Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria

ABSTRACT Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded erythrocyte membrane protein 1 (PfEMP1) that facilitates dual binding to host intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR). The PfEMP1 subtype is characterized by the presence of a particular motif (DBLβ_motif) in the constituent ICAM-1-binding DBLβ domain. The rate of natural acquisition of DBLβ_motif-specific IgG antibodies and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used an enzyme-linked immunosorbent assay (ELISA) to measure DBLβ-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLβ-specific rat antisera to inhibit the interaction between ICAM-1 and DBLβ was assessed using ELISA and in vitro assays of IE adhesion under flow. The acquisition of DBLβ_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLβ_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLβ_motif antigens. Importantly, antibodies against the DBLβ_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLβ_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLβ_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.

throcyte membrane protein 1 (PfEMP1) family. These proteins are encoded by approximately 60 var genes per P. falciparum genome and are expressed on the IE surface, where they bind to a range of host receptors (reviewed in reference 3).
Despite extensive inter-and intraclonal diversity, the PfEMP1 proteins can be classified into three major groups (A, B, and C), based on var gene sequence and chromosomal context (4,5). Group A is less diverse than the other groups, and expression of group A PfEMP1 proteins on the IE surface has repeatedly been linked to the development of severe malaria (6,7). This is consistent with the restricted serological diversity of P. falciparum parasites from patients with severe malaria (8,9). It also fits the observation that acquisition of immunity to complicated disease often precedes development of protection from uncomplicated malaria and asymptomatic parasitemia and that PfEMP1 expression is modulated by PfEMP1-specific immunity (10)(11)(12). More recently, the PfEMP1 groups have been further subdivided according to their constituent Duffy-binding-like (DBL) and cysteine-rich interdomain region (CIDR) domains, and a number of multidomain blocks, known as domain cassettes (DCs), have been identified (13)(14)(15)(16). Three of these, DC4, DC8, and DC13, have been linked to severe malaria in children (6,14,17,18). DC4 consists of three domains (DBL␣ 1.1/1.4 , CIDR␣ 1.6 , and DBL␤ 3 ) and defines a subfamily of group A PfEMP1 proteins that mediates binding to intercellular adhesion molecule 1 (ICAM-1) (15). IE adhesion to ICAM-1 appears associated with severe malaria, implicating DC4-specific antibodies in clinical protection, as they are acquired early in life by children living in areas where malaria is endemic and are associated with clinical protection from malaria (6,15,19). However, until recently, the role of IE adhesion to ICAM-1 specifically in CM has been unclear (20)(21)(22)(23)(24).
DC8 consists of four domains (DBL␣ 2 , CIDR␣ 1.1 , DBL␤ 12 , and DBL␥ 4/6 ) and is found among group B/A genes, while the two-domain (DBL␣ 1.7 , CIDR␣ 1.4 ) DC13 is found in some group A PfEMP1 proteins (14). Endothelial protein receptor C (EPCR) is the cognate receptor for DC8-and DC13-containing PfEMP1 proteins (25). Some studies have reported high transcript levels of var genes encoding EPCR-binding PfEMP1 variants in parasites from children with severe malaria, including CM, and perturbed EPCR expression in brain tissue of CM patients (26)(27)(28). While these findings point to a role for EPCR in severe malaria in general, and cerebral malaria (CM) in particular, the available evidence overall remains equivocal (29)(30)(31).
We have previously proposed that the above ambiguities may reflect that the pathogenesis of CM involves P. falciparum parasites expressing PfEMP1 capable of mediating IE adhesion to both ICAM-1 (via DBL␤) and EPCR (via CIDR␣ 1 ) (3). A few such dual receptor-binding PfEMP1 proteins were identified shortly after, although the study did not link them to CM specifically and did not document concomitant binding to both receptors (32). However, those gaps were recently closed by our demonstration of a link between CM and group A PfEMP1 proteins capable of binding ICAM-1 and EPCR simultaneously (33). This dual receptor-binding subgroup of PfEMP1 proteins is characterized by an EPCR-binding CIDR␣ 1 domain followed immediately by a DBL␤ domain featuring a specific ICAM-1-binding motif (DBL␤_motif domain) (33).
The rate of natural acquisition of IgG against the DBL␤_motif associated specifically with CM and the ability to induce such antibodies by vaccination are both unknown. The current study was therefore designed to investigate whether cross-reactive IgG antibodies specific for DBL␤_motif domains and/or their ICAM-1-binding motif are acquired following natural exposure to P. falciparum parasites and whether ICAM-1 adhesion inhibitory antibodies can be induced by immunization with specific DBL␤_motif proteins and with peptides representing the ICAM-1-binding motif therein. Confirmation of these hypotheses would support the feasibility of developing a vaccine designed specifically to prevent CM.

RESULTS
Delayed acquisition of IgG to ICAM-1-binding group A-type DBL␤ domains. Group A PfEMP1 proteins that contain a DBL␤ domain with the motif I(V/L)X 3 N(E)GG (P/A)XYX 27 GPPX 3 H (DBL␤_motif domains) (where X represents an unknown amino acid) can bind to the host endothelial receptor ICAM-1 and always feature a neighboring CIDR␣ 1 domain that enables concomitant binding to another endothelial receptor, EPCR (33). Expression of these dual receptor-binding PfEMP1 proteins is associated with CM, which is a major cause of mortality and severe morbidity among African children. The age at which most CM cases occurs varies with transmission intensity but generally occurs later than the peak prevalence of parasitemia and malaria-related severe anemia. DBL␤ domains present in group A PfEMP1 proteins, but without the above motif (DBL␤_nonmotif domains), do not bind ICAM-1 and are less conserved in the C terminus (33).
We first used ELISA to measure the levels of IgG with specificity for 14 recombinant DBL␤_motif domains (M1 to M12, M14, and M15) (Fig. 1) and 13 nonmotif DBL␤ domains (N20 to N32) in plasma from 79 Ghanaian children with different clinical presentations of P. falciparum malaria ( Table 1). The antibody reactivity to all these group A PfEMP1 proteins differed substantially among the children ( Fig. 2A) and also among the different DBL␤ domains (Fig. 2B). Overall, the plasma levels of IgG increased with age (P r Ͻ 0.001, where r is Pearson's product moment correlation), with levels of IgG specific for DBL␤_motif proteins being generally lower than those of DBL␤_nonmotifspecific IgG (P T Ͻ 0.001, where T is the Kruskal-Wallis one-way analysis of variance on ranks). However, the latter difference was due mainly to low IgG recognition of the DBL␤_motif among the younger age groups (Յ4 years of age) (Fig. 2C). Thus, the levels of IgG specific for the DBL␤_motif were significantly lower than the DBL␤_nonmotif-specific IgG levels among children aged 1 to 2 years and 3 to 4 years (P T Յ 0.001), but not in the two older age classes considered (5 to 6 years and Ͼ6 years; P T Ն 0.21).
The overall plasma levels of IgG specific for DBL␤_motif domains as well as DBL␤_nonmotif domains were similar in patients with severe and uncomplicated malaria (P U ϭ 0.4 in both cases, where U is the Mann-Whitney test for intergroup differences), while levels of DBL␤_nonmotif domain-specific IgG were lower in the children with severe malaria than in the patients with uncomplicated disease (scores, 7 and 12.5; P U ϭ 0.02, respectively).
Taken together, these results indicate that DBL␤_motif and DBL␤_nonmotif proteins are similarly immunogenic but that DBL␤_motif-specific IgG is acquired later in life than DBL␤_nonmotif-specific IgG.
Immunization with DBL␤_motif antigens induces cross-reactive, neutralizing IgG. Plasma from clinically immune individuals living in areas of stable and intense transmission of P. falciparum parasites can inhibit the interaction between ICAM-1 and a range of DBL␤_motif domains (15,33). In this study, a pool of plasma from 10 of the children (selected for reactivity with DBL␤_motif domains [ Fig. 2A and Table 1] and plasma availability) inhibited ICAM-1 binding to DBL␤_motif protein M9 (Fig. 3A). These data suggest the presence of neutralizing IgG antibodies capable of recognizing multiple DBL␤_motif-containing PfEMP1 variants (cross-reactive IgG). If such antibodies could be induced by vaccination, it would increase the feasibility of developing a broadly protective PfEMP1-based vaccine against cerebral malaria. However, our data might also reflect the presence of many different variant-specific IgG specificities, where each antibody specificity is capable of inhibiting the binding of ICAM-1 to only  Table 3). Residues that are critical for the direct interaction with ICAM-1 (red triangles) or for the architecture of the ICAM-1 binding (white triangles) are indicated. The group A PfEMP1 ICAM-1-binding motif was identified by Lennartz et al. (33). Age group (range of yrs) 1-2 (n ϭ 16) 3-4 (n ϭ 9) 5-6 (n Parasites/l (ϫ1,000) a particular DBL␤_motif domain variant (a broad repertoire of IgG with narrow specificity). It is inherently difficult to distinguish between these two alternatives in naturally acquired immunity. Nevertheless, truly cross-reactive PfEMP1-specific human antibodies have previously been demonstrated in a study employing naturally acquired monoclonal IgG specific for the VAR2CSA-type PfEMP1 involved in the pathogenesis of placental malaria (34).
To investigate the relative importance of the above nonexclusive alternatives, to further assess the functional significance of DBL␤_motif in acquired immunity, and to  (33). Shading indicates IgG level score: black, score of 4 (Ͼ100 EU); dark gray, score of 3 (76 to 100 EU); gray, score of 2 (51 to 75 EU); light gray, score of 1 (26 to 50 EU); white, score of 0 (0 to 25 EU). The DBL␤ domain numbers correspond to the numbers in Table 3. Danish controls (n ϭ 25) did not react with any of the domains (data not shown). (B) The means of IgG level scores (defined as described for panel A) of individual DBL␤ domains that contain (motif; OE) or do not contain (nonmotif; ) the ICAM-1-binding motif. Error bars indicate 95% confidence intervals. DBL␤ domain numbering is as described for panel A. (C) The means of IgG level scores (defined as described for panel A) of individual children for IgG specific for DBL␤_motif (OE) and DBL␤_nonmotif () domains. The statistical significance (Mann-Whitney rank sum test) of pairwise comparisons is shown along the top of the panel. make a first step toward PfEMP1-based vaccination specifically against cerebral malaria, we immunized four rats with DBL␤_motif proteins M1, M6, M9, and M10, respectively (see Table 3; see also Fig. S1 in the supplemental material). We used an enzyme-linked immunosorbent assay (ELISA) to test the ability of the antisera to inhibit the binding of ICAM-1 to 14 DBL␤_motif proteins (M1 to M7 and M9 to M15) and two ICAM-1-binding DBL␤_nonmotif proteins (N27 and N33). Each of the four DBL␤_motif-specific antisera inhibited binding of ICAM-1 to most of the DBL␤_motif proteins by more than 50% but had little effect on ICAM-1-binding to DBL␤_nonmotif domains (Fig. 3B). When pooled, the DBL␤_motif-specific antisera strongly inhibited (Ͼ75%) binding of ICAM-1 to all DBL␤_motif domains, with much less effect (Ͻ50%) on ICAM-1-binding to the DBL␤_nonmotif domains (Fig. 3B). We next affinity purified IgG from three of the rat antisera, using M6pep, to evaluate the involvement of IgG directly targeting the ICAM-1-binding region in the above-described inhibition. The purified M6pep-specific IgG generally inhibited ICAM-1 binding to the same degree as the antisera (Fig. 3B), with strong correlation between the antiserum and motif-specific IgG data for M6 (Spearman's rank order correlation [r s ] ϭ 0.78; P Ͻ 0.001).
Overall, these data indicate that immunization with single DBL␤_motif antigens can induce cross-reactive IgG that inhibits binding of ICAM-1 to the homologous, as well as a broad range of heterologous, DBL␤_motif domains.
DBL␤_motif-specific IgG is broadly inhibitory of IE adhesion to ICAM-1 under physiologic flow. The ability of neutralizing IgG to interfere with receptor-specific IE sequestration in vivo likely depends on the characteristics of the involved PfEMP1 proteins per se, on their expression on the IE surface, and on the shear forces at the anatomical location of the interaction of IEs with host endothelium. We have previously shown that naturally acquired IgG can inhibit ICAM-1-specific adhesion of erythrocytes infected by P. falciparum expressing DBL␤_motif-containing PfEMP1 (33). To test if such antibodies could also be elicited by immunization with recombinant PfEMP1 proteins containing the DBL␤_motif, we tested the ability of DBL␤_motif-specific IgG to inhibit the adhesion of IEs to ICAM-1 in an in vitro assay simulating physiologic flow conditions (35). M6-specific IgG significantly inhibited ICAM-1-specific adhesion of IEs expressing  Table 3. Data using a pool of rat antisera (M1, M6, M9, M10) are also shown (pool). Antisera marked with asterisks were affinity purified on a peptide (M6pep) representing the binding motif in PFD1235w_DBL␤_D4 prior to assaying. Three independent experiments were done (three technical replicates/assay). A sequence-distance tree illustrating the relatedness of the different domains is shown along the left edge of the panel.
We conclude from these experiments that immunization with DBL␤_motif antigens induces cross-reactive IgG antibodies that inhibit ICAM-1-specific adhesion of IEs that express a variety of PfEMP1 proteins containing DBL␤_motif domains. The inhibition of binding of native PfEMP1 protein to ICAM-1 under conditions of flow thus mirrors that observed with recombinant proteins in ELISA.
Immunization with peptides representing the ICAM-1-binding region induces antibodies broadly inhibiting the binding of recombinant and native DBL␤_motif domains to ICAM-1. The results above suggested that IgG targeting the ICAM-1binding region in DBL␤_motif domains is of particular importance for inhibiting the binding of group A dual receptor-binding PfEMP1 to ICAM-1. This interpretation is further supported by our recent data showing that DBL␤_motif-purified antibodies from naturally infected humans and experimentally vaccinated animals inhibit ICAM-1-specific adhesion of IEs expressing the DBL␤_motif-containing PfEMP1 protein PFD1235w (33). To assess directly if inhibitory and cross-reactive antibodies could be elicited by peptide immunization, we immunized rats with peptides representing the ICAM-1-binding region in DBL␤_motif domains (M6pep and M9pep) and tested their ability to inhibit binding of ICAM-1 to DBL␤_motif domains. Antisera from rats immunized with M6pep only, or with M6pep and M9pep, were broadly inhibitory of the binding of ICAM-1 to 10 DBL␤_motif domains (M2 to M7, M9, and M11 to M13). The peptide antisera did not affect binding to two ICAM-1-binding DBL␤_nonmotif domains (N27 and N33) (Fig. 5A). Experiments assessing the ability of the antisera to inhibit the adhesion of IEs to ICAM-1 under flow corroborated these findings. Thus, both of the above-mentioned antisera (M6pep and M6pep/M9pep) significantly inhibited the adhesion of IEs expressing the DBL␤_motif-containing PfEMP1 proteins PFD1235w (Fig. 5B) (expressing M6 native protein) and HB3VAR03 (Fig. 5C) (expressing M8 native protein) but had no effect on IEs expressing IT4VAR13 (expressing N27 native protein), which does not contain a DBL␤_motif domain (Fig. 5D). Furthermore, the single-and dual-peptide antisera yielded immunofluorescence patterns typical of IgG reacting with IE surface-expressed PfEMP1 when tested against IEs expressing either HB3VAR03 or PFD1235w but did not label IEs expressing IT4VAR13 (Fig. 5E). In contrast, the IEs expressing IT4VAR13 were labeled by an IT4VAR13-specific antiserum but not by the single-and dual-peptide antisera (Fig. 5E).
Finally, we assessed the ability of erythrocytes infected by 33 primary P. falciparum isolates ( Table 2) from Ghana (n ϭ 14) and Tanzania (n ϭ 19) to adhere to ICAM-1 under flow. We also tested the ability of pooled rat antiserum to M6pep and M9pep to inhibit the adhesion of ICAM-1-adhering isolates. Twenty-two of the isolates (3 from children with uncomplicated malaria, 14 from patients with severe malaria, and 5 from children with cerebral malaria) showed adhesion of IEs to ICAM-1 (Fig. 6A). The adhesion of 11 of these isolates (1 from a child with uncomplicated malaria, 6 from children with severe malaria, and 4 from children with cerebral malaria) was inhibited (Ͼ25%) by the anti-peptide serum pool (Fig. 6B).
We conclude that immunization with linear peptides that represent only the ICAM-1-binding region of specific DBL␤_motif domains can induce cross-reactive antibodies that are capable of inhibiting the binding of ICAM-1 to a range of recombinant and native, IE-expressed DBL␤_motif domains. Importantly, the motif antibody inhibits the binding of four of five P. falciparum isolates from cerebral malaria patients.  Table 3. The antiserum marked with an asterisk was affinity purified on a peptide (M6pep) representing the binding motif in PFD1235w DBL␤_D4 prior to assaying. An ICAM-1-specific neutralizing antibody (Continued on next page)

DISCUSSION
Cerebral malaria (CM) is one of the most severe complications of P. falciparum malaria and a leading cause of mortality (reviewed in reference 36). PfEMP1-mediated adhesion of IEs to the endothelial receptors ICAM-1 and EPCR have both repeatedly been implicated in the pathogenesis of severe malaria (15,25,33). However, a specific and direct link to the development of CM has been missing until recently, when we identified a sequence motif in PfEMP1 proteins associated specifically with the development of CM (33). This ICAM-1-binding motif (DBL␤_motif) (Fig. 1) is found in some group A PfEMP1 proteins (15), immediately downstream of an EPCR-binding CIDR␣ domain (25). In the present study, we set out to study the acquisition of DBL␤_motifspecific IgG following natural exposure and whether DBL␤_motif-specific antibodies induced by vaccination are cross-reactive and inhibit adhesion to ICAM-1.
In areas with stable transmission of these parasites, substantial protective immunity to malaria is acquired during childhood, first to severe complications and later to clinical disease. As a consequence, adults are largely protected from malaria in such areas, although sterile immunity is rarely, if ever, achieved. This sequence appears to be the consequence of an ordered acquisition of antibodies to a relatively conserved set of PfEMP1 proteins associated with severe disease, followed by antibodies to a large and diverse set of PfEMP1 proteins associated with uncomplicated malaria and asymptomatic parasitemia. Where transmission is very intense, serious and fatal malaria episodes are markedly concentrated during the first few years of life, mainly as severe malarial anemia. CM, in contrast, is rare (37). Where endemicity is lower, CM tends to be seen more often but mainly among children some years older than those that succumb to severe malarial anemia. Together these findings suggest that discrete PfEMP1 subsets are involved in severe malaria with and without cerebral involvement and perhaps even that toddlers are relatively resistant to CM for nonimmunologic reasons. This fits our demonstration here that although DBL␤ domains are generally immunogenic following natural exposure, acquisition of DBL␤_motif-specific IgG occurs later than that of DBL␤_nonmotif-specific IgG (Fig. 2) and coincides with the age bracket in which the incidence of CM peaks under transmission intensities comparable to those in our study area (38,39).
The clinical significance of acquisition of PfEMP1-specific antibodies is thought to involve their ability to interfere with sequestration of IEs in various tissues (15,40). A particularly thoroughly investigated example is the role of anti-adhesion antibodies in the acquisition of protective immunity to placental P. falciparum malaria, caused by accumulation of IEs in the intervillous spaces (41,42). Placental IE sequestration is mediated by a particular group of PfEMP1 (VAR2CSA) binding to oncofetal chondroitin sulfate A (43,44), and clinical trials of vaccines based on the VAR2CSA adhesive epitope and aimed to protect against this important cause of prenatal and infant morbidity and mortality are under way. It appears that DBL␤_motif-specific IgG can inhibit IE adhesion to ICAM-1 in a similar way (Fig. 3A) (15,33). Here, we demonstrate that this inhibition can be mediated by genuinely cross-reactive antibodies, as opposed to a broad repertoire of IgG species, each with narrow specificity for a single or very few DBL␤_motif sequences (Fig. 3). This finding is of significance, since naturally acquired protection from malaria is generally believed to be the consequence of the accumulation of a broad repertoire of a large number of antibody specificities (9,12,45). Such broadly reactive IgG can inhibit the adhesion of erythrocytes infected with parasites isolated from patients with severe malaria (6 of 13 isolates) and cerebral malaria (4 of 5 isolates) under physiologic flow conditions (Fig. 6). Furthermore, such IgG can be  Table S1 in the supplemental material for raw data. (Continued on next page) induced by peptides (M6pep and M9pep) representing just the core element of the DBL␤_motif that mediates the binding to ICAM-1 ( Fig. 5 and 6).
Approximately half the children with acute P. falciparum malaria in our study had severe disease, according to the WHO criteria (46), but we did not observe any significant differences in plasma levels of DBL␤_motif-specific IgG in children with or without severe disease. This may be related to the fact that due to the low prevalence in the area none of the study children had CM (Table 1). It is doubtful that a relationship between DBL␤_motif-specific IgG and clinical presentation of acutely ill malaria patients would be apparent, even if we had been able to include plasma samples from CM patients, due to the unavoidable variation in time between infection and presentation to hospital. It is plausible that only very large, and preferably longitudinal, studies would have the power required to document such relationships in semi-immune, naturally infected individuals.
In conclusion, our study demonstrates that CM-related DBL␤ domains are immunogenic following natural exposure and that acquisition of DBL␤_domain-specific IgG coincides with the age where CM has its peak prevalence in areas of moderate but stable P. falciparum transmission. Furthermore, we show that immunization with such domains in addition to peptides representing the minimal ICAM-1-binding region can induce IgG that can inhibit PfEMP1 binding to ICAM-1 and neutralize IE adhesion under physiologic flow. Importantly, these antibodies broadly neutralized the adhesion of erythrocytes infected by parasites isolated from four of five children with cerebral malaria. Together, these findings raise hopes that development of a vaccine specifically against CM may be possible, despite the notorious polymorphism and intraclonal diversity of the PfEMP1 family (recently reviewed in references 3 and 47).

MATERIALS AND METHODS
Plasma and parasite samples. Plasma samples (n ϭ 79) for the present study were collected in 2014 at Hohoe Municipality Hospital in the Volta Region of Ghana from children with acute malaria (Table 1) (33,48).
P. falciparum parasites were collected at this hospital (n ϭ 14) and at the Korogwe District Hospital (n ϭ 19) in Korogwe District in northeastern Tanzania (14). Clinical manifestations of malaria were classified according to the definitions and associated criteria of the World Health Organization. Patients were categorized as having cerebral malaria (CM; n ϭ 7) if they had a positive blood smear of the asexual form of P. falciparum and unrousable coma (Blantyre coma score [BCS] Յ 2) with exclusion of other causes of coma and severe illness. Patients were categorized as having severe malarial anemia (SA; n ϭ 12) if the hemoglobin level was Ͻ5 g/dl and the BCS was Ͼ2. Patients were classified as having severe malaria other than SA and CM if they presented with hyperparasitemia (Ͼ250,000 parasites/l), multiple convulsions (Ͼ2 episodes in 24 h), respiratory distress (i.e., rapid, deep, and labored breathing), or combinations of these symptoms. Patients with uncomplicated malaria (UM; n ϭ 48) had fewer than 250,000 parasites/l.
The study was approved by the Ethical Review Committee of the Ghana Health Services (file GHS-ERC 08/05/14) and by the National Ethical Review Committee of the National Institute for Medical Research, Tanzania (NIMR/HQ/R.8a/Vol.IX/559). A pool of plasma from P. falciparum-exposed Tanzanian individuals (49) and 25 nonexposed Danish individuals were used as positive and negative controls, respectively. Long-term in vitro culture-adapted and fully sequenced parasite clones 3D7, HB3, and IT4 were also studied.
Recombinant proteins. The genes encoding the DBL␤ domains used were amplified from genomic DNA or produced as synthetic genes (http://eurofins.dk) ( Table 3). Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously (15). All the proteins were purified (see Fig. S1 in the supplemental material) by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) and are referred to by the designations listed in Table 3.

FIG 5 Legend (Continued)
Fewer than 0.25 IEs/mm 2 bound to uncoated channels were observed. (E) Immunofluorescence of representative IEs with surface expression of PFD1235w (top row), HB3VAR03 (center row), and IT4VAR13 (bottom row) and labeled by sera from rats immunized with M6pep only (left column) or with both M6pep and M9pep (center column), or by a rat antiserum to N27 (right column). See Table S2 in the supplemental material for raw data. Age group (range of yrs) IgG purification. IgG antibodies specific for M6 and M6pep were affinity purified from rat antisera as described previously (51). In brief, M6 and M6pep (1 mg/ml) were dialyzed overnight against coupling buffer and coupled to HiTrap normal human serum (NHS)-activated HP columns, as described by the manufacturer (GE Healthcare). Antisera were diluted 1:1 in phosphate-buffered saline (PBS) and affinity purified on the columns, followed by elution of bound IgG in low-pH buffer (glycine-HCl, pH 2.75) and pH adjustment by Tris-HCl (1 M, pH 9.0).
Measurements of antibody-mediated inhibition of DBL␤ binding to ICAM-1. Inhibition of recombinant DBL␤ domain binding to ICAM-1 by human immune plasma and rat antisera was measured by ELISA. In brief, wells of MaxiSorp plates were coated with recombinant ICAM-1 (50) (50 l/well, 2 or 4 g/ml, 0.1 M glycine-HCl buffer, pH 2.75) by incubation overnight (4°C) and blocked with blocking buffer (1 h, room temperature). His-tagged DBL␤ proteins (0.5 to 16 g/ml final concentration) were mixed with immune plasma or antisera (1:5 final concentration) or purified IgG (10 g/ml final concentration) and added to duplicate wells (1 h, room temperature). The plates were washed, and binding was detected using HRP-conjugated anti-penta-His antibody (Qiagen) as described above. All antisera were prescreened by ELISA to verify the absence of His tag-reactive antibodies.
In vitro culture and antibody selection of P. falciparum parasites. The P. falciparum clones 3D7, HB3, and IT4 were maintained in long-term in vitro cultures and antibody selected for IE surface expression of specific PfEMP1 proteins as described previously (15,51). In brief, we used the human monoclonal IgG antibody AB01 to select 3D7 IEs for the expression of PFD1235w (53). HB3 IEs were similarly selected for surface expression of VAR03 using a rat antiserum against M8, and IT4 IEs were selected for expression of VAR13 by a rat antiserum against N27 (33). In all cases, expression of the required PfEMP1 on the surface of mature IEs purified on a VarioMACS separator was monitored by flow cytometry using PfEMP1-specific antisera, essentially as described previously (51,54). Only cultures with Ͼ60% antibody-labeled IEs were used.
In addition, primary isolates of P. falciparum parasites from 33 of the above-mentioned malaria patients were cultured in vitro for up to 28 days (median and 25th and 75th percentile, 8, 2.5, and 13 days, respectively) in Albumax (10%) (ThermoFisher Scientific), supplemented with NHS (2%), essentially as described previously (55). The genotypic identity of the isolates was routinely verified by genotyping as described previously (56), and Mycoplasma infection was regularly excluded using the MycoAlert Mycoplasma detection kit (Lonza) according to the manufacturer's instructions.
Adhesion of IEs to ICAM-1 under physiological flow in vitro. Microslides (VI 0.1 ) (Ibidi) were coated with recombinant Fc-tagged ICAM-1 protein (50) (50 g/ml, 4°C, overnight) and blocked using PBS plus 2% bovine serum albumin (BSA). Parasite suspensions, adjusted to 3% parasitemia and 1% hematocrit in RPMI 1640 supplemented with 2% NHS (pH 7.2), were flowed over the coated slides (5 min) at a shear stress of 1 dyn/cm 2 as described previously (35). The numbers of bound IE/mm 2 in five separate fields were counted, using a Leica inverted phase-contrast microscope (ϫ20 magnification). To assess the capacity of affinity-purified DBL␤-specific IgG to inhibit adhesion, IEs selected for expression of particular PfEMP1 variants were preincubated with the purified IgG (15 min, room temperature). The receptor specificity of the IE adhesion observed was verified by preincubating the ICAM-1-coated flow channels with an ICAM-1-specific antibody (40 g/ml, clone 15.2; AbD Serotec).
The inhibition of ICAM-1-adhering erythrocytes (Ն10 adherent IEs/mm 2 ) infected with primary P. falciparum field isolates was tested using pooled rat antisera (1:100 dilution) to two peptides representing the ICAM-1-binding motifs in M6 (M6pep) and M9 (M9pep). A minimum of three independent a Nomenclature as described in reference 15. b "Yes" indicates DBL␤_motif domains that bind ICAM-1 (M1 to M15, N27, and N33); "no" indicates domains that do not bind ICAM-1 (the remainder). c All domains were group A except for two group B DBL␤ domains as indicated (N27 and N33). d Also known as PF3D7_1150400. e Also known as PF3D7_0425800. f Also known as KOB85388. g Also known as ABM88750. h Also known as AAS89259. i From genomic DNA, using previously described primers (15,33), except where indicated. j Data from reference 33. k Unpublished data. l Data from reference 61.
experiments were completed for each of the tested laboratory clones (3D7, HB3, IT4), whereas each of the field isolates was tested in one experiment with five technical replicates. All assays were deidentified for the operator. Immunofluorescence microscopy of IEs labeled with PfEMP1-specific antibodies. Immunofluorescence microscopy was done essentially as described previously (57). Briefly, aliquots (50 l) of erythrocytes infected by parasites expressing PFD1235w, HB3VAR03, or IT4VAR13 were adjusted to 5% parasitemia and resuspended in PBS containing 1% Ig-free BSA (Sigma-Aldrich). Antisera were added (1:50 dilution), and the suspensions were incubated on ice (1 h). Following three washes, cells were resuspended and labeled with anti-rat-fluorescein isothiocyanate (FITC) secondary antibody (1:500) and incubated on ice for 1 h. Cells were washed three times, and thin smears were made. Nuclei were visualized by adding 5 l ProLong Gold antifade mountant (ThermoFisher Scientific) prior to the addition of coverslips. Immunofluorescence was visualized with a Nikon Eclipse TE2000 microscope equipped with a ϫ63 objective.
Bioinformatics. Multiple alignments of DBL␤ domains known to bind ICAM-1 were made using MUSCLE v.3.7 software (58), and sequence distance trees were made with MEGA software (59). A WebLogo 3 sequence logo (60) of the ICAM-1-binding motif was generated based on alignment of the included DBL␤_motif domains (Table 3) with the consensus motif I(V/L)X 3 N(E)GG(P/A)XYX 27 GP PX 3 H (15,33).
Statistics. We used Pearson's product moment correlation (r) or Spearman's rank-order correlation (r s ) to evaluate parameter association and one-way analysis of variance (F), the Kruskal-Wallis one-way analysis of variance on ranks (T), and the Mann-Whitney test (U) to test for intergroup differences.