Cross-reactivity Studies of an Anti-Plasmodium vivax Apical Membrane Antigen 1 Monoclonal Antibody: Binding and Structural Characterisation

doi:10.1016/j.jmb.2006.12.028

J. Mol. Biol. (2007) 366, 1523–1537

Cross-reactivity Studies of an Anti-Plasmodium vivax Apical Membrane Antigen 1 Monoclonal Antibody: Binding and Structural Characterisation Sébastien Igonet 1 †, Brigitte Vulliez-Le Normand 1 †, Grazyna Faure 1 Marie-Madeleine Riottot 1 , Clemens H.M. Kocken 2 , Alan W. Thomas 2 and Graham A. Bentley 1 ⁎ 1

Unité d'Immunologie Structurale, CNRS URA 2185, Département de Biologie Structurale et Chimie, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris cedex 15, France 2

Department of Parasitology, Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the crossreactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed. © 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: apical membrane antigen 1; Plasmodium; vaccine candidate; monoclonal antibody; crystal structure

Introduction Apical membrane antigen 1 (AMA1) is a type I integral membrane protein present in all charac† S.I. and B.V.N. contributed equally to this work. Abbreviations used: AMA1, apical membrane antigen 1; CDR, complementarity-determining region; FR, variable domain framework region; GIA, growth inhibition assay; mAb, monoclonal antibody; H, antibody heavy chain; L, antibody light chain. E-mail address of the corresponding author: [email protected]

terised species of the malaria parasite, Plasmodium.1 It comprises an ectoplasmic region of about 500–550 residues (depending on species), a single transmembrane segment and a small cytoplasmic domain that is highly conserved between species. AMA1 is stored in the microneme organelles after synthesis but is subsequently translocated to the parasite surface via the rhoptry neck just prior to, or during, host cell invasion.2,3 During the invasion process, the ectoplasmic region is cleaved from the surface, but the functional significance of this proteolytic maturation event is not clearly understood. 4,5 Genetic disruption experiments have underlined the importance of AMA1 for parasite viability6 and,

0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

1524 moreover, the stage-specific expression and localisation of AMA1 point to a crucial role in invasion process. Antibodies raised against the ectoplasmic region of AMA1 can block parasite invasion in vitro, further emphasising the functional importance of this surface protein.7–12 AMA1 is expressed in both sporozoites (pre-erythrocytic stage) and merozoites (erythrocytic stage), and invasion-blocking antiAMA1 antibodies have similar effects on hepatocyte and erythrocyte invasion, suggesting an essentially identical role of the protein in both these phases of the Plasmodium life-cycle.13 Two principal mechanisms for invasion inhibition by anti-AMA1 antibodies have been proposed: direct steric blockage of AMA1 function and cross-linking. 7,12,14 Steric obstruction of function by certain invasion-inhibitory antibodies is implied by the observation that their derived monovalent Fab fragments can also be effective inhibitors, as in the case of the inhibitory monoclonal antibodies (mAbs) R31/C27 and 4G2.12 Cross-linking and the consequent inhibition of AMA1 dispersion over the parasite surface from the apical end have also been linked to invasion inhibition.12,14 Indeed, both steric and cross-linking mechanisms appear to operate concurrently in the polyclonal humoral response.12 By contrast, agglutination of parasites by polyclonal anti-AMA1 antibodies has not been observed,12 making this an unlikely mechanism of invasion inhibition. Immunisation with AMA1 induces antibodymediated in vivo protection against parasite challenge in simian15–17 and rodent systems.18,19 In addition, passive transfer of anti-AMA1 antibodies can also facilitate protection.18,19 Irreversibly reduced AMA1, however, is not recognised by antibodies raised against the native antigen; neither can it induce protective immunity.19 An effective immune response thus depends on conformational epitopes maintained by a set of eight disulfide bridges conserved in all plasmodial AMA1 homologues.20,21 Because of its essential role in the Plasmodium life-cycle and its ability to induce protective immunity in animal models, the ectoplasmic region of AMA1 is a leading malaria vaccine candidate and is currently in the early stages of clinical trials.22,23 Some overall serological cross-reactivity between the most prevalent human malaria parasites, Plasmodium falciparum and Plasmodium vivax, has been reported, 24 and some evidence exists for cross-species immunity in humans;25 however, data relating to this are very limited at present. Given the complex cross-species interactions between malaria parasites in humans,26 malaria vaccines that are effective against the most prevalent species at least would be very useful. Accordingly, there is an interest in exploring the existence and potential of cross-species epitopes in malaria vaccine candidates such as AMA1. The three-dimensional structures of the complete ectoplasmic region of AMA1 from P. vivax (PvAMA1)27 and the domains I–II construction of the P. falciparum homologue have been determined

Cross-reactive Antibody to Apical Membrane Antigen 1

recently by X-ray crystallographic analysis.28 In addition, NMR solution structures have been determined for the individual domains II and III.21,29 These results provide a structural basis for analysing polymorphism and protecting B-cell epitopes in AMA1, factors that can be key to understanding the function of the protein and to its eventual optimisation as a vaccine candidate. Polymorphism, which has been studied extensively in the P. falciparum homologue, PfAMA1, is under strong diversifying selection pressure from the immune response of the host.30,31 Although polymorphism extends over the entire ectoplasmic primary structure of PfAMA1, the distribution of polymorphic sites is highly biased to one side of its three-dimensional structure,28,32 implying that a significant fraction of the surface, contributed to by all three domains, is subject to functional constraints. The three-dimensional location of function-blocking B-cell epitopes can give fine details on functionally important regions. To date, the epitopes of very few species-specific mAbs have been localised on AMA1 but function-blocking epitopes have nonetheless been mapped to all three domains of the ectoplasmic region.27,33,34 Thus, functionally sensitive regions extend over a large surface of the protein, which suggests that plasmodial AMA1 could have more than one binding partner, as shown for the homologue from another apicomplexan parasite, Toxoplasma gondii.35 In the framework of vaccine development, there is an interest in identifying both function-blocking epitopes and those leading to cross-linking of AMA1 molecules and the subsequent inhibition of their dispersion over the parasite surface during host cell invasion.12,14 It is important to analyse the impact of polymorhism on the response and to ascertain if epitopes on the recombinant antigen are, indeed, accessible on the parasite surface. With these objectives in mind, we have generated a number of mAbs against the recombinant ectoplasmic region of the P. vivax homologue, PvAMA1, to obtain a broader view of the structure and protection mechanisms of epitopes present in this antigen. One of these, F8.12.19, shows significant crossspecies reactivity although no function-blocking effect was detected in ex vivo parasite growth inhibition assays. Here, we present the crystal structures of the Fab fragment of F8.12.19 complexed to PvAMA1 and to the cross-reactive antigen PfAMA1, showing that it binds to domain III. We discuss these results from the perspective of immune protection and cross-species reactivity of the antibody response to AMA1.

Results Polyclonal anti-PfAMA1 and PvAMA1 responses Western blot analysis of rabbit antisera raised against recombinant PfAMA1 and PvAMA1 ecto-

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Cross-reactive Antibody to Apical Membrane Antigen 1

plasmic regions demonstrates that there is very limited cross-reactivity of the anti-PfAMA1 antiserum with PvAMA1 and vice versa (Figure 1). Moreover, when the PfAMA1 is reduced and alkylated, cross-reactivity of anti-PvAMA1 antiserum is not enhanced by possible exposure of linear epitopes. This indicates that use of the complete ectoplasmic region of AMA1 in vaccination procedures induces only low-level cross-species reactivity in the polyclonal response. Characterisation of mAb F8.12.19 The murine mAb F8.12.19, raised against the recombinant ectoplasmic region of PvAMA1 in BALB/c mice, has isotype IgG1-κ. The VL domain derives from the Vκ germ-line gene IGKV4-72 and joining (J) gene segment IGKJ4; the IGHV5-4 VH germ-line gene, the IGHD3-2 diversity (D) minigene and the IGHJ3 JH gene segment gave the highest level of sequence identity to the VH domain. The gene nomenclature is according to Martinez-Jean et al.36 and Giudicelli et al.37 Non-synonymous mutations in the V-encoding germ-line genes lead to nine amino acid sequence changes: Ile→Ser-L2 (FR-L1), Ser→Arg-L26 (CDR-L1), Pro→Ser-L40 (FRL2), Tyr→His-L49 (FR-L2), Asn→His-L94 (CDR-L3), Thr→Ile-H28 (CDR-H1), Gly→Asn-H54 (CDR-H2), Pro→Val-H60 (FR-H3), Met→Ile-H89 (FR-H3). (Residue numbering is according to Kabat et al.38). In addition, there is a two-nucleotide mutation in the H101 codon of the germ-line JH minigene from GCT (Ala) to GGC (Gly). One synonymous somatic mutation occurs in the VL gene: Asn-L53 (CDRL2); three synonymous somatic mutations occur in the VH gene: Cys-H22 (FR-H1), Leu-H78 (FR-H3) and Glu-H85 (FR-H3). Immunofluorescence assays revealed that F8.12.19 recognises mature P. vivax schizonts, showing that

the epitope is accessible on the methanol-fixed parasite, and that it cross-reacts with AMA1 from Plasmodium cynomolgi, Plasmodium berghei, Plasmodium knowlesi and P. falciparum. The fluorescence signal from the closely related species, P. cynomolgi and P. knowlesi, was similar to that from P. vivax, whereas the response from P. falciparum and P. berghei was less but still significant (data not shown). The cross-reactivity of F8.12.19 with PfAMA1 was quantified by measuring the kinetics of binding and dissociation of the recombinant ectoplasmic region by surface plasmon resonance. With the antibody covalently immobilised on the sensor chip, the recombinant ectoplasmic regions of PvAMA1 (strain Sal I) and PfAMA1 (strain FVO) as analyte were compared on the same sensor chip channel. Using the 1:1 Langmuir binding model to fit the experimental association and dissociation curves, the kinetic kon and koff constants were calculated to be 1.6 × 105 M− 1s− 1 and 1.0 × 10− 4 s− 1, respectively, for the cognate antigen PvAMA1, and 2.2 × 104 M− 1s− 1 and 1.7 × 10− 3 s− 1, respectively for PfAMA1 (Figure 2). The apparent dissociation constant (KD = koff/kon) is thus 0.6 nM for PvAMA1 and 77 nM for PfAMA1, consistent with the immunofluorescence assays on P. falciparum schizonts. F8.12.19 recognises a conformational epitope on PvAMA1 since binding, measured by ELISA, is largely abrogated when the antigen is treated with SDS and β-mercaptoethanol (data not shown). This is also true of other anti-PvAMA1 mAbs we have produced (unpublished results), whereas the control monoclonal antibody anti-His Cterm (Invitrogen) recognises the linear histidine tag at the C terminus of the recombinant antigen under the same reducing conditions. Western blot data using individual recombinant PfAMA1 domains also indicated that F8.12.19 recognises a conformational epitope located in domain III of PfAMA1 (data not shown). These data demonstrate cross-species reactive mAbs can be isolated. F8.12.19 did not demonstrate significant levels of parasite growth inhibition, as assayed in an ex vivo growth inhibition assay (GIA) with P. cynomolgi schizont-infected red cells, harvested from an infected rhesus monkey. It cannot be ruled out, however, that low levels of inhibitory effects are evoked by this mAb, as the complicated ex vivo assay allows for detection of only relatively large inhibitory effects. General description of the structure

Figure 1. Slot blot analysis of polyclonal anti-AMA1 rabbit sera. Slot blots were prepared and incubated as described in Materials and Methods. The top row contains PvAMA1 ectoplasmic region, the middle row PfAMA1 ectoplasmic region and the bottom row reduced and alkylated (R/A) PfAMA1 ectoplasmic region. Lane 1 is anti-PfAMA1 antiserum and lane 2 is anti-PvAMA1 antiserum. Minimal cross-reactivity is observed between anti-PfAMA1 antiserum and PvAMA1, as well as between anti-PvAMA1 antiserum and PfAMA1. Exposure of linear epitopes in PfAMA1 by reduction/alkylation does not enhance cross-reactivity with anti-PvAMA1 antiserum.

Crystallographic data and refinement statistics for the final atomic models of the PvAMA1 and PfAMA1 complexes with Fab F8.12.19 are given in Tables 1 and 2, respectively. The crystals of the two complexes, which were obtained using very similar buffer conditions, are closely isomorphous to each other. The complete light and heavy chains of the Fab fragment could be traced in both structures, even for the C termini of both chains and the segment H130-H137 of the heavy-chain constant

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Cross-reactive Antibody to Apical Membrane Antigen 1

Figure 2. Surface plasmon resonance runs showing the interaction of the recombinant ectoplasmic region of AMA1 as analyte and IgG F8.12.19 covalently immobilised on the Biacore sensor chip. The runs were corrected for non-specific binding by subtraction of the curves obtained by passage of the same protein solution through a blank channel (no immobilised IgG) on the same senor chip. The curves labelled buffer show the results for passage of the running buffer alone. Analyte was injected for 60 s for the association phase. This was followed by injection of the running buffer alone at the same flow-rate to give the dissociation phase. The response in resonance units (RU) is plotted as a function of time (in s.). (a) PvAMA1 at concentrations 5.0 μg/ml, 2.5 μg/ml, 1.25 μg/ml and 0.62 μg/ml, as indicated. (b) PfAMA1 at concentrations 50 μg/ml, 25 μg/ml, 12.5 μg/ml and 6.2 μg/ml, as indicated.

domain, which are often disordered in Fab crystal structures. These latter regions are stabilised by intermolecular contacts within the crystal lattice. Table 1. Summary of crystal parameters

Space group Unit-cell parameters a = b (Å) c (Å) Z (molecules/unit cell) VM (A3 Da− 1)a Solvent volume (%) Beamline (ESRF) Wavelength (Å) Resolution range (Å) No. unique reflections Redundancy Rsymm (overall) (%) Completeness (overall) (%) I/σ(I) (overall) (%)

PvAMA1

PfAMA1

P63

P63

171.8 44.7 6 1.9 34 ID14-2 0.933 20–2.5 (2.6–2.5) 26,533 10.0 14.1 (93) 98.1 (88.0) 14.8 (1.8)

171.9 44.2 6 2.2 45 ID14-4 0.949 20–2.8 (3.0–2.8) 18,102 (2786) 21.2 (16.2) 25.3 (116) 96.0 (80.4) 12.1 (2.7)

Rsymm = redundancy-independent R-factor (intensities).55 a Assuming one molecule of PvAMA1(domains I, II and III)Fab or PfAMA1(domains II and III)-Fab complex per asymmetric unit.

The elbow angle, defined as the angle subtended by the pseudo 2-fold axes relating VH to VL and CH to CL, respectively, is 134° in both structures. The Fab coordinates of the two complexes superimpose upon each other with a r.m.s. difference of 0.37 Å between the α-carbon positions. A total of 156 solvent positions were included in the final model of the PvAMA1 complex but none was placed in the lower-resolution structure of the PfAMA1 complex. Table 2. Refinement statistics

Resolution (Å) R-value (working set) Rfree No. reflections (total) No. reflections for R value (test) No. protein atoms No. solvent atoms r.m.s deviation from ideal Bond lengths (Å) Bond angles (deg.)

PvAMA1

PfAMA1

20–2.5 0.192 0.250 24,858 1558 3606 150

20–2.9 0.215 0.278 16,037 1137 3605 0

0.02 1.93

0.02 1.95

Cross-reactive Antibody to Apical Membrane Antigen 1

All hypervariable regions of F8.12.19 are well defined in the electron density and can be assigned to the following canonical conformations (except CDR-H3): CDR-L1, class 1; CDR-L2, class 1; CDR-L3, class 1; CDR-H1, class 1; CDR-H2, class 3.39 CDR-H3 comprises 14 residues (residues Asp-H95 to TyrH102 according to the Kabat convention38) and is thus significantly longer than the mean length of 8.7 noted for murine antibodies.40 CDR-H3 forms a kinked base as predicted from key residues in this region:41 the main-chain carbonyl group of Arg-H94 forms a bifurcated hydrogen bond to the main-chain amide groups of Gly-H101 and Tyr-H102, and Nε of Trp-H103 forms a hydrogen bond to the carbonyl group of Phe-H100F. The conformation of CDR-H3 is maintained largely by a number of hydrogen bonds between side-chain and main-chain atoms within this CDR, together with buried water molecules that contribute to an intricate network of bridging polar interactions: the side chain of Arg-H94 interacts with the main-chain carbonyl groups of Asp-H95 and Gly-H96; Asp-H95 forms hydrogen bonds to the main-chain amide groups of Gly-H96, Gly-H100B, Tyr-H100C, Gly-H100D and Gly-H100E, either directly or via two buried water molecules; Oγ of Ser-H100A interacts with the carbonyl group of ProH97 (Figure 3). We therefore anticipate that the CDRH3 conformation is well defined in the free antibody and that it undergoes little change upon binding the antigen. CDR-H3 has five glycine residues but only Gly-H100B has ϕ-ψ angles that fall outside favourable regions of the Ramachandran plot for residues with side chains. Interestingly, residue H101 cannot

1527 be other than glycine because the presence of any side-chain would lead to steric hindrance with other residues in CDR-H3; the CDR-H3 conformation we observe thus accounts for the two-nucleotide mutation at the Ala codon in the germ-line JH minigene to the Gly codon. Although the complete recombinant PvAMA1 ectoplasmic region (residues 43–487) and domains II and III of PfAMA1 (residues 303 to 544) were complexed with Fab F8.12.19 in the protein preparations used for crystallisation, only a small but contiguous segment of domain III from these two homologues could be identified in the electron density: the 34-residue segment from Ile421 to Lys454 in PvAMA1 and the equivalent 34-residue segment from Ile479 to Arg512 in PfAMA1 (Figure 4). This part of the antigen could be identified readily in the electron density by the cystine knot formed with Cys432-Cys449 and Cys434-Cys451 in PvAMA1 and Cys490-Cys507 and Cys492-Cys509 in PfAMA1. Additional electron density, observed in the final difference maps of both PvAMA1 and PfAMA1, did not coincide with the complete, superimposed PvAMA1 structure (PDB entry 1W8K) (Figure 5). This region is connected to modelled part of the antigen via the Cys388-Cys444 bridge in PvAMA1 (443Cys-502Cys in PfAMA1) but the electron density was too disordered to propose an unambiguous interpretation. No other region of significant electron density occurred in the maps. Superposition of the complete structure of the PvAMA1 ectoplasmic region onto the domain III

Figure 3. A stereo view of CDR-H3 of F.8.12.19 from residues Cys-H92 to Gly-H104 in the PvAMA1 complex. Hydrogen bonds that are important for maintaining the conformation of this hypervariable region are shown as dotted lines. The two solvent molecules that contribute to the stability of the loop through bridging contacts are shown as red spheres (labelled Wat).

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Cross-reactive Antibody to Apical Membrane Antigen 1

Figure 4. A stereo view of the Fv fragment of F8.12.19 (VL in orange, VH in purple) and the bound antigen segments from PvAMA1 (blue) and PfAMA1 (cyan). Only the main chain is shown for the bound antigens. Superposition was made by optimising the fit of the α-carbon atoms of the Fab components only (i.e. without antigen); only the Fv fragment of the PvAMA1 complex is shown. (a) A view along the antigen-binding groove with the Fv structure shown in ribbon form. (b) An orthogonal view from above the antigen-binding site with the Fv shown as a van der Waals' surface.

segment in the two complexes shows that the intact antigen cannot be accommodated in the crystal lattice of either structure. Severe steric hindrance would occur between symmetry-related molecules, particularly domain I around the crystallographic origin in the case of the complete PvAMA1 ectoplasmic region. Steric hindrance in crystal packing of the domain II–III construction of PfAMA1 would also occur but would be less severe. Such constraints on the crystal packing and the absence of electron density for much of the AMA1 ectoplasmic region would require (i) that much of the antigen structure be disordered through distortion induced by steric hindrance, or (ii) that parts of the antigen be removed by proteolysis prior to crystal formation, or (iii) that both these factors intervene. We suggest that proteolysis is responsible, in part at least, because the recombinant PvAMA1 ectoplasmic region expressed in Pichia pastoris is prone to partial cleavage.32 We had previously identified two partial cleavage sites in domains II and III, respectively, in PvAMA1, but the polypeptide fragments are maintained together covalently by the network of cystine bridges.27 Both cleavages occur in disordered loops in the crystal structure of the complete PvAMA1

ectoplasmic region and thus do not affect the overall integrity of the native conformation. This is confirmed by our observation, from SDS/polyacrylamide gels performed under reducing conditions, that the recombinant protein recovered from crystals of the PvAMA1 ectoplasmic region was partially proteolysed. The extent of proteolysis is variable between different antigen expressions and we have been unsuccessful in controlling degradation with protease inhibitors during expression and purification. Kennedy and collaborators have detected a partial cleavage site in the recombinant PfAMA1 ectoplasmic region expressed in P. pastoris that is equivalent to the site we found in domain II in PvAMA1.9 We therefore suggest that the lattice packing leads to the selection of only those complexes containing antigen sufficiently proteolyzed to alleviate steric hindrance, although it is clear that proteolysis would have to include other cleavage sites in addition to the two that we have identified in PvAMA1. Indeed, the PvAMA1-Fab F8.12.19 crystals took over two months to appear and could not be produced systematically. Although crystals of the Fab complex with PfAMA1 appeared within one week, this can be attributed to the lower

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Cross-reactive Antibody to Apical Membrane Antigen 1

Figure 5. Stereo view of the difference density in the PfAMA1 complex contoured at the 2.5 r.m.s. level. This shows the only significant residual density in the Fourier maps (located on the left-hand side of the Figure) that could not be modelled unambiguously. For clarity, only the antigen is shown. A similar region of uninterpreted density is present in the difference map of the PvAMA1 complex.

stability in the PfAMA1 construction devoid of domain I, since the N-terminal region interacts with domains II and III. In line with this, we have been unsuccessful in obtaining crystals with the complete PfAMA1 ectoplasmic region (domains I to III). The bound antigen segments Ile421–Lys454 of PvAMA1 and Ile479–Arg512 of PfAMA1 in the two crystal structures show some structural divergence from the free antigen. The r.m.s. difference between the α-carbon positions of the complete PvAMA1 ectoplasmic region (PDB entry 1W8K) and the bound PvAMA1 segment is 2.5 Å (25 out of 34 residues included) and 2.2 Å for the PfAMA1 complex (26 out of 34 residues included). By comparison, the P. vivax and P. falciparum antigen segments in the complexes differ by 0.9 Å from each other (Figure 6). Much of the difference from the free antigen occurs in the loop formed by the PvAMA1 residues 440–444 that connects the two β strands of the hairpin between residues 436 and 449. In the intact antigen, the connecting loop interacts with domain II via five hydrogen bonds that strongly influence its conformation. Similarly, the intradomain III cystine bridge (Cys388–Cys444 in PvAMA1, Cys443–Cys502 in PfAMA1), connecting the complexed segment to the disordered region of AMA1 in these two structures, also influences the conformation of this loop in the intact antigen. In the two complexes, on the other hand, these contacts are absent and this part of the structure shows con-

siderable mobility, as revealed by the elevated temperature factors. When PvAMA1 residues 440– 444 and the two C-terminal residues of the segment (453–454) are excluded from the comparison between the free and complexed antigen, the r.m.s. difference between α-carbon positions reduces to 0.8 Å for the PvAMA1 complex and 0.6 Å for the PfAMA1 complex. Since these seven excluded residues are distant from the F8.12.19 antigenbinding site, the differences in antigen structure do not affect the conformation of the epitope in the intact PvAMA1 and PfAMA1. Antigen–antibody interactions The mAb F8.12.19 recognises a discontinuous epitope that is divided into three segments comprising 11 residues: Lys427, Glu428, Cys432, Pro433, Cys434, Glu435, Pro436, Glu437, Asn450, Cys451, Val452 in the cognate antigen PvAMA1, and Lys485 (427), Asp486(428), Cys490(432), Pro493(433), Cys492(434), Asp493(435), Pro494(436), Glu495 (437), Lys508(450), Cys509(451), Val510(452) in the cross-reacting antigen PfAMA1 (interatomic contacts < 3.8 Å; for PfAMA1, the equivalent PvAMA1 residue position in parentheses; sequence differences are in italics). The epitope lies in a shallow groove formed by CDR-L3 and CDR-H3 on one side and CDR-H1 and CDR-H2 on the other (Figure 4). The total solvent-accessible surface buried at the antigen–antibody interface is 1437 Å2 for the PvAMA1

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Cross-reactive Antibody to Apical Membrane Antigen 1

Figure 6. A stereo view of the PvAMA1 (blue) and PfAMA1 (cyan) antigen segments in the respective Fab F8.12.19 complexes superimposed optimally onto the corresponding segment of the complete PvAMA1 ectoplasmic region (red). (PDB entry 1W8K). Only the mainchain atoms and cystine bridges (yellow) are shown. Two orthogonal views are shown in (a) and (b), respectively.

complex and 1399 Å2 for the PfAMA1 complex; contributions from the antigen, antibody and individual CDRs are listed in Table 3. The SC (shape complementarity) index is 0.71 and 0.68 for the PvAMA1 and PfAMA1 complexes, respectively.42 Thus, by these criteria, F8.12.19 achieves higher complementarity with the cognate antigen. Superposition of the complete PvAMA1 structure onto the complexed segment Ile421-Lys454 shows that no additional residues of the cognate antigen

Table 3. Buried surface-accessible area for the antigen, total antibody and individual CDRs (Å2)

Antigen Antibody (total) CDR-L1 CDR-L2 CDR-L3 CDR-H1 CDR-H2 CDR-H3

PvAMA1

PfAMA1

724 713 19 0 171 139 203 192

725 674 54 0 187 129 190 166

should be within contact distance from F8.12.19; the crystal structure therefore reveals the complete epitope recognised by the monoclonal antibody (Figure 7). Contacts made by the antibody to AMA1 are contributed by CDR-L3, CDR-H1, CDRH2 and CDR-H3 in both complexes. Eleven hydrogen bonds and one salt-bridge are formed between PvAMA1 and the antibody (Table 4), with three buried water molecules intervening by bridging contacts across the interface of the complex. Fewer polar contacts are achieved in the PfAMA1 complex; the salt-bridge is retained but only seven hydrogen bonds are formed as a result of sequence differences between the two antigens at the interface. The contribution of solvent to antibody–antigen interactions in the PfAMA1 complex could not be assessed because of the low resolution of the diffraction data. The structural basis of cross-reactivity is discussed in the next section. Of the nine non-synonymous changes occurring in the variable domains, only Gly-H54→Asn makes direct contacts to AMA1, creating a hydrogen bond to Glu428 of PvAMA1 but not to Asp486, the equivalent residue in PfAMA1 (see Table 4).

Cross-reactive Antibody to Apical Membrane Antigen 1

1531

Figure 7. A stereo view of a model of F8.12.19 complexed to the complete PvAMA1 ectoplasmic region based on superposition of the PvAMA1 structure (PDB entry 1W8K) onto the complexed PvAMA1 antigen segment. Only the Fv moiety of F8.12.19 is shown for clarity (ribbon form with VL in orange and VH in purple). PvAMA1 is shown as an α-carbon trace with domain I in green, domain II in blue and domain III in red. The cystine bridges in PvAMA1 are shown in yellow. This view is identical with that of Figure 4(a).

The structural basis of cross-reactivity by F8.12.19 The epitope is highly conserved between PvAMA1 and the other Plasmodium homologues examined by immunofluorescence assays. The aligned sequences from the cross-reacting species corresponding to the antigen segment 421–454 of PvAMA1 and the residues forming the epitope are shown in Figure 8. Polar interactions, which are very important in defining the antigen specificity of an antibody, essentially involve invariant residues, residues with conservative differences and main-chain atoms of AMA1 (see Table 4). Only PvAMA1 residues 428

Table 4. Polar antigen-antibody interactions in the PvAMA1-FabF8.12.19 complex PvAMA1 Lys427(Nζ) Lys427(Nζ) Glu428(Oε1) Glu428(Oε2) Cys432(O) Cys432(O) Glu435(N) Glu435(Oε1) Glu437(Oε1) Asn450(Nδ2) Val452(N) Val452(O)

PfAMA1

Antibody

Lys485(Nζ) Lys485(Nζ) – – Cys490(O) Cys490(O) Asp493(N) Asp493(Oδ1) Glu495(Oδ2) – – Val510(O)

Asp-H31(O) Asp-H52(Oδ2) Asn-H54(N) Asn-H54(Nδ2) Tyr-H33(OH) Tyr-H58(OH) Tyr-H35(OH) Tyr-H33(N) Tyr-H100C(OH) Gly-H100B(O) Ser-L92(O) His-L94(N)

Contacting atoms are given in parentheses. Antigen residues in bold are those that differ in sequence between the two species.

(Glu in PvAMA1, Asp in P. cynomolgi AMA1 and PfAMA1), 435 (Glu in PvAMA1, Asp in PfAMA1), 437 (Glu in PvAMA1, Thr in P. berghei AMA1) and 450 (Asn in PvAMA1, Lys in PfAMA1) could lead to different side-chain interactions in the other Plasmodium homologues. The crystal structure of the PfAMA1 cross-reaction complex shows how these residues can be readily accommodated at the antibody–antigen interface for the different homologues, since they are located at the periphery of the antigen-binding site. The most significant sequence difference is between Asn450 of PvAMA1 and Lys508 of PfAMA1. The hydrogen bond formed between Asn450 (PvAMA1) and the antibody is lost in the PfAMA1 complex and Lys508 (PfAMA1) contributes non-polar contacts only, with the amino group of this side-chain being exposed to the solvent. The conservative difference Glu435 (PvAMA1)/Asp493(PfAMA1) preserves the same polar contacts to the antibody. The conservative difference Glu428(PvAMA1)/Asp486(PfAMA1), by contrast, leads to a loss of two hydrogen bonds in crystals of the PfAMA1 complex; although modelling shows that Asp486(PfAMA1) could form the same hydrogen bonds to the antibody as does Glu428(PvAMA1), polar contacts are formed instead with a neighbouring molecule in the crystal lattice. These differences in polar interactions and the reduced antibody–antigen complementarity in the PfAMA1 complex lead to a loss of about 100-fold in the measured affinity constant determined by surface plasmon resonance. In the case of P. knowlesi AMA1, all epitope residues are the same as in

1532

Cross-reactive Antibody to Apical Membrane Antigen 1

Figure 8. The PvAMA1 epitope recognised by F8.12.19 and the crossreacting homologues from other Plasmodium species examined by immunofluorescence assays. The Plasmodium species are P. vivax (Pv), P. knowlesi (Pk), P. cynomolgi (Pc), P. falciparum (Pf) and P. berghei (Pb). The aligned sequences shown correspond to the segments found in the crystal structures of the two complexes. Residue numbering (row Res no) corresponds to PvAMA1. The antibody-contacting residues (row epitope), corresponding to interatomic distances <3.8 Å, are indicated by asterisks (*). Contacting residues from the antigen (row interaction) are indicated as involving polar side-chain groups (s), polar main-chain groups (m), both side-chain and main-chain polar groups (+) and non-polar groups (•). Black-shaded residues are invariant and gray-shaded residues are conservative differences.

PvAMA1 and F8.12.19 should bind to this homologue with the same affinity. The effect of polymorphisms on the recognition of different strains of P. vivax by F8.12.19 cannot be assessed because data for this species are restricted essentially to domain I.32 Polymorphism studies in P. falciparum, on the other hand, are more complete and show that the epitope includes two dimorphic sites: Lys/Leu485 (Lys427 in PvAMA1) and Asp/ Ala493 (Glu435 in PvAMA1). For PfAMA1 from the P. falciparum FVO strain that we have studied here, the dimorphic residues that contact F8.12.19 are Lys485 and Asp493,32 thus representing a very conservative difference with respect to the P. vivax Sal I strain. From modelling studies, we predict that the other dimorphic residues possible in other strains, Leu485 and Ala493, can be accommodated at the antibody–antigen interface, but these two apolar side-chains would lead to a loss of two and one hydrogen bonds, respectively (see Table 4).

Discussion The two crystal structures of the complex formed by the cross-reactive anti-PvAMA1 mAb F8.12.19 with the ectoplasmic regions of PvAMA1 and PfAMA1, respectively, show that the epitope is discontinuous and is located on domain III between residues Lys427 and Val452 of the cognate antigen PvAMA1 and the equivalent segment, Lys485 to Val510, of the cross-reacting antigen, PfAMA1. The conformation of this region is largely determined by the cystine knot formed by the pair of disulphide bridges Cys432–Cys449 and Cys434–Cys451 of PvAMA1, and Cys490–Cys507 and Cys492–Cys509 in PfAMA1, in line with our observation that F8.12.19 does not recognise the reduced antigen. Indeed, the loop region between residues Cys432 and Glu437 in PvAMA1, and Cys490 and Glu494 in PfAMA1, which are maintained by the cystine knot, protrudes into the central region of the antigenbinding site and forms the major component of this discontinuous epitope. Surprisingly, only the segment comprising residues Ile421 to Lys454 of PvAMA1, and Ile479 to

Arg512 of PfAMA1, could be built into the electron density maps. No other trace of the two antigens was found except for a short ill-defined region that could not be modelled unambiguously. We have argued above that PvAMA1 and PfAMA1 probably undergo proteolysis because superimposing the complete ectoplasmic region of PvAMA1 onto the complexed segment in the antigen-binding site leads to steric hindrance between neighbouring complexes in the crystal lattice, thus precluding crystallisation of a complex with the intact antigen. Although this might suggest that the Fab moiety dominates in the formation of crystals, we have not been successful so far in crystallising the antibody fragment alone; however, this could arise from subtle influences from some of the intermolecular contacts that occur between the antigen and the CH1 of the Fab. The crystal structure of the PvAMA1 and PfAMA1 complexes reported here, and our previous studies on PvAMA1,32 underline the lability of the recombinant ectoplasmic region to proteolytic degradation. In spite of antigen degradation in the crystallised Fab complexes, superposition of the structure of the complete PvAMA1 ectoplasmic region onto the traced antigen fragment in both cases shows that no additional residues contact F8.12.19. The structure determinations have thus revealed the complete epitope recognised by the mAb. The F8.12.19 epitope is accessible on the membrane-bound antigen present in methanol-fixed parasites because immunofluorescence studies show that the mAb binds to mature P. vivax schizonts. With these assays, we also showed that F8.12.19 cross-reacts with AMA1 homologues from P. falciparum, P. cynomolgi, P. knowlesi and P. berghei, an observation corroborated in the case of P. falciparum by kinetic binding and crystal structure studies. The crystallographic analysis has provided a structural basis for understanding the cross-reactive behaviour of this mAb, showing that the antibody contacts both main-chain atoms as well as side-chains that are largely invariant or highly conserved. The reactivity profile that we have observed with polyclonal rabbit antisera directed against PfAMA1 and PvAMA1 demonstrates that there is very little cross-reactivity between these antisera and the respective ortholo-

1533

Cross-reactive Antibody to Apical Membrane Antigen 1

gous antigens. We have, nonetheless, been able to isolate a murine monoclonal antibody that recognises a conserved cross-species epitope of Plasmodium AMA1. Further identification of cross-reactive mAbs and their epitopes could help to identify regions of AMA1 that are conserved across species and that could be considered as part of future multispecies vaccines. Polymorphisms, which are considered to arise through immune pressure from the host,43 are located on all three domains, suggesting that protective epitopes are distributed over the entire antigen. However, divergent conclusions concerning the importance of domain III in the immune response have emerged from different studies. Antibodies that were affinity-purified from the immune serum of donors living in endemic regions using a recombinant domain III construction were shown to inhibit parasite growth.21 In line with these observations, immunisation with a peptidomimetic based on the segment comprising residues 446–490 in domain III of PfAMA1 induced polyclonal and monoclonal antibodies that cross-reacted with parasites.34 Of note, the polyclonal antibodies and two of the monoclonal antibodies produced in this latter study showed growth-inhibitory activity. Other studies, however, concluded that domains I and II induced the most significant amounts of inhibitory antibodies (B.W. Faber and A.W.T., unpublished results)8,44 and that only a small fraction of the immune response was against domain III.43 Of the ten anti-PvAMA1 monoclonal antibodies that we have produced, three show clear competition with F8.12.19 in binding to the antigen, suggesting that domain III forms an important target in the immune response to the recombinant antigen, at least in immunisations of mice (unpublished results). Our motivation for studying the anti-AMA1 mAb F8.12.19 has been to contribute towards an overall characterisation of dominant AMA1 epitopes in the context of vaccine development. The humoral immune protection induced by AMA1 operates by two distinct mechanisms: direct steric inhibition of function (receptor binding and proteolytic maturation) and inhibition of AMA1 dispersion over the parasite surface during invasion by crosslinking.12,14 While functional inhibition by steric hindrance can be tested for directly by using individual mAbs, inhibition by cross-linking can be detected only in a polyclonal context. We observed no major parasite growth-inhibitory effects in the presence of F8.12.19, suggesting that this mAb does not react with regions of AMA1 involved directly in the functional processes mentioned above; however, we cannot exclude the possibility that the magnitude of inhibition by F8.12.19 falls below the observable threshold of this delicate ex vivo GIA, which requires the use of P. cynomolgi. Nonetheless, immunofluorescence assays show that the epitope is accessible on the surface of parasites, leaving open the possibility of immune protection, in concert with other dominant epitopes, by AMA1 cross-linking.

The presence of polymorphic sites in dominant Bcell epitopes can reduce the effectiveness of AMA1 as a vaccine.9 PfAMA1 has two dimorphic sites present in the F8.12.19 epitope but our modelling studies suggest that these sequence differences with respect to the FVO strain antigen that we have cocrystallised with F8.12.19 can be accommodated at the antibody–antigen interface. The present lack of polymorphism data for domain III of PvAMA1 does not allow a similar evaluation for this homologue. Nonetheless, the extensive cross-species reactivity observed with F8.12.19 mimics polymorphic (or intraspecies) variations and shows that the humoral immune response can sometimes tolerate antigenic changes. Although antibodies induced by recombinant AMA1 show reduced recognition of heterologous parasites, the level of invasion inhibition can still remain significant. For example, rabbit antibodies induced by strain 3D7 PfAMA1 gave an in vitro growth-inhibition level for FVO parasites that was 20% of that for the homologous parasite.9 This shows a significant, if reduced, protective capacity is still present against a heterologous strain. In this example, the heterologous antigen differs from the immunising strain at 24 polymorphic sites, a difference that is close to the maximum found in field strains. Of note, this example used polyclonal antibodies where both steric blocking and AMA1 cross-linking mechanisms of immune protection should be operating. This underlines the importance of a thorough epitope characterisation of this leading malaria vaccine candidate.

Materials and Methods Production of recombinant antigens Cloning, expression and purification of the PvAMA1 (Sal I strain) ectoplasmic region has been described elsewhere.45,46 The recombinant protein comprises the sequence between residue 43, the first residue after the predicted prosequence (numbering beginning with the first residue of the signal sequence), and residue 487, the last residue before the expected transmembrane region. Production of a synthetic gene for the PfAMA1 (FVO strain) ectoplasmic region, with codons optimised for expression in P. pastoris, has been reported.10,47 Coding sequences for residues 25–544 (prosequence and domains I, II and III), residues 303 to 544 (domains II and III), residues 303–442 (domain II) and residues 419–544 (domain III) were PCR-amplified and cloned into the pPICZαA vector in-frame with the N-terminal secretion sequence and the C-terminal myc/His6 markers. The KM71H strain (Muts phenotype) of P. pastoris was used for secreted methanol-induced expression. The recombinant proteins were purified by Ni-affinity and ion-exchange chromatography following published protocols.10,46 Production of polyclonal antisera, monoclonal antibodies and Fab F8.12.19 Polyclonal antisera were raised in rabbits against the purified PfAMA1 and PvAMA1 ectoplasmic regions as

1534 described.10,45 The mAb F8.12.19 was obtained by the hybriboma technique.48 BALB/c mice were immunised by administering 10 μg of the recombinant PvAMA1 ectoplasmic region in the presence of Freund's adjuvant, followed by three booster injections of the same dose at 15day intervals. A final injection of 10 μg of PvAMA1 without adjuvant was given three days before sacrifice, and the hybridomas thus obtained were hybridised and cloned by limiting dilution. mAb F8.12.19 (isotype IgG1,κ, as shown by reactivity with isotype-specific antibodies) was precipitated from ascites fluid with ammonium sulphate and purified by ion-exchange chromatography (DEAE-Sephacel). Fab fragments were produced by cleavage with papain (1:50 (w/w) protease to substrate) and purified on a DEAE-Sephacel column followed by passage over a MonoQ ion-exchange column using a NaCl gradient to separate isoforms. The major isoform thus obtained was used for all crystallisation trials. Western blotting Slot blots were prepared using an Immunetics Miniblotter 45 (Cambridge, MA,), fitted with a PVDF membrane, by incubating 110 μl of protein solution (0.5 mg/ml in PBS for PfAMA1 ectoplasmic region; 0.25 mg/ml for PvAMA1 ectoplasmic region and reduced/alkylated PfAMA1) per slot for 90 min at room temperature. The protein solutions were then aspirated, the PVDF membrane was washed briefly in PBS and blocked overnight at room temperature in 0.3% (w/v) BSA, 0.05% (v/v) Tween 20 in PBS. Serum incubations were performed in the Miniblotter perpendicular to the protein slots, with rabbit serum diluted 1:5000 (v/v) and secondary antibody (goat anti-rabbit, alkaline-phosphatase conjugated, Pierce) diluted 1:1000 (v/v). Colour was developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate. Nucleotide sequencing of mAb F8.12.19 A total mRNA fraction was purified from about 107 F8.12.19 hybridoma cells by guanidinium thiocyanate extraction and used as a substrate for cDNA synthesis. cDNA for VL was amplified and sequenced by PCR using primers MuIgκVL5′-G and MuIgκVL3′-1 (Ig primer sets, Novagen) and cDNA for VH was amplified using primers MuIgVH5′-F and MuIgVH3′-2 (Ig primer sets, Novagen). The nucleic acid sequence was determined by GENOME express (France) using the resulting PCR products. Sequences were analysed using the program IMGT/V-Quest.37,49

Cross-reactive Antibody to Apical Membrane Antigen 1

fade was added before examination using a fluorescence microscope. Kinetic studies of PvAMA1-F8.12.19 interactions by surface plasmon resonance Kinetic studies of PvAMA1 and PfAMA1 binding to mAb F8.12.19 were made by surface plasmon resonance measurements using the Biacore® 2000 system (Biacore AB, Uppsala, Sweden). mAb F8.12.19 (50 μg/ml in a 10 mM sodium acetate buffer, pH 4.5) was covalently coupled to a CM5 sensor chip via primary amine groups according to the manufacturer's description (Biacore®), giving a final signal of 9776 RU (corresponding to a protein surface concentration of ∼ 10000 pg/mm2). One independent flow-cell on the same sensor chip was used for blank control measurements. PvAMA1 and PfAMA1, each at four different concentrations in PDS and 0.005% P20 (pH 7.4), was injected into the sensor chip flow-cell at a rate of 20 μl/min. The flow-cell surfaces were regenerated after each run by passing through 5 μl of 20 mM HCl. The kinetic constants, kon and koff, were evaluated using the program BIA-EVALUATION (version 3.1, Biacore) by fitting to a simple two-component association/dissociation model of AMA1 binding to immobilised F8.12.19. The same flow-cell was used for all mAb-binding experiments and background subtractions were made from the signal measured when passing the same AMA1 solution through the control flow-cell. Parasite growth-inhibition assay (GIA) In vitro parasite growth inhibition assays were performed essentially as described for P. vivax,45 using P. cynomolgi schizont-infected red blood cells isolated from an infected rhesus monkey. Briefly, parasites were washed, white blood cells were removed by Plasmodipur filtration (Euro Diagnostica BV, Arnhem, The Netherlands) and parasites were cultured overnight (standard Plasmodium culture conditions) in 96-well plates in the presence of 1 mg/ml of mAb F8.12.19 (done in triplicate). After overnight culture, thin films were prepared from the wells and stained with Giemsa. Parasitemia, estimated by counting ring-stage parasites, was determined as a measure of invasion. Inhibition was calculated by comparing the ring-stage parasitemia to the level of parasitemia under the same conditions in the absence of mAb and in the presence of a negative control mAb.

Immunofluorescence assays

Crystallisation of Fab F8.12.19 complexes

Immunofluorescence assay slides were prepared by making thin films with parasite material (mature blood stages) from infected primates (with parasites P. vivax strain ONG, P. cynomolgi M strain), infected mice (P. berghei ANKA strain) or in vitro culture (P. falciparum NF54 strain, P. knowlesi H strain). Slides were fixed for 1 min with methanol (−20 °C) and air-dried. Monoclonal antibodies were diluted to 1 μg/ml in 1% (v/v) FCS/PBS and the thin films were incubated with 100 μl antibody for 1 h at room temperature in a moist environment. Slides were washed four times with PBS and then incubated with anti-mouse IgG-FITC, 1:100 dilution in 1% FCS/PBS for 1 h at room temperature in a moist environment. Slides were washed as above, dried gently with tissue and one drop of anti-

Crystallisation trials were made by the vapourdiffusion, hanging drop technique under the following conditions. Complex with PvAMA1 PvAMA1 and Fab-F8.12.19 were mixed in a 1:1 stoichiometric ratio and left to incubate for 4 h at room temperature to form the antibody–antigen complex before mixing with crystallisation screening buffers. Crystals used for diffraction measurements were obtained as follows. The crystallisation buffer in the reservoir comprised 10% (w/v) PEG 6000 and 0.1 M sodium acetate buffered to pH 4.4. The crystallisation drop was prepared

1535

Cross-reactive Antibody to Apical Membrane Antigen 1 by adding 0.8 μl of the crystallisation buffer to 0.8 μl of the PvAMA1-Fab complex, giving a final protein concentration of 3.9 mg/ml. The crystallisation boxes were left at 17 °C and crystals of size suitable for diffraction appeared after about 2 months. Complex with PfAMA1 Crystallisation trials were carried out with the domains II–III and domains I–II–III PfAMA1 constructions but only that corresponding to domains II–III gave crystals. The Fab fragment was incubated in small stoichiometric excess with the recombinant protein (1.2:1) before adding crystallisation buffers. Crystallisation drops were prepared by mixing 0.8 μl of protein with 0.8 μl of reservoir buffer comprising 12% PEG 6000 and 0.1 M sodium acetate (pH 4.6). The final protein concentration was 3.2 mg/ml. Crystals appeared after five days at 17 °C. Crystal diffraction measurements Crystals were conserved in liquid nitrogen after very briefly transferring to a cryo-protecting buffer consisting of the crystallisation buffer to which a volume of glycerol had been added to bring the latter to a final concentration of 20% (v/v). Diffraction measurements were made at the E.S.R.F synchrotron, Grenoble, on the beam-lines ID14-2 and ID14-4. Data were measured at cryogenic temperatures (100 K) using an ADSC Q4 CCD detector and intensities were integrated using the program XDS.50 Structure determination and refinement Crystallographic calculations were made with the CCP4 program suite.51 The three-dimensional crystal structure of the PvAMA1-FabF8.12.19 complex was solved by molecular replacement using the coordinates of the following known structures as search models: PvAMA1 ectoplasmic region (Protein Data Bank (PDB) entry 1W8K), the variable-domain dimer (VH+ VL) from PDB entry 2IGF, and the constant-domain dimer (CH+ CL) from PDB entry 2IGF. Rotation and translation function calculations, using AMoRe (CCP4 version)51 gave clear solutions for both the variable- and constant-domain models of the Fab when used separately as search models. These results were confirmed by a two-body search with the variable and constant-domain dimer solutions, which placed the two components on a common crystallographic origin and gave the correct pairing of the light and heavy polypeptide chains. No solution could be found for PvAMA1 by molecular replacement calculations. Electron density maps phased by the initial Fab model showed the presence of significant difference density at the antigen-binding site of F8.12.19 that did not extend significantly beyond this region to account for the complete recombinant antigen. Refinement began by building in the sequence of F8.12.19 and a polypeptide chain into the residual electron density at the antigenbinding site. The programs REFMAC552 and ARP/ WARP53 were used to refine the atomic parameters; the model was inspected and adjusted manually between each refinement round using the program O.54 A total of 34 contiguous amino acid residues (421–454) could be built into antigen density. No other region of PvAMA1 could be identified in the electron density maps.

The crystal structure of the PfAMA1 complex, which is closely isomorphous with that of the PvAMA1 complex, was solved by molecular replacement using the refined Fab F8.12.19 coordinates. This returned a clear solution in the rotation and translation functions, and the electron density maps obtained after an initial round of refinement showed additional density in the antigen-binding site. This density closely resembled the antigen structure in the PvAMA1 complex and, as expected from the isomorphism between the two crystal structures, included only part of domain III of PfAMA1. The final model includes residues 479–512 from domain III of PfAMA1. Figures of molecular structures were made with MacPyMol‡. Data bank accession codes The VH and VL nucleotide sequences have been deposited in GenBank with accession codes DQ890518 and DQ890518, respectively. Coordinates of the F8.12.19PvAMA1 and F8.12.19-PfAMA1 complexes have been deposited in the Protein Data Bank with accession codes 2j4w and 2j5l, respectively.

Acknowledgements This work was funded by the European Commission (contracts QLK2-CT-1999-01293 and QLK2-CT2002-01197), the European Malaria Vaccine Initiative, the Pasteur Institute, the Centre National de la Recherche Scientifique and the Biomedical Primate Research Centre. S.I. received bursaries from the Ministère de l'Education Nationale, de la Recherche et de la Technologie and from the Fondation pour la Recherche Médicale. We thank Martin A. Dubbeld for excellent technical assistance and Juan Carlos Pizarro for aid in antibody sequencing.

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Edited by R. Huber (Received 6 October 2006; received in revised form 11 December 2006; accepted 13 December 2006) Available online 16 December 2006