-binding DBLβ domains in the group B and group C PfEMP1 proteins that have been linked to uncomplicated malaria [22; 23; 24; 25].\u003c/p\u003e \u003cp\u003eThe above studies have thus established associations between specific PfEMP1 subgroups and specific clinical presentation of \u003cem\u003eP. falciparum\u003c/em\u003e malaria. In addition, the pathogenesis of CM appears to involve the presence of dual-binding IEs within brain microvasculature endothelial cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Broadly cross-reactive monoclonal antibodies targeting DBLβ\u003csub\u003emotif\u003c/sub\u003e of most, if not all, group A and B/A DBLβ ICAM-1-binding domains would be a powerful tool in elucidating the molecular basis of CM pathogenesis and might have potential in anti-CM therapy. IgG that targets DBLβ\u003csub\u003emotif\u003c/sub\u003e domains and inhibits their binding to ICAM-1 is detectable in the plasma of naturally \u003cem\u003eP. falciparum\u003c/em\u003e-exposed individuals, and can be induced by immunization of rats with recombinant DBLβ\u003csub\u003emotif\u003c/sub\u003e protein [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Importantly, however, the antibodies do not target all variants. We have previously described a mouse monoclonal antibody (24E9) that binds the convex surface of DC4DBLβ and interferes with adhesion of DC4-positive IEs to ICAM-1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but does not interfere with the adhesion of IEs positive for other (non-DC4) CM-associated group A or B/A PfEMP1 proteins (unpublished data). The current study was therefore designed to investigate whether broadly cross-reactive and ICAM-1 adhesion inhibitory monoclonal antibodies could be induced by immunization with combinations of multiple DBLβ\u003csub\u003emotif\u003c/sub\u003e proteins.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of additional DBLβ\u003csub\u003emotif\u003c/sub\u003e domains\u003c/h2\u003e \u003cp\u003eWe have previously defined a 53-amino acid sequence motif (DBLβ\u003csub\u003emotif\u003c/sub\u003e) that contains the determinants for ICAM-1 binding of group A and B/A PfEMP1 proteins [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further examine the diversity of this motif, we extracted 62,000 DBLβ sequences from the Pf3K database of \u003cem\u003eP. falciparum\u003c/em\u003e genomes collected worldwide [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We identified 3,042 DBLβ\u003csub\u003emotif\u003c/sub\u003e domains, of which 578 were unique, but similar, to the 15 previously published DBLβ\u003csub\u003emotif\u003c/sub\u003e variants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The sequences could be grouped into three major groups (I-III) containing a total of 18 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To test their capacity for binding to ICAM-1, we produced nine different recombinant proteins (underlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]Their sequences were obtained from different clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and selected to be distant from previously identified ICAM-1 binding sequences (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and to be common in the Pf3K dataset. All nine proteins bound ICAM-1 (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Position sequence similarity matrix (PSSM) analysis showed that the nine new and the 15 previously published [15; 22; 25] proteins showed 77% sequence similarity within the motif region and 58% sequence similarity if the sequences up- and down-stream of the motif were included (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003emAb02 is cross-reactive against motif-containing PfEMP1\u003c/h3\u003e\n\u003cp\u003eParasites that express DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing PfEMP1 proteins on the IE surface adhere to both ICAM-1 and EPCR (dual binders) and have been linked to sequestration of IEs in the microvasculature of the brain [22; 26; 30]. In an attempt to generate a monoclonal antibody selectively recognizing all or most DBLβ\u003csub\u003emotif\u003c/sub\u003e, we immunized with a total of eight different DBLβ\u003csub\u003emotif\u003c/sub\u003e domains (MA01, MA05, MA07, MA16, MA17, MA20, MA22, MA23, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), two at a time. Of several monoclonal antibodies generated from these mice, mAb02 (IgG1, κ light chains) reacted at high titres with all 24 DBLβ\u003csub\u003emotif\u003c/sub\u003e domains tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, left) and not with any of 13 ICAM-1-binding DBLβ domains from Group B or C DBLβ and not containing DBLβ\u003csub\u003emotif\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, right). mAb02 also reduced the binding of all 24 DBLβ\u003csub\u003emotif\u003c/sub\u003e domains to ICAM-1 in a concentration-dependent manner but had no effect on the binding of two non-motif ICAM-1-binding DBLβ domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Of note, mAb02 reduced ICAM-1 binding of both DC4 and non-DC4 group A and B/A DBLβ\u003csub\u003emotif\u003c/sub\u003e domains, unlike the previously described monoclonal antibody 24E9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which only binds DBLβ domains in DC4. The two monoclonal antibodies bound with similar picomolar range affinity to a typical DC4 DBLβ\u003csub\u003emotif\u003c/sub\u003e domain (MA06, PFD1235W, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters derived from surface plasmon resonance (SPR) experiments on mAbs interaction with PFD1235W DBLβ\u003csub\u003emotif\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[nM]\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e [M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e [s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e [pM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emAb02::MA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.96 (\u0026plusmn;\u0026thinsp;0.02) x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.13 (\u0026plusmn;\u0026thinsp;0.01) x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026ndash;1 interaction model\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24E9mAb::MA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33 (\u0026plusmn;\u0026thinsp;0.01) x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.84 (\u0026plusmn;\u0026thinsp;0.01) x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026ndash;1 interaction model\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eBinding kinetics of immobilized mAb02 and 24E9 monoclonal antibodies binding to MA06 (PFD1235W\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eDBLβ D4). \u003csup\u003e1\u003c/sup\u003eMaximum concentration tested\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003emAb02 recognises a linear epitope\u003c/h3\u003e\n\u003cp\u003emAb02 recognised both reduced and non-reduced DBLβ\u003csub\u003emotif\u003c/sub\u003e proteins (MA06, MA10, MA17) in Western blot and showed no reactivity with the control protein NB35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results were corroborated by dot blots employing different reduced and non-reduced DBLβ-derived peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The dot blot data further indicated that the DBLβ\u003csub\u003emotif\u003c/sub\u003e epitope targeted by mAb02 was the highly conserved sequence GGPGYYNTEVQKKDR (shaded grey in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This inference was verified by peptide ELISA, as mAb02 efficiently recognized Peptide 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), representing the ICAM-1-binding epitope of Group A and B/A DBLβ domains (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In contrast, 24E9 did not recognize Peptide 10, in accordance with our earlier data that 24E9 recognizes a conformational epitope that involves the Peptide 10 sequence, but also additional amino acids outside it [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The Peptide 10 sequence maps to the disordered regions of the ICAM-1 binding site in the crystal structure (5MZA) of DBLβ\u003csub\u003emotif\u003c/sub\u003e (MA01, PF11_0521) bound to ICAM-1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), and is highly conserved among our panel of verified DBLβ\u003csub\u003emotif\u003c/sub\u003e ICAM-1 binders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the 3,042 DBLβ\u003csub\u003emotif\u003c/sub\u003e domains in the Pf3K database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\n\u003ch3\u003emAb02 reacts with native PfEMP1 variants and inhibits IE adhesion to ICAM-1\u003c/h3\u003e\n\u003cp\u003eBoth mAb02 and 24E9 bound to BM057- and 3D7-IEs s expressing DC4-containing PfEMP1 with DBLβ\u003csub\u003emotif\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, only mAb02 bound to HB3-IEs expressing the non-DC4, but DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing PfEMP1 protein HB3VAR03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). IEs expressing DBLβ\u003csub\u003emotif\u003c/sub\u003e adhere to ICAM-1, a phenotype linked to sequestration of IEs in the microvasculature of the brain [31; 32]. The adhesion of BM057-IEs to ICAM-1 under physiological flow conditions (wall shear stress of 1 dyn/cm\u003csup\u003e2\u003c/sup\u003e) was inhibited by mAb02 in a concentration-dependent manner, with efficiency similar to that previously reported for 24E9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCross-reactive DBLβ\u003c/b\u003e \u003csub\u003e \u003cb\u003emotif\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eantibodies induced during\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003einfections\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe plasma of adults and children living in areas with stable \u003cem\u003eP. falciparum\u003c/em\u003e transmission contains DBLβ\u003csub\u003emotif\u003c/sub\u003e-reactive IgG [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We measured levels of IgG specific for a typical DBLβ\u003csub\u003emotif\u003c/sub\u003e (Peptide 10) and the entire DBLβ domain containing it (MA06) in 94 plasma samples from Beninese children with CM (N\u0026thinsp;=\u0026thinsp;21), non-CM severe malaria (nonCM-SM; N\u0026thinsp;=\u0026thinsp;33) and uncomplicated malaria (UM; N\u0026thinsp;=\u0026thinsp;40) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A high proportion of the samples reacted with Peptide 10 (42/94; 45%) and/or with MA06 (57/94, 61%). Overall, the Peptide 10 and MA06 responses were weakly correlated (r\u0026thinsp;=\u0026thinsp;0.22; P\u0026thinsp;=\u0026thinsp;0.03). This was almost entirely due to the CM samples (r\u0026thinsp;=\u0026thinsp;0.45; P\u0026thinsp;=\u0026thinsp;0.04), as these responses were not significantly correlated when considering either the SM samples (r\u0026thinsp;=\u0026thinsp;0.02; P\u0026thinsp;=\u0026thinsp;0.91) or the UM samples (r\u0026thinsp;=\u0026thinsp;0.21; P\u0026thinsp;=\u0026thinsp;0.19) separately. The IgG reactivity to Peptide 10 and MA06 did not depend significantly on age (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM (n\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003enCSM (n\u0026thinsp;=\u0026thinsp;33)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCM\u0026thinsp;+\u0026thinsp;SM (n\u0026thinsp;=\u0026thinsp;54)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eUM (n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42 (30; 48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (18; 54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.5 (21.75; 49.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e36 (16.25; 48)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMales/Females (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.1/42.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.5/45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.6/44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.5/37.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHaemoglobin (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.3 (3.3; 6.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6 (3.4; 5.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.6 (3.4; 6.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e8.6 (7.15; 10.35)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlantyre score (0\u0026ndash;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (2; 2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (5; 5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (2; 5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParasite density (p/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,840 (942.5; 976,00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39,112 (3,280; 240,000.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16,727.5 (1,900; 185,333.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e60,933.5 (6,582; 158,000)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMortality rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eValues are medians (25th; 75th percentile). CM: Cerebral malaria, nCSM: non-cerebral severe malaria, SM: severe malaria, UM: Uncomplicated malaria. NA: Not applicable (no coma).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCM is caused by cerebral sequestration of IEs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These IEs often express structurally and serologically related PfEMP1 proteins that can bind both ICAM-1 and EPCR (dual binders) [22; 30; 35]. The ICAM-1 binding site of these PfEMP1 resides within a particular sequence motif (DBLβ\u003csub\u003emotif\u003c/sub\u003e) that can be found among PfEMP1 proteins belonging to group A and B/A. Using blood-brain barrier (BBB) organoids, we recently showed that DBLβ\u003csub\u003emotif\u003c/sub\u003e-positive IEs are selectively taken up by brain endothelial cells \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The uptake depends on ICAM-1 and results in breakdown of the BBB and swelling of the BBB organoids [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This suggests that internalization of DBLβ\u003csub\u003emotif\u003c/sub\u003e-positive IEs is involved in the pathogenesis of CM [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, we describe a monoclonal antibody, mAb02, which selectively recognizes group A and B/A PfEMP1 proteins carrying DBLβ\u003csub\u003emotif\u003c/sub\u003e. Importantly, the antibody recognizes an epitope in DBLβ\u003csub\u003emotif\u003c/sub\u003e that is conserved among such domains both within and outside DC4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This sets it apart from a previously described monoclonal antibody, 24E9, which only binds to DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing domains within DC4 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 24E9 was derived from mice immunized with a single DC4 DBLβ domain (MA06, PFD1235w; DBLβ3_D4), whereas mAb02 was obtained after sequential immunization with eight different DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing DBLβ domains. The latter domains were chosen after analysis of 62,000 DBLβ domains in the Pf3K database, and selected to capture the global diversity of the 3,042 DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing domains identified there, within as well as outside DC4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Together, these data indicate that the immunization strategy used here can focus the immune response on broadly conserved and conformational epitope(s) shared by the immunogens. Of biological significance, mAb02 recognized native PfEMP1 proteins on the surface of erythrocytes infected by three different parasite lines (3D7, BM057, HB3) that had been selected for expression of DBLβ\u003csub\u003emotif\u003c/sub\u003e PfEMP1s including one (HB3), which was not recognized by the 24E9 monoclonal antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, sub-nanomolar concentrations of mAb02 significantly inhibited the adhesion of BM057-IEs to ICAM-1 binding under physiologically realistic flow conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The target epitope of mAb02 mapped to a continuous linker region between two α-helices (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which contain amino acid residues strictly conserved among DBLβ\u003csub\u003emotif\u003c/sub\u003e domains recognised by mAb02 and known to be critical for the ICAM-1 interaction and the architecture of the ICAM-1-binding site [22; 28]. This indicates that mAb02 functions by steric hindrance and masks the ICAM-1 binding site of DBLβ\u003csub\u003emotif\u003c/sub\u003e domains. The clinical significance of the DBLβ\u003csub\u003emotif\u003c/sub\u003e epitope recognized by mAb02 is supported by the observation that levels of IgG specific for a representative DBLβ\u003csub\u003emotif\u003c/sub\u003e epitope (Peptide 10) and the DBLβ within which it resides (MA06) correlated significantly among Beninese children with CM, but not among sympatric children with either non-CM severe malaria or uncomplicated malaria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It suggests that the CM children were infected by parasites expressing PfEMP1 variants containing a DBLβ\u003csub\u003emotif\u003c/sub\u003e epitope.\u003c/p\u003e \u003cp\u003eIn conclusion, we have identified a monoclonal antibody (mAb02) that targets a functionally important epitope defining DBLβ domains of dual-binding PfEMP1 proteins implicated in the pathogenesis of CM [22; 26; 30; 31]. This epitope is therefore a promising candidate for inclusion in a vaccine designed to protect against CM. In addition, mAb02 itself may be exploited to develop biological therapeutics for cerebral malaria, an approach that is currently receiving a lot of attention in the fight against malaria [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e All methods were carried out in accordance with relevant guidelines and regulations. All animal procedures were approved by The Danish Animal Procedures Committee (Dyrefors\u0026oslash;gstilsynet) as described in permit no. 2013-15-2934-00920, and all experiments were done according to the guidelines described in Danish act LBK 1306 (23/11/2007) and BEK 1273 (12/12/2005). The mice immunizations were conducted in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines and reported according to ARRIVE guidelines. The studies involving collection of human plasma approved by the DRFMT Comit\u0026eacute; National d\u0026rsquo;Ethique pour la Recherche en Sante, No. 87/MS/DC/SGM/DRFMT/CNERS/SA Cotonou, Benin.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eRecombinant proteins and peptides\u003c/h3\u003e\n\u003cp\u003eDNA sequences encoding DBLβ domains MA01 to MA24 (group A, DBLβ\u003csub\u003emotif\u003c/sub\u003e), NB31 to NB36 and NC37 (ICAM-1 binding group B and C, non-DBLβ\u003csub\u003emotif\u003c/sub\u003e), and NA24, NA28 and NA30-33 (non-ICAM-1 binding group A, non-DBLβ\u003csub\u003emotif\u003c/sub\u003e) were PCR-amplified from \u003cem\u003eP. falciparum\u003c/em\u003e genomic DNA or produced as synthetic constructs (Eurofins). Amplicons were cloned into a modified pET15b vector and expressed as HIS-tagged proteins (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) in \u003cem\u003eE. coli\u003c/em\u003e Shuffle C3029 (New England Biolabs) as described [15; 22; 25; 27]. All proteins were purified by immobilized metal ion affinity chromatography using HisTrap HP 1 mL columns (GE Healthcare).\u003c/p\u003e \u003cp\u003eDBLβ\u003csub\u003emotif\u003c/sub\u003e domains used for immunization of mice (MA1, MA5, MA7, MA16, MA19, MA21, MA22, MA24; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were further purified using Fc-tagged ICAM-1 coupled to a HiTrap NHS-activated HP column as described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The DBLβ proteins were eluted using 0.1 M Glycine-HCl buffer (pH 2.75) and neutralized in 1 M Tris-HCl buffer (pH 10). Proteins used for surface plasmon resonance (SPR) analysis (see below) were purified using size-exclusion gel filtration purification column HiPrep 16/60 Sephacryl S-300 HR (Cytiva). Recombinant Fc-tagged ICAM-1 was expressed in HEK293 cells and purified on a HiTrap Protein G HP (GE Healthcare) as described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePeptides representing the ICAM-1 binding region, or smaller sites within of DBLβ domains found in MA01, MA06, MA09 or MA10 (PF11_0521, PFD1235W D4, DD2VAR32 or KM364031 respectively, table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were obtained from Schafer-N (Denmark).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunization, hybridomas and monoclonal antibody production\u003c/h2\u003e \u003cp\u003eThe study involved 6-week-old female Balb/c ByJR mice purchased from Janvier Labs that were immunized with two different group A ICAM-1 binding DBLβ domains (20 \u0026micro;g of each domain in Addavax 1:1/mouse) at each of four time points, i.e., with a total of eight different domains. The following DBLβ domains was used: MA07 and MA16 (intramuscularly (i.m.), day 0), MA21 and MA22 (i.m., day 13), MA19 and MA05 (i.m., day 31), and MA24 and MA01 (intraperitoneally, Day 52). The mice were euthanized using isoflurane and cervical dislocation and bled out on day 55, and the spleen were taken out. Single splenocytes were fused to immortalized myeloma cells (SP2/0-Ag14) cells using polyethylene glycol (PEG) and selected using semisolid medium HAT-containing medium according to manufacturer\u0026rsquo;s protocol (ClonaCell-HY hybridoma cloning kit, StemCell Technologies). Hybridomas were picked after two weeks and moved to individual wells of flatbottomed 96-well plates (Nunc) and after further culture for one-week, undiluted cell supernatants were tested for DBLβ-reactive antibodies by ELISA (see below). Cells from positive wells were single cell-sorted using a BD FACS Melody Cell Sorter and cultured as above. Positive clones were adapted to DMEM medium containing 1% L-glutamine (Sigma), 1% Pen/Strep (Sigma) and 10% heat-inactivated HY-clone FBS ultra-low fetal calf serum (GE Healthcare) and subsequently cultured in one-litre Cell-Line bioreactor flask (Sigma-Aldrich) following manufacturer\u0026rsquo;s instructions. Monoclonal antibodies were purified from filtered (0.22 \u0026micro;m) supernatants on a Protein G HP column (GE Healthcare). Bound IgG was eluted in low-pH buffer 0.1 M Glycine-HCl buffer (pH 2.75) and directly neutralized using 1 M Tris-HCl (pH 9.0) to neutral pH, and subsequently buffer-exchanged to PBS using centrifugal VivaSpin columns (Sigma Aldrich).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHuman plasma samples\u003c/h2\u003e \u003cp\u003ePlasma samples from children with CM (n\u0026thinsp;=\u0026thinsp;21), severe non-CM malaria (SM; n\u0026thinsp;=\u0026thinsp;33), or uncomplicated \u003cem\u003eP. falciparum\u003c/em\u003e malaria (n\u0026thinsp;=\u0026thinsp;40), as defined by the World Health Organization [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] were collected in 2019 at three hospital centers in Cotonou, Benin. Plasma samples were collected at two hospitals in Cotonou, Southern Benin. The samples were collected during malaria transmission season from June-September 2019 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Blood samples were collected at day of hospitalization and children below 5 years of age were subjected to clinical investigation and screened by rapid diagnostic test for malaria (Malaria Pf/Pan or DiaQuick). Clinical categorizations were determined according to WHO definitions\u003csup\u003e1\u003c/sup\u003e. Patients categorized with cerebral malaria presented with unarousable coma (Blantyre coma score\u0026thinsp;\u0026le;\u0026thinsp;2) an no other cause of coma. Non-cerebral severe malaria patients presented with hyper parasitemia (\u0026gt;\u0026thinsp;50,000), and/or severe anemia (hemoglobin\u0026thinsp;\u0026lt;\u0026thinsp;5 g/dl) and no coma. Uncomplicated malaria patients presented with fever, headache or myalgia without signs of severe disease (eg organ dysfunction). Parasitaemia was quantified by microscopy. All samples were collected upon informed consent obtained from parent or legal guardian\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHybridoma screening ELISA\u003c/h2\u003e \u003cp\u003eMaxiSorp plates (Sigma-Aldrich) were coated with recombinant DBLβ proteins (50 \u0026micro;l; 2 \u0026micro;g/ml) in 0.1 M Glycine-HCl buffer (pH 2.75) overnight at 4\u0026deg;C, washed and blocked as described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Two screenings of hybridoma supernatants were done, the first using a pool of the DBLβ domains used for immunization and the second using individual MA01-M24 pool as coating antigen (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). 40 \u0026micro;l hybridoma supernatant (containing mAbs) were incubated in wells 1 h while shaking at room temperature. After wash, mAbs were bound with HRP-conjugated anti-mouse IgG (1:3000 in blocking buffer (1% Triton X-100, 10% BSA, 0.01% phenol red in PBS); 50 \u0026micro;l/well for 1 h), followed by wash with an additional washing step, using 200 \u0026micro;l PBS. And the bound antibodies were detected using 3,3',5,5'-Tetramethylbenzidine (TMB) PLUS2 substrate (50 \u0026micro;l/well). The enzymatic reaction was stopped with 0.2 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (50 \u0026micro;l/well), and the OD values read at 450 nm using a HiPo Microplate Photometer MPP-96 (Biosan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMonoclonal and human antibody peptide ELISA\u003c/h2\u003e \u003cp\u003eMaxiSorp plates were coated overnight at 4\u0026deg;C with Peptide 7 or Peptide 10 (10 \u0026micro;M, 50 \u0026micro;l/well) dissolved in 50 mM sodium bicarbonate buffer (pH 9.5), or MA06 (18 nM, 50 \u0026micro;l/well, dissolved in 0.1 M Glycine-HCl buffer pH 2.75). mAb02 (323 nM, 50 \u0026micro;l/well) or human plasma (1:100, 50 \u0026micro;l/well) were added following a washing and blocking step and incubated for 1 h. Bound antibody was detected using HRP-conjugated anti-mouse or anti-human IgG (1:3,000 in blocking buffer; 50 \u0026micro;l/well) and developed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCompetition ELISA\u003c/h2\u003e \u003cp\u003eThe ability of mAb02 to inhibit binding of DBLβ to ICAM-1 was tested using Maxisorp plates coated overnight (2 \u0026micro;g/ml, 50 \u0026micro;l/well, 4\u0026deg;C) with recombinant ICAM-1 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Following blocking and washing the DBLβ domains were diluted in blocking buffer according to their ICAM-1-binding affinity tested in prior titration experiments (final concentrations; MA10: 5nM; MA01, MA11, MA23: 9nM; MA02, MA04, MA05, MA06, MA14, MA17, MA22: 18nM; MA03, MA07, MA09, MA16, NB33 and ND34: 36 nM; MA12, MA18: 73 nM; MA15, MA19, MA21, MA24: 145 nM; MA08, MA13, MA20: 582 nM). The diluted DBLβ domains were mixed 1:1 with mAb02 in 2-fold increasing concentrations (final mAb02 concentration 0.8 to 51.6 nM) and immediately added to the ICAM-1-coated wells and incubated while shaking for 1 h. Following a washing step, bound DBLβ domains were detected with HRP-conjugated anti-PentaHIS antibody (1:3000 in blocking buffer; 1 h, Qiagen) as described above. The inhibitory capacity of mAb02 was determined relative to control wells with DBLβ, but without mAb02.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePeptide dot blotting\u003c/h2\u003e \u003cp\u003ePeptides (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were prepared in PBS with or without 50 mM DTT (heated 5 min at 95\u0026deg;C) and dotted (5 \u0026micro;g/peptide) onto 0.45 \u0026micro;M nitrocellulose membranes. The membranes were dried, blocked in TBS-T blocking buffer (TBS\u0026thinsp;+\u0026thinsp;0.05% Tween20, 5% skimmed-milk) for 1 h and washed three times 5 min in TBS-T. mAb02 (10 \u0026micro;g/ml) in TBS-T plus 5% BSA was added to the membrane and incubated for 1 h. Following a washing step in TBS-T, HRP-conjugated anti-mouse IgG (1:3000 in TBS-T\u0026thinsp;+\u0026thinsp;5% BSA) (Agilent) was added and incubated for 1 h. A final TBS-T wash was performed before the membrane was developed using KPL LumiGLO Reserve Chemiluminescent Substrates (Sera Care) and pictures captured using a BioRad ChemiDoc system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003ePurified DBLβ proteins treated with or without DTT (95\u0026deg;C; 5 min) were run in a 4\u0026ndash;12% BisTris gel (NuPAGE, Novex) using MOPS SDS running buffer (Invitrogen). Western blots were prepared on nitrocellulose membranes (0.45 \u0026micro;m) by wet transfer using standard methods. Binding sites were blocked in TBS-T containing 5% skimmed milk for 1 h before three times 5 min washes in TBS-T. Blot were probed with mAb02 (5 \u0026micro;g/ml in TBS-T, 5% BSA) for 1 h and subsequently washed. Bound antibody was detected using HRP-conjugated anti-mouse IgG (1:3000 in TBS-T\u0026thinsp;+\u0026thinsp;5% skimmed milk) (Agilent). Three times 5 min washes between steps were in TBS-T. The membrane was developed using KPL LumiGLO Reserve Chemiluminescent substrate as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe DBLβ\u003csub\u003emotif\u003c/sub\u003e sequence of the ICAM-1-binding 3D7 PfEMP1 protein PFD1235w (also known as PF3D7_0425800) was used to BLASTp-extract 578 similar sequences from assemblies of Illumina whole genome sequencing data, as described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This analysis included sequence data from worldwide \u003cem\u003eP. falciparum\u003c/em\u003e genomes made available by MalariaGen [22; 29], supplemented by sequence data from PlasmoDB, NCBI, and the Broad Institute, but no new sequences were generated in this study. The average amino acid sequence identity of DBLβ domains was calculated using the Praline multiple sequence alignment tool using default settings [40; 41; 42; 43]. Frequencies and distribution of DBLβ\u003csub\u003emotif\u003c/sub\u003e-containing DBLβ domains for each geographical region was estimated from counting motif occurrence in the \u003cem\u003evar\u003c/em\u003e sequence contigs assembled from \u003cem\u003eP. falciparum\u003c/em\u003e genomes. Relatedness of the motifs was inferred by using the maximum likelihood method and the IQ-tree web server software algorithm [44; 45; 46]. The tree was visualized using the FigTree software [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA WebLogo3 sequence logo [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] was generated based on alignment of 3,042 protein sequences with the ICAM-1-binding motif (I[V/L]x3N[E]GG[P/A]xYx27GPPx3H) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein structure visualization of sequence similarity was done using the ConSurf server using standard settings [49; 50; 51; 52], with the 5mza PDB file used as input [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All protein structure visualization was done using PymMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schr\u0026ouml;dinger, LLC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSurface plasmon resonance (SPR)\u003c/h2\u003e \u003cp\u003eAll SPR experiments were performed at 25\u0026deg;C on a Biacore T200 instrument equipped with a Series S CM5 sensor chip (Cytiva, Uppsala, Sweden). SPR running buffers and amine-coupling reagents (N-ethly-N\u0026rsquo;-(3-dimethlyaminoproply) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanol-amine HCl) were purchased from Cytiva. PBS-P with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e-Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (11.9 mM; pH 7.4), NaCl (137 mM), KCl (2.7 mM), and surfactant P20 (0.05% v/v) was used as running buffer. Anti-mouse antibody (Mouse Antibody Capture Kit, BR-1008-38, Cytiva, Uppsala, Sweden; 30 \u0026micro;g/ml) was immobilized at 11,000 relative units (RU) on flow cells 1 to 4 of the CM5 chip using standard EDC/NHS coupling chemistry and following the manufacturer\u0026rsquo;s protocol. For kinetic experiments, mAb02 and 24E9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] at 5 \u0026micro;g/ml (flow rate 10 \u0026micro;l/min, contact time 15 s) were captured at low-density (60\u0026ndash;80 RU) in flow cells 2 (Fc2) and 3 (Fc3) of the anti-mouse antibody-coated CM5 chip. Flow cell 1 (Fc1) was used as a reference cell for subtraction of systematic instrumental drift. Serial threefold dilutions starting at 3.7 nM of MA06 (PFD1235W DBLβ D4) in SPR running buffer were injected sequentially in all flow cells at a flow rate of 60 \u0026micro;l/min and 600 s dissociation time. Regeneration solution (10 mM Glycine-HCl pH 1.7) was injected for 12 s at a flow rate of 20 \u0026micro;l/min after each injection to remove both captured antibodies and bound DBLβ proteins. Data processing and kinetic fitting were done using the BiaEvaluation software (v. 3.2.1, Cytiva, Uppsala, Sweden). The raw sensorgrams were double referenced (referring to the subtraction of the data over the reference surface and the average of the buffer injections from the binding responses), and the association and dissociation phases were fit using a 1:1 interaction model yielding single values for k\u003csub\u003ea\u003c/sub\u003e, k\u003csub\u003ed\u003c/sub\u003e, and the equilibrium dissociation constant KD (k\u003csub\u003ed\u003c/sub\u003e/k\u003csub\u003ea\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMalaria parasites, antibody selection of IEs, and detection of native PfEMP1\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. falciparum\u003c/em\u003e parasite lines 3D7, BM057, HB3 were cultured in vitro and antibody-selected for IE surface expression of specific PfEMP1 proteins as described [15; 18]. We used the human mAb AB01 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] to select 3D7 for PFD1235w-positive IEs, 24E9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to select BM057 for JN037695-positive IEs, and a rat antiserum against HB3VAR03-DBLβ [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] to obtain HB3VAR03-positive IEs. Native PfEMP1 on the IE surface was detected by flow cytometry, essentially as described [18; 54]. In brief, late-stage IEs were purified on a magnet-activated cell sorting column (MACS) and incubated with mAb02, 24E9, an HB3VAR21-specific monoclonal antibody (150 \u0026micro;g/ml; negative mAb), negative mouse IgG (100 \u0026micro;g/ml) or rat serum (2.5 \u0026micro;L) depleted of human IE reactivity. IE-bound antibody was detected with goat-anti-mouse IgG PE (Abcam), fluorescein-conjugated goat-anti-mouse IgG (1∶100 in PBS\u0026thinsp;+\u0026thinsp;2% BSA; Vectorlabs), or fluorescein-conjugated goat-anti-rat IgG (1∶100 in PBS\u0026thinsp;+\u0026thinsp;2% BSA; Vectorlabs). Samples were DNA-stained using ethidium bromide (20 \u0026micro;g/ml) or Hoechst 33342 (10 \u0026micro;g/ml, Thermo Fischer) and run on a CytoFlex S4 flow cytometer (Beckman Coulter). Data were analysed using FlowJo software (Becton Dickinson). IEs were gated based on DNA stain, forward, and side scatter to exclude cell debris and uninfected erythrocytes.\u003c/p\u003e \u003cp\u003eThe genotypic identity of all parasite lines was routinely verified by genotyping [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and mycoplasma infection was excluded using the MycoAlert mycoplasma detection kit (Lonza) according to manufacturer\u0026acute;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAssay for adhesion of IEs to ICAM-1 under flow\u003c/h2\u003e \u003cp\u003eMicroslides VI\u003csup\u003e0.1\u003c/sup\u003e (ibidi) were coated overnight at 4\u0026deg;C with recombinant ICAM-1-Fc (50 \u0026micro;g/ml), then blocked (1% BSA in PBS) for one hour at 4\u0026deg;C. The microslides were connected to a syringe pump (Alladin, WPI) to generate a wall shear stress of 1 dyn/cm\u003csup\u003e2\u003c/sup\u003e mimicking physiological flow conditions found in the microvasculature. Infected erythrocytes (3\u0026ndash;5% parasitemia or 1% haematocrit) were resolved in RPMI-1640 supplemented with 2% normal human serum and flowed over the microslide for 5 mins. The number of bound IEs per square millimetre for five separate fields was counted at 20 x magnification for a minimum of three independent experiments in triplicate were done. To inhibit ICAM-1 binding, IEs were pre-incubated with mAb02 (0.065-65 nM) for 15 minutes prior to the assay. Mouse IgG (65 nM) was included as a negative IgG control. The ICAM-1-specific antibody 15.2 (258 nM) and the DC4-DBLβ\u003csub\u003emotif\u003c/sub\u003e-specific monoclonal antibody 24E9 (65 nM) were included as additional controls.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAvailability of Data and Materials\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and the supplementary information.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Mette Ulla Madsen for excellent technical assistance and Maria Rosaria Bassi for her excellent help with the mouse work.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Lundbeck Foundation (R313-2019-322), the Novo Nordisk Foundation (NNF170C0026778), and the Faculty of Health and Medical Sciences, University of Copenhagen. The funder had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eATRJ, ND, and LB designed the study and the included experiments. ND, LB, RWO, JJR, BLM, MRW, and YA conducted the experiments. AM, NTN and RT collected the human samples. ND, LH and ATRJ wrote the manuscript. All authors reviewed the manuscript and approved the submitted version.\u003c/p\u003e\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eW. World Health Organization, World Malaria Report 2022. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHviid, L. \u0026amp; Jensen, A. T. 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Detection of antibodies to variant antigens on \u003cem\u003ePlasmodium falciparum\u003c/em\u003e-infected erythrocytes by flow cytometry. \u003cem\u003eCytometry\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 329\u0026ndash;336 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnounou, G. et al. Biased distribution of msp1 and msp2 allelic variants in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e populations in Thailand. \u003cem\u003eTrans. R Soc. Trop. Med. Hyg.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, 369\u0026ndash;374 (1999).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6252569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6252569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe frequently fatal outcome of cerebral malaria has been linked to the adhesion and accumulation in the cerebral microvasculature of infected erythrocytes (IEs), which express a particular type of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e erythrocyte membrane protein 1 (PfEMP1). This type, found in the A and B/A subsets of PfEMP1, contains a particular structural motif (DBLβ\u003csub\u003emotif\u003c/sub\u003e) and has dual affinity for the host vascular receptors ICAM-1 and EPCR. Here, we report the functional characterization of a mouse monoclonal antibody, mAb02, raised against eight different DBLβ\u003csub\u003emotif\u003c/sub\u003e domains. The antibody selectively recognizes DBLβ\u003csub\u003emotif\u003c/sub\u003e-positive PfEMP1 proteins and inhibits their binding to ICAM-1. It also recognizes IEs expressing DBLβ\u003csub\u003emotif\u003c/sub\u003e-positive PfEMP1 proteins on their surface and inhibits their adhesion to ICAM-1. The mAb02 epitope is located in a disordered region of the ICAM-1-binding site of DBLβ\u003csub\u003emotif\u003c/sub\u003e and includes residues directly involved in the interaction between DBLβ\u003csub\u003emotif\u003c/sub\u003e and ICAM-1, as well as residues that are important for the positioning of the interacting residues. Our study shows that mAb02 targets a broadly conserved epitope that is only found in PfEMP1 proteins binding to ICAM-1 and EPCR and implicated in the pathogenesis of cerebral malaria (CM). This suggests the potential of mAb02 in the development of monoclonal antibody-based intervention against CM and for identification of IEs with capacity to causing CM.\u003c/p\u003e","manuscriptTitle":"A monoclonal antibody selectively recognizing PfEMP1 proteins associated with cerebral malaria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 19:28:10","doi":"10.21203/rs.3.rs-6252569/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-27T03:06:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T13:34:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T06:56:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T12:28:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T08:04:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T16:59:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T21:15:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T13:06:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179107045507005981268297653165603338292","date":"2025-05-01T11:13:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310734528163302462180150558236263386624","date":"2025-04-30T12:16:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30445727576199084753359869080218315423","date":"2025-04-30T11:12:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85610370209555119935636000248917504849","date":"2025-04-29T16:46:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200454568854183757662709313070909560186","date":"2025-04-28T17:11:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81211487695039836012970883987888102519","date":"2025-04-28T11:46:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178786982920554897724527597827611903297","date":"2025-04-28T10:43:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140166916650496231966254391313001699100","date":"2025-04-28T10:33:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T10:20:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T09:51:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-25T17:51:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T17:32:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-18T11:11:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"441774ef-1bc3-49f6-9f38-99ec852e8d89","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47946378,"name":"Biological sciences/Immunology"},{"id":47946379,"name":"Health sciences/Molecular medicine"},{"id":47946380,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-10-13T16:02:24+00:00","versionOfRecord":{"articleIdentity":"rs-6252569","link":"https://doi.org/10.1038/s41598-025-18465-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-06 15:57:14","publishedOnDateReadable":"October 6th, 2025"},"versionCreatedAt":"2025-05-05 19:28:10","video":"","vorDoi":"10.1038/s41598-025-18465-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18465-1","workflowStages":[]},"version":"v1","identity":"rs-6252569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6252569","identity":"rs-6252569","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}