Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to ∼1 million deaths and ∼100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this ∼300 kDa hypervariable protein’s domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

PfEMP1 can also be grouped according to 23 domain cassettes (DC), the most frequent ones DC1 to 3, spanning the entire protein while the others include 2-4 domains [6].
A robust, highly specific, sensitive functional methodology has been thoroughly described for tailor-made vaccine development aimed at PfEMP1 (ipso facto severe malaria), recognising variable and conserved HABPs (cHABPs) in relevant invasion molecules by working with ,15 to 20 mer-long peptides [11]. cHABPs are immunologically silent since they do not induce immune responses; however, when their critical binding residues have been properly modified [12][13][14] they become highly immunogenic and protection-inducing modified HABPs (mHABPs).

Ethics Statement
The present study was approved by the Fundación Instituto de Inmunología's animal ethics committee. The capture of Aotus monkeys (International Union for Conservation of Nature and Natural Resources (IUCN) status: least concern), the pertinent maintenance, immunization challenge and research procedures have been authorized by the official Colombian environmental authority in the Amazonian region (CORPOAMAZONIA, resolutions 0066/Sep/2006, 0028/May/2010, 0632/Jun/2010 and 0042/Jan/2011 and previous authorizations beginning in 1982).
The US Committee on the Care and Use of Laboratory Animals' guidelines were followed for all animal handling procedures, in turn complying with Colombian regulations for PfEMP1-based amino-acid sequence synthetic peptides' RBC and C32 cell binding activity (black bars represent specific binding activity slope); above 2% (dotted line) were considered HABPs [11][12][13][14]. Blue shows HABPs chosen for immunization and red those containing canonical or homologous Using PfEMP1 to Deal a Blow at Severe Malaria PLOS ONE | www.plosone.org biomedical research (resolution 8430/1993 and law 84/1989). Monkeys at the station were numbered, sexed, weighed, given a physical-clinical exam and kept temporally in individual cages, prior to all experimental procedures. They were kept in controlled conditions regarding temperature (25u-30u centigrade) and relative humidity (83%), similar to those present in their natural environment. The monkeys' diet was based on a supply of fruit typical of the amazon region (i.e. such primates' natural diet), vegetables and a nutritional supplement including vitamins, minerals and proteins. Environmental enrichment included visual barriers to avoid social conflict, feeding devices, some branches and vegetation, perches and habitat. Any procedure requiring animal handling was practiced by trained veterinary personnel and animals were submitted to sedation and analgesia procedures to reduce stress when necessary [15]. The monkeys were cared for by expert veterinarians and biologists and supervised weekly by CORPOAMAZONIA veterinarians.
All individuals were released back into the Amazon jungle after the experimental procedures and 30-40 days of quarantine and clinical evaluation in optimal health conditions, as approved by CORPOAMAZONIA and in the presence of its officials.

Peptide Synthesis and Radiolabelling
All peptides were synthesised using standard t-Boc solid-phase peptide synthesis (SPPS) strategy [16]. A tyrosine residue was added to the C-terminus of peptides lacking it to allow radiolabelling, as widely described [14].
Polymeric peptides were obtained for immunisation purposes by adding CG to N-and -C termini, as previously described [14].

Binding Assays with PfEMP1 Peptides
PfEMP1 binding to endothelial cells (C32 cells) and RBC was performed according to previously described protocols [14]. Peptides having binding activity greater than or equal to 2% (0.02 ratio) were considered high-activity binding peptides (HABPs), according to previously-established criteria [11].

Animals and Immunisation
Groups of 4-10 Aotus monkeys proving IFA negative for P. falciparum blood stage, kept in our monkey colony in the Amazon jungle (Leticia, Colombia) according to National Institute of Health guidelines for animal handling and Colombia Ministry of Health laws (resolution 8430 of 1993 and law 84 of 1989) and directly supervised by CORPOAMAZONIA officials [17] and legal permits and authorization for capture and housing by the Colombian Ministry of the Environment have been in force for more than 30 years and there has been strong collaboration with the Colombian Association of Indian Authorities (ATICOYA, ASITAM and AZCAITA, representing ,40 Indian communities) (pertinent documentation available on request), CORPOAMA-ZONIA 0266 (Dec/2010) being the most recent authorization.
Aotus monkeys were subcutaneously immunised twice or three times with 250 mg polymerised peptide (on days 1, 20 and 40) which had been previously homogenised with Freund's complete adjuvant for the 1 st dose and Freund's incomplete adjuvant for the 2 nd and 3 rd doses. Controls received only Freund's adjuvant and saline solution on the same days. Blood samples were taken on day 1 before (P 0 ) the first immunisation and 20 days after the 2 nd (II 20 ) and 3 rd (III 20 ) immunisations for immunological analysis [17].
Samples were sequentially treated for 15 min with 200 ml of the appropriate immune serum dilution followed by an anti-goat anti-Aotus IgG F (ab) 2 fragment conjugated with fluorescein isothiocyanate. Slides were washed with TBH supplemented with 50 ll Tween 20 per 100 ml between each sequential incubation. All incubations were performed at room temperature in a humidified chamber. Monolayers were counterstained by adding one drop of ethidium bromide per well to enable parasitised erythrocytes to be visualised. After a few seconds, slides were washed with distilled water, mounted and read at 100x in oil immersion.

Western Blot Analysis
FVO strain culture Pf-schizont lysate was electrophoretically separated and transferred to nitrocellulose membranes. Each nitrocellulose strip was individually incubated with Aotus monkey sera diluted 1:200 in blocking solution, washed several times and incubated with goat anti-Aotus IgG, F(ab) 2 fragment alkaline phosphatase (AP) conjugated at 1:1,000 dilution and developed with NBT/BCIP [18].

Challenge and Parasitaemia Assessment
Immunised and control Aotus monkeys were intravenously infected 20 days after the last immunisation with 100,000 P. falciparum FVO-strain infected RBC, a dose known to be 100% infective for these monkeys [17].
Protection was defined as the complete absence of parasites in blood during the 15 days of the experiment. Non-protected monkeys developed patent parasitaemia on day 5 or 6, reaching . 5% levels between days 8 and 10. They then received treatment with antimalarial drugs and were kept in quarantine until ensuring complete cure, to be returned into the jungle later on [17].
Parasitaemia was measured daily for each monkey, starting on day 5 after challenge, using immunofluorescence for reading parasitised RBC percentage on Acridine Orange-stained slides [17].

CD Analysis
Peptide structures in solution were acquired by circular dichroism measurement in water and 30% TFE mix. The spectra were obtained on a JASCO J-810 spectrometer at room temperature. Data was assessed at 190 to 260 nm wavelength using 20 nm/min scan rate and 1 nm band with. The data was collected using Spectra Manager Software and analysed using SELCOM3, CONTILL and CDSSTR database [19].
(GACxPxRRxxLC) binding motif. Left, schematic representation of PfEMP1 domains showing H-bonds between HABPs (arrows); head structure recombinant fragments containing NTS and DBL1a (fuchsia), CDR1a (green), DBL3X (orange) and DBL6e (blue), 3D structure determined by X-ray crystallography. (B) Sequence logos for amino acid conservation in corresponding HABPs according to their frequency in .100 strains; each amino acid height reflects their relative frequency (%) and thus their contribution to conservation. doi:10.1371/journal.pone.0088420.g001 Figure 2. Humoral immune response and protective efficacy induced by PfEMP1 HABPs derived peptides in Aotus monkeys. Aotus monkeys' humoral immune responses and protective immunity induced by PfEMP1-derived peptides, according to our serial numbering system with corresponding amino acid sequence (modifications in bold). Reciprocal IFA antibody titres in bleeding 20 days after second (II 20 ) and third (III 20 ) immunisation and number of protected monkeys in experimental challenge [12,14] 6584 and 6622) were dissolved in 500 ml TFE-d3/H20 (30/70 v/v). The basic NMR structure determination protocol [20] was as follows: proton spectra were assigned by DQF-COSY, TOCSY and NOESY; TOCSY and DQF-COSY spectra were then used to identify individual spin systems (amino acids) and NOESY (400 ms mixing time) was used for determining peptide primary and secondary structure. TOCSY spectra recorded at different temperatures (285-315 K) were used to obtain amide temperature coefficients for predicting hydrogen bonds (-DdHN/DTppb/K), as thoroughly described beforehand [14,21].

Structural Calculation
Peptide structure was determined by Accelrys software. NOE peaks, selected from 400 ms NOESY data sets, were integrated and converted into distance restraints. These restraints were grouped as strong, medium and weak (1.8-2.5 Å , 2.5-3.5 Å , and 3.5-5.0 Å distance restraints, respectively). Hydrogen bond constraints were introduced for slow exchange rate peptide NH, distance ranges involving likely NH-O hydrogen bonds were set at 1.8-2.5 Å . A family of 50 structures was obtained using Distance Geometry (DGII) software and then refined using simulated annealing protocol (DISCOVER software) to select those having reasonable geometry and fewer violations.

HAPB Superimposition on Crystallised DBL Protein Fragments
The 3D structure of DBL domains from PDB 2XU0 [22], 3CML [23] and 2WAU databases [9] was used for selecting peptide regions presenting high activity binding peptides (HABP) based on aminoacid sequence alignment between strains. InsightII biopolymer molecular software (Accelrys Inc.) was used for such superimposition using backbone superimposition based on RMSD criteria as well as H-bond measurement between HABPs forming the niche which is important for binding site receptors.
Such striking data showed that the canonical GACxPxRRxxLC motif localised in HB4 (;HBb) [4][5][6], in the ''head structure'', is the critical sequence inducing strain-transcending full protective immunity when appropriately modified, as in 24196 (6510), whilst 37822 (modified from homologous 6573) localized in DBL3X induced partial protective immunity. A homologous sequence (PxRRxxxC) present in DBL4e domain is contained in 6593 (not used for immunisations) ( Figure 1A) and a shorter PxRRxxLx sequence was found in DBL6e N-terminus in some strains [28], confirming this motif's presence in nearly all DBL domains [5].
IE usually express only one PfEMP1 at a time but the parasite switches var gene expression, by a mechanism involving a var intron re-localization regulated by an 18 bp nuclear binding element that regulates actin polymerization [29] and leads to the change in host-cell receptor specificity and serotype [30,31], evading the immune response [32]. Such polymorphism could partly explain the partial protective immunity obtained, despite mHABPs being properly modified [12] and high antibody titres being induced (Figure 2) but it has been also demonstrated that PfEMP1 specifically induces a large panel of immune suppression mechanisms among these the early production of human c interferon [33], but the domain (s) involved in such scape mechanisms remains to be identified.
Thus native 6510 and 6573, parents of strain-transcending protective immunity inducing 24196 and 37822, respectively, containing the GACxPxRRxxLC motif, displayed an almost completely unordered and similar structure in DBL1a and DBL3X since 6510 superimposition onto 6573 gives 0.65 rmsd ( Figure 4E) explaining in part the cross protective immunity; in sharp contrast with strain-transcending non-protective antibodyinducing HABPs having helix structures (6505, 6506, 6583,6584 and 6622) ( Figure 4A, C, F) and partially protection inducing 6512 (unordered) and 6621 (a helical and partially unordered, by CD and X-ray crystallography), suggesting an association between structure and immunogenicity and protection [12][13][14].
Modifying H-bond-establishing Residues among cHABPs Induced Strain-transcending Immunity 3D analysis of 6510 ( 128 GACAPYRRLHVC 139 DQNLE-Q*IE* 147 ) showed that C139 HN established an H-bond with Oe 1 from E168 present in 6512 HABP N-terminus ( 168 EGQ-SITQDYPKYQATYGDSP 187 ) forming the niche where the A1 blood group terminal a-1,3 linked N-acetylgalactosamine (Gal-NAc) [34] bound through residues Q145 and E147 (asterisk in Figure 4B) [34], suggesting that modifying these H-bondestablishing residues among cHABPs via T139C replacement in 24196 was fundamental [12] for inducing fully-protective, straintranscending antibody immunity (Figure 2 and 3C). Antibodies against these mHABPs might thus have been blocking IE to UE for rosette formation, thereby impeding IE agglutination and microvascular obstruction, associated with CM, making 24196 essential for severe malaria control in some individuals, as will be discussed later on.  Figure 4D, dot on top). Replacing 6573 R1268 by F in 37822 ( 1262 GA-NIDPF 1268 RQMLTLY 1275 ) induced strain-transcending immunity, controlling parasitaemia at ,1% throughout the experiment, due to these cHABPs' tremendous genetic variability means that blocking this highly polymorphic CSPG binding site could be relevant for PAM control and other severe malaria-associated problems where CSPG is involved.
These large functional-structural and immunological studies show that strain-transcending complete protective immunity against severe malaria can be fulfilled through previously defined principles [11][12][13][14] modifying the GACxPxRRxxLC conserved motif (canonical in the PfEMP1 ''head structure'') binding to endothelial cells. This, in turn, leads towards a fully-protective, multi-epitope, multi-stage, minimal subunit-based, chemicallysynthesised definitive antimalarial vaccine [11][12][13][14]. Figure S1 Summary of sequential and medium range NOEs of 6583, 6584 and 6622. Summary of sequential and medium range NOEs determined in H2O/TFE-d3 (70%/30%). NOE intensity is indicated by bar height. The numbers inside the diagram are the 3J coupling constants. D represents residues involved in an Hbond.