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 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 (25°–30° 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), CORPOAMAZONIA 0266 (Dec/2010) being the most recent authorization.

Aotus monkeys were subcutaneously immunised twice or three times with 250 µg 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 (P₀) the first immunisation and 20 days after the 2^nd (II₂₀) and 3^rd (III₂₀) immunisations for immunological analysis [17].

PfEMP1 Detection by Immunofluorescence

Modifications of Staalsoe’s method (Cytrometry 35∶329) were used. Late trophozoite- and schizont-enriched FCB-2 P. falciparum cultures (5–10% parasitaemia) or highly infected Aotus-adapted P. falciparum FVO strain-enriched schizonts or late trophozoites were spun for 5 min at 1,800 rpm and left for a further 20 min to form sediment, washed three to four times with Tris-buffered Hanks’ solution (TBH) (10 ml 0.15M Tris buffer, pH 7.2, 90 ml 0.9% NaCl, and 100 ml Hanks’ solution) and then diluted to give 1% suspension.

Samples were sequentially treated for 15 min with 200 µl 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].

NMR Spectroscopy

8 or 10 milligrams of each peptide (6583, 6584 and 6622) were dissolved in 500 µl 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 (-ΔδHN/ΔTppb/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.

Strain-transcending Protection-inducing mHABP 3D Structure

The 3D structure of the rosette-forming blood group A-binding Palo Alto VarO strain (Uganda) “head structure” containing the NTS-DBL1α-CIDR1γ region [34], the A4 strain (Brazil) chondroitin-sulphate-proteoglycan (CSPG) binding DBL3X domain [23], [35] and the 3D7 strain (unknown origin) DBL6ε domain binding to CSA has been determined by X-ray crystallography [9].

(reminder: our HABPs are based on Indochina Dd2 sequence). Our group determined that native HABP 6505 displayed a perfect α-helix structure by ¹H-NMR (Figure 4A, yellow) [14]; when superimposed onto the NTS-DBL1α 3D structure (Figure 4A, green) it gave a 0.16 rmsd and 6583 and 6584 (Figure 4B, fuchsia and dark blue) had α-helix conformation (Figure S1 and Table S1), giving 0.43 and 0.52 rmsd, respectively, when superimposed onto the DBL3X sequence [23], [35]. It has thus been thoroughly demonstrated that chemically-synthesised HABPs display the same 3D structure as biologically-derived recombinant proteins [11], [12].

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Figure 4. Structural characterization of HABPs present in crystallized Duffy binding like domains (DBL).

DBL domain 3D structure determined by X-ray crystallography A) Head structure: DBL1α (PDB 2XU0) (pink), C) DBL3X (PDB 3CML) (yellow), F) DBL6ε (PDB 2WAU) (pale blue). ¹H-NMR-determined structure localisation, displaying the perfect fit of HABP 6505 (yellow) superimposed onto DBL1α, 6583 (dark blue) and 6584 (purple) onto DBL3X and 6622 (grey) onto DBL6ε. B, D, G). H-bonds between HABP residues and their corresponding sequence on top, displaying relevant residues in binding to A blood group trisaccharides and CSPG (asterisk and black dot, respectively). E) Superimposed conserved binding motif fragments from 6510 and 6573. H) CD spectra for corresponding HABPs.

https://doi.org/10.1371/journal.pone.0088420.g004

Circular dichroism (CD) revealed that 6621 and 6622 had a helical structure (Figure 4H), while SELCON3 deconvolution analysis revealed 0.785, 0.836 and 0.898 turn and unordered structure composition for 6510, 6512 and 6573.

6510 (located in the NTS-DBL1α fragment subdomain S1 between cysteine 3 and 4) contained a short αH1 helix and short 3₁₀ H1 helix; partially unordered HABP 6573 (DBL3X subdomain S1) only displayed a short α-helix [23], the rest being unordered.

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 DBL1α 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 antibody-inducing HABPs having helix structures (6505, 6506, 6583,6584 and 6622) (Figure 4A, C, F) and partially protection inducing 6512 (unordered) and 6621 (α helical and partially unordered, by CD and X-ray crystallography), suggesting an association between structure and immunogenicity and protection [12]–[14].

Modifying H-bond-establishing Residues among cHABPs Induced Strain-transcending Immunity

3D analysis of 6510 (¹²⁸GACAPYRRLHVC¹³⁹DQNLEQ*IE*¹⁴⁷) showed that C139 HN established an H-bond with Oε₁ from E168 present in 6512 HABP N-terminus (¹⁶⁸EGQSITQDYPKYQATYGDSP¹⁸⁷) forming the niche where the A1 blood group terminal α-1,3 linked N-acetylgalactosamine (GalNAc) [34] bound through residues Q145 and E147 (asterisk in Figure 4B) [34], suggesting that modifying these H-bond-establishing residues among cHABPs via T139C replacement in 24196 was fundamental [12] for inducing fully-protective, strain-transcending 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.

By the same token, 6573 (¹²⁵⁷HGDTNGACIPPR¹²⁶⁸QTQNLCVG¹²⁷⁵), containing also conserved binding motif GACxPxRRxxLC (Figure 1A), established an H-bond between R1268 HNε and E1456 Oε2 present in 6583 (¹⁴⁵⁵KE¹⁴⁵⁶W¹⁴⁵⁷GEQFCIE¹⁴⁶⁴RLR^•YEQNIRE¹⁴⁷⁴); 6583 established another H-bond between E1464 Oε1 with OH in 6584 Y1511 (¹⁵⁰¹CKRK^•CEKYKKY¹⁵¹¹ISEKKQE¹⁵¹⁸W¹⁵¹⁹) to form a tripartite binding site for CSPG (Figure 4D, dot on top). Replacing 6573 R1268 by F in 37822 (¹²⁶²GANIDPF¹²⁶⁸RQMLTLY¹²⁷⁵) 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.

Two HABPs were localised in DBL6ε, consisting of 7 variable blocks (VB) having limited polymorphism. Completely 3₁₀-helix structure 6622 (²⁴⁶⁴KKWWDMNKYHIWESML²⁴⁷⁹) determined by ¹H-NMR (Figure 4F, grey ribbon) is localised in VB4; partially α-helical 6621 (²⁴⁴⁶SDKIGKILGDGVGQN²⁴⁶⁰EKR²⁴⁶³) (Figure 4F, red ribbon) localised in one of the elbows of DBL6ε domains, in VB4 [28], [36], established a H-bond between N2460 O (6621) and K2464 HN (6622), forming a niche for a non-identified RBC receptor binding (Figure 4G).

D2456S replacement in 38070 (6621) induced high Ab titres and partial protective immunity (∼1.5% parasitaemia), again confirming inter-HABP H-bond breaking’s relevance in immune induction. 38070 displayed binding motifs and registers (grey) characteristic of HLADRβ1*0301 allele (YNKNKLKIDHRIKIGE), an allele found ∼15% frequency in monkeys and humans.

6622-derived 38074 (MWVDQKRKEWQEIK), inducing non-protective antibodies, displayed the HLADRβ1*0802 binding motif and register (grey); this HABP is in highly polymorphic region (Figure 1B).

Recent studies have found predominant transcription of domain cassette DC8 (UPSB promoter followed by NTSβ-DBLα2-CIDRα1-DBLβ12-DBLγ4) and DC13 (encoding DBLα1.7-CIRDα1.4) (both containing the GACxPxRRxxLC motif) in blood samples from 70% of 88 Tanzanian children suffering severe malaria. This stresses the importance of 24196 (containing this motif) in inducing strain-transcending complete protective immunity against severe malaria in HLADRβ1*04 individuals, suggesting that more HABPs from other IE membrane-expressed molecules (like histidine-rich protein-II-derived 24230 (6800) under HLADRβ1*07 control [11]–[14], [37], STEVOR [38], [39]; RIFIN etc.) are needed to obtain definitive full protection against severe malaria.

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]–[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, chemically-synthesised definitive antimalarial vaccine [11]–[14].