Editor's evaluation: Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats (2021)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines. Editor's evaluation This paper combines simulations and experimental biophysical approaches to probe the molecular basis of antibody recognition of key repeat peptides found on the surface of Plasmodium vivax, a parasite that causes malaria. Currently, we know surprisingly little about the mechanism by which antibodies recognize these peptides. Thus, this work provides crucial molecular insights into a recognition process of considerable biomedical significance, which might ultimately inform rational design of novel and more effective antimalarial vaccines. https://doi.org/10.7554/eLife.72908.sa0 Decision letter eLife's review process Introduction Malaria is a major public health concern, with an estimated 409,000 deaths in 2019 (World Health Organization, 2020). Human malaria is caused by Plasmodium parasites, with the majority of cases attributed to Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) (Lover et al., 2018). Pv is the predominant Plasmodium spp. in circulation for a majority of countries outside of Africa (~75% of cases in South and North America, ~50% of cases in the Southeast Asia region, and ~30% in the Eastern Mediterranean region; World Health Organization, 2020). Despite overall lower mortality compared to Pf malaria, Pv infection can cause debilitating disease, including fever, myalgia, chronic anemia, reduced birthweight, and increased risk of neonatal death (Alexandre et al., 2010; Bardají et al., 2017; Genton et al., 2008). Circumsporozoite protein (CSP) is the most abundant protein on the surface of all Plasmodium sporozoites and is necessary for parasite development and infection (Cerami et al., 1992; Ménard et al., 1997; Nguitragool et al., 2017). CSP contains an unusual central region consisting of multiple, short amino acid repeats whose sequence depends on the Plasmodium species (Chenet et al., 2012; Rich et al., 2000; Tahar et al., 1998). The PvCSP central region is composed of nonapeptides GDRA(A/D)GQPA and ANGAGNQPG characteristic of strains VK210 and VK247, respectively (Arnot et al., 1985; Rosenberg et al., 1989; Figure 1A). Unlike the 4-amino acid (aa)-long motifs of Pf and Plasmodium berghei (Pb) CSP, which are rich in asparagine and proline residues, PvCSP repeats are longer and consist primarily of glycine and alanine residues (~50% of all residues). Moreover, ~26% of the PvCSPvk210 central repeat region consists of charged residues, including arginine and aspartic acid residues. Both VK210 and VK247 strains have worldwide distribution (Cheng et al., 2013; Kain et al., 1992; Soares et al., 2020), and VK210 appears to be a major target of the humoral immune response in studied populations (González et al., 2001; Kim et al., 2010; Soares et al., 2020). Figure 1 Download asset Open asset Comparison of PvCSPvk210 and PvCSPvk247 repeat sequences. (A) Schematic representations of PvCSPvk210 and PvCSPvk247 sequences, each including an N-terminal domain, central repeat region, and C-terminal domain. Colored blocks represent repeat motifs. The sequence of peptides used in circular dichroism (CD) spectroscopy studies is shown below. (B) Superimposition of the CD spectra obtained for PvCSPvk210 (top panel) and PvCSP247 peptides (bottom panel). PvCSPvk210 peptides 210-1, 210-2, 210-3, 210-4, and 210-5 are colored from navy to light blue; PvCSPvk247 peptides are depicted in green (247-1), yellow (247-2), and orange (247-3), respectively. The central region of CSP is immunodominant, and antibodies targeting the repeats can inhibit sporozoite infection (Imkeller et al., 2018; Kisalu et al., 2018; Kisalu et al., 2018; Mishra et al., 2012; Murugan et al., 2020; Oyen et al., 2017; Oyen et al., 2020; Pholcharee et al., 2020; Pholcharee et al., 2021; Potocnjak et al., 1980; Tan et al., 2018; Triller et al., 2017; Wang et al., 2020). The PfCSP central repeat is a major component of the most advanced malaria vaccine to date, RTS,S/AS01 (Adepoju, 2019; Draper et al., 2018), and PvCSP or PvCSP-derived peptides are present in various clinical vaccine candidates against Pv (Mueller et al., 2009; Mueller et al., 2015). Effective pre-erythrocytic stage vaccines are highly desirable to reduce the number of primary infections, and in the case of Pv, to consequently prevent the establishment of hypnozoites that can lead to multiple relapses (Krotoski et al., 1982). Although not much is known about the molecular basis of PvCSP recognition by inhibitory antibodies, a positive statistical association between the level of antibodies against the repeat region of PvCSP and protection has previously been established (Yadava et al., 2014). Anti-PvCSP species-specific monoclonal antibodies (mAbs) 2F2 and 2E10.E9 were generated in mice after immunization with radiation-attenuated Pv sporozoites of strains VK210 and VK247, respectively (Nardin et al., 1982). Incubation of Pv sporozoites with mAbs 2F2 and 2E10.E9 results in significantly reduced sporozoite infectivity, and thus despite a limited molecular understanding of their recognition, both antibodies have been valuable research tools in studies of Pv sporozoites (Cabrera-Mora et al., 2015; Gimenez et al., 2017; Miyazaki et al., 2020; Roth et al., 2018; Teixeira et al., 2014). Interestingly, a recent study reported that sporozoites attenuated with low concentrations of mAb 2F2 were significantly reduced in size and had lower DNA content, indicating post-hepatocyte-invasion antibody inhibition of liver stage development (Roth et al., 2018). Here, we present a detailed molecular analysis of the PvCSP repeat region and its recognition by inhibitory mAbs 2F2 and 2E10.E9. Molecular dynamics (MD) simulations on PvCSP-derived repeat peptides indicate that in the absence of interacting mAbs, the PvCSP repeat is largely disordered. Our structural studies reveal how mAbs 2F2 and 2E10.E9 lock PvCSP repeat peptides in a predominant coiled conformation, with antibody germline-encoded aromatic residues contributing significantly to the antigen contacts. Moreover, we describe how mAb 2E10.E9 engages in head-to-head homotypic interactions when targeting PvCSP in a similar manner as previously described human mAbs against PfCSP (Imkeller et al., 2018; Oyen et al., 2018; Pholcharee et al., 2021) and a murine mAb against PbCSP (Kucharska et al., 2020). Results PvCSP repeat peptides are structurally disordered and behave like harmonic springs Due to the variety of PvCSP repeat sequence motifs, we created an extensive list of peptides for circular dichroism (CD) spectroscopy and MD simulations studies (Supplementary file 1, Figure 1A) to examine the structural propensities of 18- and 27-aa-long motifs from the PvCSPvk210 and PvCSPvk247 repeats. CD spectra of all analyzed peptides were indicative of a lack of secondary structure, with minima at ~200 nm (Figure 1B). For a finer dissection of potential minor secondary structure propensities, we performed all-atom MD simulations on 27-aa peptides derived from PvCSPvk210 and PvCSPvk247 (Supplementary file 1, Figure 2, Figure 2—figure supplement 1, Figure 2—figure supplement 2). All analyzed peptides were highly disordered in solution and adopted a large ensemble of conformations (Figure 2A), similar to peptides derived from PfCSP and PbCSP (Kucharska et al., 2020). Secondary structure was present mainly in the form of transient hydrogen-bonded β-turns (Figure 2B). The propensity of individual residues to form turns varied from 0 to ~60%, with PvCSPvk210 peptides displaying slightly lower averaged turn propensity (~15–20%) than PvCSPvk247 peptides (~23–26%) (Figure 2C). PvCSPvk210 peptides containing (GDRAAGQPA)2 motifs (210-7 and -9), as well as the last repeat of the 247-3 peptide (GNGAGGQAA), were the only motifs with a significant propensity to form helices (>20%). Figure 2 with 2 supplements see all Download asset Open asset Conformational flexibility of PvCSPvk210 and PvCSPvk247 peptides. (A) Superposition of the conformations of peptides resulting from molecular dynamics (MD) simulations at every 2 ns and aligned to the conformational median structure. (B) Example snapshots of the highest (peptides 210-7, 210-8, 210-9, and 247-3) or the second highest (peptides 210-6, 247-1, 247-2) propensity β-turn for each peptide. Color coding of atoms: oxygen (red), nitrogen (blue), and hydrogen (white). For clarity, only the hydrogen atom involved in the H-bonded turn is shown. H-bond between C=O of residue i and N–H of residue i + 3 is shown as a gray line. (C) Secondary structure propensity at each residue, averaged over 20 replicas and computed using the Dictionary of Secondary Structure for Proteins (DSSP) criteria (Nagy and Oostenbrink, 2014). (D) Elastic modulus of peptides computed from MD simulations. The reversible work or free energy (ΔG) for extension and compression of the peptide is plotted as a function of equilibrium end-to-end distances (dEE) (solid line). The data is fitted to the quadratic function of elastic potential energy (black dashed line). For each peptide, the estimated values of k (elastic modulus), d0 (equilibrium dEE), and R2 (regression coefficient to indicate quality of fit) are shown. Shading represents standard error of mean. In order to estimate the elastic modulus of these peptides with intrinsically low secondary structure propensities, peptides were modeled as Hookean springs (Figure 2D), in which the force needed to extend or compress a spring by some distance is proportional to that distance, and the underlying energy function is quadratic (Equation 1). The PMF or free energy governing changes in the peptide’s end-to-end distance was computed and fit to a quadratic function. The excellent data fits (R2 = 0.91–0.97) suggest that in aqueous solution, peptides derived from the PvCSP repeats behave like harmonic springs. On average, the elastic modulus was ~3–5 cal/(mol Å2), with the highest values (stiffest peptide) observed for peptide 247-2 (4.9 ± 0.2 cal/(mol Å2)) and lowest (most flexible peptide) observed for peptide 247-1 (3.2 ± 0.2 cal/(mol Å2)). Molecular basis of 2F2 recognition of the PvCSPvk210 central repeat To gain insights into how inhibitory mAb 2F2 binds to a largely disordered, elastic repeating PvCSPvk210 epitope, we performed isothermal titration calorimetry (ITC) with PvCSPvk210 repeat peptides representative of the different sequence motifs contained within the central repeat region (Supplementary file 1, Figure 3A, Figure 3—figure supplement 1). 2F2 Fab binds to all tested peptides containing two repeats with similar affinity, with a slight preference for peptide 210-3 (GDRADGQPAGDRAAGQPA; 13.8 nM) over 210-2 (GDRAAGQPAGDRAAGQPA; 37.5 nM) (Figure 3A), with other peptides displaying intermediate affinities (17.1–19.1 nM). Peptides 210-3 and 210-2 differ in the fifth residue of the first repeat, which is an aspartic acid for peptide 210-3 and an alanine for peptide 210-2 (GDRA(D/A)GQPA). Figure 3 with 4 supplements see all Download asset Open asset 2F2 Fab binding to PvCSPvk210 repeat peptides. (A) Affinities of 2F2 Fab for peptides 210-1, 210-2, 210-3, 210-4, and 210-5 as measured by isothermal titration calorimetry (ITC). Open circles represent independent measurements. Mean binding constant (KD) and binding stoichiometry (N) values are shown above the corresponding bar. Error bars represent SEM. (B) Upper panel: sequences of peptides used in ITC experiments with variable residues underlined. Dark gray denotes the core epitope of the peptide resolved in all the X-ray crystal structures, and light gray shading indicates residues resolved in the corresponding X-ray crystal structures. Bottom panel: comparison of the conformations of PvCSP210 peptides in X-ray crystal structures. PvCSPvk210 peptides are colored from navy to light blue, with the residues adopting one turn of a 310-helix depicted in pink. (C) Top and side views of the 210-4 peptide (light blue) in the binding groove of the 2F2 Fab shown as surface representation (heavy chain [HC] shown in green and kappa chain [KC] shown in white). (D) Comparison of the conformations adopted by the core epitope of peptides 210-1, 210-2, 210-3, 210-4, and 210-5 when bound to 2F2. (E) Detailed interactions between Fab 2F2 and peptide 210-4. H-bonds and salt bridges are shown as black dashes, peptide 210-4 is shown in light blue, HC is shown in green, and KC is shown in gray. Fab residues are annotated with H or K letters to indicate heavy and kappa light chain, respectively. Next, we co-crystalized the 2F2 Fab with five different peptides derived from PvCSPvk210 (210-1, 210-2, 210-3, 210-4, and 210-5, Supplementary file 1, Figure 3, Figure 3—figure supplement 2) to gain molecular insights into the binding mode and cross-reactivity of mAb 2F2 binding to PvCSPvk210. The crystal structures were solved to resolutions ranging from 1.97 Å to 2.67 Å (Table 1). 2F2 recognizes the core epitope (2DRA(D/A)GQPAGD11) of all PvCSPvk210 peptides in an almost identical coil conformation peptide backbone root-mean-square deviation (RMSD 0.10–0.28 Å) with two consecutive β-turns observed for residues 7QPAGD11 (Supplementary file 2) forming one turn of a 310-helix, thus consistent with the moderate secondary structure propensities observed for the unliganded PvCSPvk210 repeats (Figure 3B). The co-crystal structures also provide molecular insights into the cross-reactivity of 2F2 to different types of PvCSPvk210 repeat motifs (1GDRA(D/A)GQPA9). The sidechains of D/A5 point up and away from the 2F2 paratope and do not significantly contribute to the 2F2 Fab-peptide interaction, helping to explain the similar binding affinities to the different peptides containing this variation (Figure 3E). 2F2 also binds the 210-5 peptide containing a unique repeat C-terminal of the central region (1GDRAAGQPAGNGAGGQAA18); however, the electron density of residues 12–18 C-terminal of the bound core peptide motif is weak in the co-crystal structure, thus providing limited structural insight into this peptide region and suggesting it does not make strong interactions with 2F2 (Figure 3—figure supplement 2). Table 1 X-ray crystallography data collection and refinement statistics. 2F2-210-12F2-210-22F2-210-32F2-210-42F2-210-52E10-247-22E10-247-32E10-247-4BeamlineAPS 23-ID-BAPS 23-ID-DAPS 23-ID-BAPS 23-ID-BAPS 23-ID-DAPS 23-ID-BAPS 23-ID-BAPS 23-ID-DWavelength (Å)1.0331671.0331671.0331671.0331671.0331671.0331671.0331671.033200Space groupP 1C 2C 2P 1C 2P 31P 21P 1Cell dimensions71.5, 81.4, 82.392.9, 60.4, 158.392.6, 60.8, 81.471.7, 82.3, 82.893.4, 60.5, 159.1142.4, 142.4, 91.356.4, 144.4, 60.554.5, 66.3, 142.3α, β, γ (o)94.6, 114.1, 111.690, 101.5, 9090, 101.6, 9095.3, 113.8, 111.590, 101.2, 9090, 90, 12090, 102.8, 90100.4, 92.3, 91.7Resolution (Å)*29.48–2.20 (2.25–2.20)29.21–2.54 (2.65–2.54)29.71–1.97(2.02–1.97)29.61–2.67 (2.77–2.67)29.69–2.27 (2.34–2.27)29.55–3.19 (3.30–3.19)29.48–2.68 (2.78–2.68)29.34–2.71 (2.81–2.71)No. molecules in the asymmetric unit (ASU)32132224No. observations264,344 (16,542)181,285 (21,112)101,891 (4384)155,413 (16,123)471,275 (38,772)693,999 (70,530)184,740 (18,748)552,431 (55,667)No. unique observations75,088 (4440)28,642 (3484)31,038 (1962)43,179 (4542)40,512 (3722)34,411 (3469)26,439 (2627)53,126 (5316)Multiplicity3.5 (3.7)6.3 (6.1)3.3 (2.2)4.7 (1.5)11.6 (10.3)20.1 (20.3)7.0 (7.1)10.4 (10.5)Rmerge (%)†15.5 (95.2)16.4 (144.5)6.5 (55.7)13.9 (62.8)34.2 (175.0)36.1 (370.6)21.6 (158.6)22.4 (123.5)Rpim (%) ‡7.5 (32.4)10.6 (97.6)6.1 (46.9)7.8 (30.8)15.3 (83.0)8.2 (84.0)8.8 (63.7)7.3 (39.8)< I/σ I>5.2 (1.5)6.7 (1.5)8.3 (1.5)4.7 (1.5)9.4 (1.5)13.2 (1.7)9.2 (1.6)7.4 (1.6)CC1/20.965 (0.527)0.994 (0.529)0.996 (0.603)0.987 (0.669)0.927 (0.435)0.998 (0.756)0.993 (0.578)0.995 (0.729)Completeness (%)97.5 (97.1)99.8 (100.0)98.7 (90.5)98.3 (97.8)99.9 (100.0)99.8 (99.6)99.9 (100.0)100.0 (100.0)Refinement statisticsReflections used in refinement74,67228,60431,03043,16040,50134,41126,43953,126Reflections used for R-free37311432155221622026173213142067Non-hydrogen atoms10,5806872359510,50469416818688513,707Macromolecule10,1236864341810,34468416818684513,620Water4338177160100-3487Heteroatom------6-R §work/R¶free17.9/22.020.2/24.918.6/23.518.6/22.819.2/23.818.0/21.020.7/23.920.9/24.6Rms deviations from idealityBond lengths (Å)0.0070.0020.0150.0020.0030.0100.0100.010Bond angle (°)0.870.511.330.570.711.391.251.22Ramachandran plotFavored regions (%)97.597.997.397.998.694.697.197.5Allowed regions (%)2.32.12.71.91.45.42.92.5B-factors (A2)Wilson B-value39.164.735.547.050.091.042.154.7Average B-factors45.775.045.751.053.7111.071.077.0Average macromolecule45.775.945.951.153.7111.071.477.2Average heteroatom------69.8-Average water molecule44.061.441.141.954.3-37.843.3 * Values in parentheses refer to the highest resolution bin. † Rmerge = ∑hkl ∑i | Ihkl, i -< Ihkl > | / ∑hkl< Ihkl > . ‡ Rpim = ∑hkl [1/(N ∑i | Ihkl, i -< Ihkl > | / ∑hkl< Ihkl > . = | | | / | of data were used for the The recognition of PvCSPvk210 peptides by 2F2 is by residues in heavy chain regions 1, 2, and 3, and kappa chain regions 1 and The PvCSPvk210 one turn of a 310-helix is in the by residues of the antibody (Figure and Figure 3—figure supplement and and is by H-bonds between and the backbone of and and and the of (Figure 3E). The on the Fab on HC and on and on the 210-4 peptide. residues are between and 3 and do not with the light chain (Figure and 2F2 binds to PvCSPvk210 peptides using both and residues. germline-encoded aromatic residues form significant interactions with the contributing a of of surface Figure Figure 3—figure supplement and To accommodate arginine residues present in the sequence of the PvCSPvk210 the 2F2 paratope has an overall potential (Figure 3—figure supplement and a central in the Fab-peptide forming H-bonds with both heavy and light chain residues of the antibody (Figure and contributing and of respectively. Molecular basis of mAb 2E10.E9 recognition of the PvCSPvk247 central repeat Unlike PvCSPvk247 contains one predominant repeat with the of the first and the last repeat in the central region (Figure 1A). 2E10.E9 Fab binds peptides derived from PvCSPvk247 with ranging from to as measured by on the binding stoichiometry derived from the peptide binding of one 27-aa peptides 247-1, and 247-3 are to two to (Figure Figure supplement 1). Figure 4 with 2 supplements see all Download asset Open asset 2E10.E9 Fab binding to PvCSPvk247 repeat peptides. (A) Affinities of 2E10.E9 Fab for peptides 247-1, and as measured by isothermal titration calorimetry (ITC). Open circles represent independent measurements. Mean binding constant (KD) and binding stoichiometry (N) values are shown above the corresponding bar. Error bars represent SEM. (B) Upper panel: sequences of peptides used in ITC with variable residues underlined. Dark gray denotes the core epitope of the peptide resolved in all X-ray crystal structures, and light gray shading indicates residues resolved in the corresponding X-ray crystal structures. Bottom panel: comparison of the conformations of PvCSP247 peptides in X-ray crystal structures, with peptides and depicted in and respectively. (C) Top and side views of the 247-3 peptide in the binding groove of the 2E10.E9 Fab shown as surface representation (heavy chain [HC] shown in and kappa chain [KC] shown in white). (D) Comparison of the conformations adopted by the core epitope of peptides and when bound to 2E10.E9. (E) Detailed interactions between Fab 2E10.E9 and peptide H-bonds are shown as black dashes, peptide 247-3 is shown in HC is shown in green, and KC is shown in gray. The Fab residues are annotated with H or K letters to indicate heavy and kappa light chain, respectively. obtained co-crystal structures of the 2E10.E9 Fab in with different 247-3 and at Å and 247-2 at Å resolution (Table 1, Figure Figure 3—figure supplement and Figure supplement 2). In the 2E10.E9 co-crystal structure, two to one peptide, which is in with the stoichiometry established by ITC (Figure The core epitope of PvCSPvk247 peptides contains eight residues and a similar coil conformation in all analyzed peptides when bound to 2E10.E9 backbone with only isolated turns (Figure Supplementary file 2). Although 2E10.E9 Fab binds peptides 247-2 and 247-3 containing the first and the last repeat motifs, the electron density of residues unique to these repeats was in the co-crystal structures This that the antibody does not with these variable residues that are outside the core (Figure Figure supplement 2). 2E10.E9 with the PvCSPvk247 peptides using 1, 2, and 3, and and 3, with the N-terminal of the peptide between and germline-encoded aromatic residues, including and a central in peptide recognition, forming H-bonds with and and contributing to the paratope of this Fab on the 2E10.E9 on peptide 247-3) (Figure 3—figure supplement and Interestingly, and make a that in an to form an H-bond with residue of the peptide and the by aromatic sidechains (Figure of the PvCSPvk247 peptides is central to the interaction, forming H-bonds with residues and (Figure 2E10.E9 Fab-Fab interactions upon PvCSPvk247 repeat binding In the 2E10.E9 peptide co-crystal structure two to one peptide, we observed multiple between the two 2E10.E9 (Figure the two 2E10.E9 in a head-to-head binding mode at an angle (Figure between the two are and mainly of both Fab and as well as the of Fab (Figure and Fab-Fab interactions contribute of on Fab on Fab The between 2E10.E9 is by five between residues of both Fab and and two H-bonds between of Fab and and of Fab (Figure Comparison of the 2E10.E9 variable sequences to the and that only one of the residues involved in Fab-Fab has been this homotypic Fab-Fab primarily germline-encoded in the of binding its repeating epitope (Figure Figure Download asset Open asset Fab-Fab interactions in Fab peptide 2E10.E9 that recognize the 247-2 peptide each other an consisting of mainly of heavy chain region of both Fab and as well as kappa chain region of Fab The heavy chain of Fab and is colored light and blue, respectively. The kappa chain of Fab and is colored light and respectively. 2, and 3 are colored light and respectively. 2, and 3 are shown in light and respectively. The 247-2 peptide is depicted in dashed forming Fab-Fab are with the of the Fab or in (C) of monoclonal antibody 2E10.E9 with its residues involved in homotypic green residues involved in homotypic interactions that form repeat flexibility upon binding of inhibitory antibodies to PvCSP Next, we the binding of mAbs 2F2 and 2E10.E9 to PvCSPvk210 and Both mAbs to their PvCSP sequences, as in experiments (Figure and mAb 2E10.E9 a compared to mAb which to the lower overall binding of this mAb to PvCSPvk247 compared to a binding for the mAb Next, ITC as both 2F2 and 2E10.E9 PvCSP with stoichiometry indicative of multiple Fab interacting with a PvCSP 2F2 Fab recognizes PvCSPvk210 with compared to the 2E10.E9 the binding data (Figure and with light of the binding stoichiometry with a molecular of and for the 2F2 and 2E10.E9 respectively (Figure These to 2F2 bound to one of PvCSPvk210 and to 2E10.E9 bound to one of Although both ITC and the of large by multiple binding to one PvCSP the stoichiometry that from these independent is slightly different between the two which we to the in for PvCSP and to the two experiments performed at Figure with 1 supplement see all Download asset Open asset of 2F2 and 2E10.E9 to PvCSPvk210 and of of 2F2 and Fab and lower to and 2E10.E9 and Fab and lower to as measured by are shown in black and fits in Data shown are representative of independent measurements. titration calorimetry (ITC) analysis of 2F2 Fab binding to PvCSPvk210 (C) and 2E10.E9 Fab binding to PvCSPvk247 (D) at data of PvCSPvk210 in the with 2F2 Fab in the data of PvCSPvk247 in the with 2E10.E9 Fab in the and of of corresponding to the Results from with light for the Fab panel) and 2E10.E9 panel) the are in of the of the i