Characterization of the thermodynamics of 2A10-antigen binding

We began our analysis by performing isothermal titration calorimetry (ITC) to understand the interaction between 2A10 and CSP. For ease of expression we used a recombinant CSP (rCSP) construct described previously which was slightly truncated with 27 repeats compared to 38 in the 3D7 reference strain [12,24]. ITC experiments were run on the purified 2A10 antibody and the purified 2A10 antigen-binding fragment (F_AB) fragment to test the thermodynamic basis of the affinity of 2A10 F_AB towards CSP. Experiments were also performed on the 2A10 F_AB fragment with the synthetic peptide antigen (NANP)₆, which is a short segment of the antigenic NANP-repeat region of CSP (Table 1; Fig 1). The binding free energies (ΔG) and dissociation constants (K_D) were found to be -49.0 kJ/mol and 2.7 nM for the full 2A10 antibody with CSP, -40 kJ/mol and 94 nM for the 2A10 F_AB with CSP, and -36.4 kJ/mol and 420 nM for the 2A10 F_AB with the (NANP)₆ peptide.

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Table 1. Thermodynamic parameters for interactions between 2A10 F_AB, 2A10 and antigens.

https://doi.org/10.1371/journal.ppat.1006469.t001

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Fig 1. ITC data for interactions between 2A10 F_AB and antigens.

(A) Titration of 2A10 F_AB with (NANP)₆. (B) Titration of 2A10 F_AB with rCSP. (C) Titration of 2A10 (complete antibody) with rCSP. The upper panels represent baseline-corrected power traces. By convention, negative power corresponds to exothermic binding. The lower panels represent the integrated heat data fitted to the independent binding sites model.

https://doi.org/10.1371/journal.ppat.1006469.g001

Surprisingly, we did not observe a typical 1:1 antibody/F_AB domain:antigen binding stoichiometry (Table 1). We found that each (NANP)₆ peptide was bound to by ~2 F_AB fragments (2.8 repeats per F_AB domain). With the rCSP protein we observed that ~11 F_AB fragments could bind to each rCSP molecule, (2.5 repeats per F_AB domain. Finally, when the single-domain F_AB fragment is replaced by the full 2A10 antibody (which has two F_AB domains), we observe binding of 5.8 antibodies per rCSP molecule (4.7 repeats per antibody). Therefore all complexes exhibit approximately the same binding stoichiometry of two F_AB fragments/domains per ~5 repeat units. These results suggest that the antigenic region of CSP constitutes a multivalent antigen and that repeating, essentially identical, epitopes must be available for the binding of multiple F_AB domains.

It is not possible to separate affinity from avidity in this system, although it is apparent that there is a substantial benefit to the overall strength of binding between the antibody and antigen through the binding of multiple F_AB domains. The F_AB:rCSP complex and the 2A10:rCSP complex had similar enthalpy and entropy of binding (Table 1), but the 2A10:rCSP complex had a lower overall ΔG binding, corresponding to a lower dissociation constant (2.7 nM vs. 94 nM for F_AB:rCSP). The observation that this antibody-antigen (Ab-Ag) interaction is primarily enthalpically driven is consistent with the general mechanism of Ab-Ag interactions [25]. It is clear that the dissociation constant (K_d) of a single F_AB domain to the (NANP)₆ peptide is substantially higher (420 nM), and that the avidity, the accumulated strength of the multiple binding events between the F_AB domains of the antibody and the CSP repeat, is the basis for the lower K_d value observed in the 2A10:rCSP complex. Thus, the characteristic repeating pattern of the epitope on the CSP antigen allows multiple weak interactions with 2A10 F_AB domains to accumulate, which yields a complex with a high avidity dissociation constant in the low nM range.

Structural analysis of the (NANP)-repeat region and the 2A10 F_AB

To better understand the molecular basis of the multivalent interaction between 2A10 and rCSP, we performed structural analysis of the components. Previous work indicated that the NANP-repeat region of CSP adopts a flexible rod-like structure with a regular repeating helical motif that provides significant separation between the N-terminal and the C-terminal domains [26]. Here, we performed far-UV circular dichroism (CD) spectroscopy to investigate the structure of the (NANP)₆ peptide. These results were inconsistent with a disordered random coil structure (S1 Fig). Rather, the absorption maximum around 185 nm, minimum around 202 nm and shoulder between 215 and 240 nm, is characteristic of intrinsically disordered proteins that can adopt a spectrum of states [27].

The lowest energy structures of the (NANP)₆ repeat were predicted using the PEP-FOLD de novo peptide structure prediction algorithm [28]. The only extended state among the lowest energy structures that was consistent with the reported spacing of the N-and C-terminal domains of CSP [26], and which presented multiple structurally similar epitopes was a linear, quasi-helical structure, which formed a regularly repeating arrangement of proline turns (Fig 2A). The theoretical CD spectrum of this conformation was calculated (S1 Fig), qualitatively matching the experimental spectra: the maximum was at 188 nm, the minimum at 203 nm and there was a broad shoulder between 215 and 240 nm. To investigate the stability of this conformation, we performed a molecular dynamics (MD) simulation on this peptide, which showed that this helical structure could unfold, and refold, on timescales of tens of nanoseconds, supporting the idea that it is a low-energy, frequently sampled, configuration in solution (S1 Movie, S2 Fig). We also observed the same characteristic hydrogen bonds between a carbonyl following the proline and the amide nitrogen of the alanine, and the carbonyl group of an asparagine and a backbone amide of asparagine three residues earlier, that are observed in the crystal structure of the NPNA fragment [29]. Thus, this configuration, which is consistent with previously published experimental data, is a regular, repeating, extended conformation that would allow binding of multiple F_AB domains to several structurally similar epitopes.

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Fig 2. Structures of the (NANP)₆ peptide (A), the 2A10 F_AB fragment (B) and the model of the F_AB fragment-(NANP)₆ complex (C).

(A) The calculated structure of the (NANP)₆ peptide is a helical structure containing the same hydrogen bonds between a carbonyl following the proline and the amide nitrogen of the alanine, and the carbonyl group of an asparagine and a backbone amide of asparagine 3 residues earlier (highlighted in red) that are observed in [29]. (B) Electron density (blue mesh; 2mF_o-dF_c at 1 σ) of the 2A10 F_AB fragment viewed from above the antigen-binding site. Light chain is shown as yellow sticks, heavy chain as cyan. (C) A calculated model of the (NANP)₆:2A10 F_AB fragment complex. The CDR2 regions of each chain are shown in red, the CDR3 regions of each chain are shown in blue.

https://doi.org/10.1371/journal.ppat.1006469.g002

To better understand the interaction between the 2A10 and the (NANP)-repeat region, we solved the crystal structure of the 2A10 F_AB fragment in two conditions (S1 Table), yielding structures that diffracted to 2.5 Å and 3.0 Å. All of the polypeptide chains were modeled in good quality electron density maps (Fig 2B), except for residues 134–137 of the light chain. This loop is located at the opposite end of the F_AB fragment to the variable region and not directly relevant to antigen binding. The 2.5 Å structure contained a single polypeptide in the asymmetric unit, whereas the 3.0 Å structure contained three essentially identical chains. Superposition of the four unique F_AB fragments from the two structures revealed that the variable antigen binding region is structurally homogeneous, suggesting that this region might be relatively pre-organized in the 2A10 F_AB. This is consistent with the observation that antibodies typically undergo relatively limited conformational change upon epitope binding [25]. Indeed, a recent survey of 49 Ab-Ag complexes revealed that within the binding site, the heavy chain Complementarity Determining Region (CDR)-3 was the only element that showed significant conformational change upon antigen binding and even this was only observed in one third of the antibodies [30].

Attempts to obtain a crystal structure of a complex between 2A10 F_AB and the (NANP)₆ peptide were unsuccessful; unlike binary Ab-Ag interactions, in which the Ab will bind to a single epitope on an antigen and produce a population of structurally homogeneous complexes that can be crystallized, in this interaction we are dealing with an intrinsically-disordered peptide, the presence of multiple binding sites on the peptide, and the possibility that more than one 2A10 F_AB domain can bind the peptide. Therefore it is difficult to obtain a homogeneous population of complexes, which is a prerequisite for crystallization. Attempts to soak the (NANP)₆ peptide into the high-solvent form of 2A10 F_AB, in which there were no crystal packing interactions with the binding-loops, caused the crystals to dissolve, again suggesting that the heterogeneity of the peptide and the presence of multiple epitopes produces disorder that is incompatible with crystal formation.

Modeling the interaction of the 2A10 F_AB with the NANP-repeat region and testing the model through site-directed mutagenesis

Although it was not possible to obtain a crystal structure of the 2A10-(NANP)₆ peptide complex, the accurate structures of the 2A10 F_AB fragment, the (NANP)₆ peptide, and the knowledge that antibodies seldom undergo significant conformational changes upon antigen binding [30], allowed us to model the interaction, which we tested using site directed mutagenesis. Computational modeling of Ab-Ag interactions has advanced considerably in recent years and several examples of complexes with close to atomic accuracy have been reported in the literature [31]. Using the SnugDock protein-protein docking algorithm [31], we obtained an initial model for binding of the peptide to the CDR region of the 2A10 F_AB fragment (Fig 2C). We then performed, in triplicate, three 50 ns MD simulations on this complex to investigate whether the interaction was stable over such a time period (S2 Movie, S3 Fig). These simulations confirmed that the binding mode that was modeled is stable, suggesting that it is a reasonable approximation of the interaction between these molecules. To experimentally verify whether our model of the 2A10 F_AB:(NANP)₆ peptide interaction was plausible, we performed site directed mutagenesis of residues predicted to be important for binding. Our model predicted that the interaction with (NANP)₆ would be mainly between CDR2 and CDR3 of the light chain and CDR2 and CDR3 of the heavy chain (Fig 2C).

In the light chain (Fig 3A and 3B), Y38 is predicted to be one of the most important residues in the interaction; it contributes to the formation of a hydrophobic pocket that buries a proline residue and is within hydrogen bonding distance, via its hydroxyl group, to a number of backbone and side-chain groups of the peptide. Loss of this side-chain abolished binding. Y56 also forms part of the same proline-binding pocket as Y38, and loss of this side-chain also resulted in an almost complete loss of binding. R109 forms a hydrogen bond to an asparagine residue on the side of the helix; mutation of this residue to alanine results in a partial loss of binding. Y116 is located at the center of the second proline-binding pocket; since loss of the entire side-chain through an alanine mutation would lead to general structural disruption of the F_AB fragment, we mutated this to a phenylalanine (removing the hydroxyl group), which led to a significant reduction in binding. Finally, S36A was selected as a control: the model indicated that it was outside the binding site, and the ELISA data indicated that had no effect on (NANP)_n binding.

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Fig 3. Detailed view of the (NANP)₆:2A10 F_AB interface and site directed mutagenesis.

(A) A model of the light chain:(NANP)₆ interface. (B) ELISA results showing the effect of mutating light chain interface residues; error bars are based on technical replicates from one of two independent experiments. (C) A model of the heavy chain:(NANP)₆ interface. (D) ELISA results showing the effect of mutating heavy chain interface residues; error bars are based on technical replicates from one of two independent experiments.

https://doi.org/10.1371/journal.ppat.1006469.g003

Within the heavy chain (Fig 3C and 3D), mutation of N57 to alanine led to complete loss of binding, which is consistent with it forming a hydrogen bond to a side-chain asparagine but also being part of a relatively well packed region of the binding site that is mostly buried upon binding. T66 is located on the edge of the binding site and appears to provide hydrophobic contacts through its methyl group with the methyl side-chain of an alanine of the peptide; mutation of this residue resulted in a partial loss of binding. Interestingly, mutation of E64, which is location in an appropriate position to form some hydrogen bonds to the peptide resulted in a slight increase in binding, although charged residues on the edge of protein:protein interfaces are known to contribute primarily to specificity rather than affinity [32]. Specifically, the cost of desolvating charged residues such as glutamate is not compensated for by the hydrogen bonds that may be formed with the binding partner. Y37 is located outside the direct binding site in the apo-crystal structure; the loss of affinity could arise from long-range effects, such as destabilization of the position of nearby loops. In general, the effects of the mutations are consistent with the model of the interaction.

The multivalency of the CSP repeat region

The binding mode of the F_AB fragment to the (NANP)₆ peptide is centered on two proline residues from two non-adjacent NANP-repeats (Fig 3A and 3C). These cyclic side-chains are hydrophobic in character and are buried deeply in the core of the F_AB antigen binding site, into hydrophobic pockets formed by Y38 and Y56 of the light chain and the interface between the two chains. In contrast, the polar asparagine residues on the sides of the helix are involved in hydrogen bonding interactions with a number of polar residues on the edge of the binding site, such as N57 of the heavy chain. Due to the twisting of the (NANP)₆ repeat, the binding epitope of the peptide is 2.5–3 alternate NANP repeats, with a symmetrical epitope available for binding on the opposite face (Fig 4A). Thus, this binding mode is consistent with the stoichiometry of the binding observed in the ITC measurements, where we observed a stoichiometry of two 2A10 F_AB fragments per (NANP)₆ peptide. To investigate whether this binding mode was also compatible with the indication from ITC that ~10.7 2A10 F_AB fragments, or six antibodies (containing 12 F_AB domains) could bind the CSP protein (Table 1), we extended the peptide to its full length. It is notable that the slight twist in the NANP helix results in the epitope being offset along the length of the repeat region, thereby allowing binding of ten 2A10 F_AB fragments (Fig 4B). Six 2A10 antibodies can bind if two antibodies interact by a single F_AB domain and the other four interact with both F_AB domains. The observation that the F_AB fragments bind sufficiently close to each other to form hydrogen bonds also explains the observation from the ITC that the complexes with rCSP, which allow adjacent F_AB fragment binding, have more favorable binding enthalpy, i.e. the additional bonds formed between adjacent F_AB fragments further stabilize the complex and lead to greater affinity (Table 1). Thus, the initially surprising stoichiometry that we observe through ITC appears to be quite feasible based on the structure of the NANP-repeat region of the rCSP protein and the nature of the rCSP-2A10 complex. It is also clear that the effect of antibody binding to this region would be to prevent the linker flexing between the N- and C-terminal domains and maintaining normal physiological function, explaining the neutralizing effect of the antibodies.

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Fig 4. The multivalency of the NANP repeat region of the CSP protein.

(A) An (NANP)₆ peptide results in the presentation of two symmetrical epitopes, formed by alternating repeats (cyan and magenta), allowing binding by two F_AB domains, in keeping with the stoichiometry observed by ITC. (B) The full 27-mer repeat region results in the presentation of at least 10 separate epitopes and the twist of the helix results in displacement along the length of the repeat region, which allows binding of up to 10 separate F_AB fragments, consistent with 4 antibodies bound by both F_AB domains, and two bound by a single F_AB domain.

https://doi.org/10.1371/journal.ppat.1006469.g004

Identification of endogenous (NANP)_n specific B cells to determine the BCR repertoire

We next set out to determine the implications of our structure for the B cell response to CSP. Because the CSP protein could conceivably cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T-independent. As a tool to test this hypothesis we used (NANP)_n-based tetramers to identify and phenotype antigen specific B cells in mice immunized with P. berghei sporozoites expressing the repeat region of the P. falciparum CSP (P. berghei CS^Pf) [15]. The tetramers are formed by the binding of 4 biotinylated (NANP)₉ repeats with streptavidin conjugated phycoerythrin (PE) or allophycocyanin (APC). To validate our tetramer approach, mice were immunized with either P. berghei CS^Pf or another line of P. berghei with a mutant CSP (P. berghei CS^5M) that contains the endogenous (P. berghei) repeat region, which has a distinct repeat sequence (PPPPNPND)_n. (NANP)_n-specific cells were identified with two tetramer probes bound to different conjugates to exclude B cells that are specific for the PE or APC components of the tetramers which are numerous in mice [33]. We found that mice immunized with P. berghei CS^Pf sporozoites developed large tetramer double positive populations, which had class switched (Fig 5A and 5B). In contrast, the number of tetramer double positive cells in mice receiving control parasites was the same as in unimmunized mice; moreover these cells were not class switched and appeared to be naïve precursors indicating that our tetramers are identifying bona-fide (NANP)_n-specific cells (Fig 5B and 5C). Further analysis of the different populations of B cells showed that most B cells present at this time-point were GL7⁺ CD38^- indicating that they are GC B cells in agreement with results from a recent publication [34] (Fig 5B and 5D). Given that T cells are required to sustain GC formation beyond ~3 days these data indicate that a T-dependent response can develop to CSP following sporozoite immunization [35].

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Fig 5. CSP-specific B cells enter the germinal center following sporozoite immunization.

BALB/C mice were immunized with either 5 x 10⁴ P. berghei CS^5M (expressing the endogenous P. berghei CSP repeat) or 5 x 10⁴ P. berghei CS^Pf (expressing the circumsporozoite protein from P. falciparum) live spoorzoites under CQ cover. 12 days later the B cell response was analyzed by flow cytometry and putative (NANP)_n-specific cells were identified using PE and APC conjugated tetramers. (A) Representative flow cytometry plots showing the identification of (NANP)_n-specific (Tetramer⁺) cells. (B) Representative flow cytometry plots showing the proportion of Tetramer⁺ cells that have class switched and entered a GC. (C) Quantification of the number of class switched Tetramer⁺ cells under different immunization conditions. (D) Quantification of the number of GC Tetramer⁺ cells under different immunization conditions. Data from a single representative experiment of 2 repeats, analyzed by one-way ANOVA with Tukey’s post test.

https://doi.org/10.1371/journal.ppat.1006469.g005

The B cell response to the (NANP)_n repeat has both T-independent and T-dependent components

Our previous data showing GC formation among (NANP)_n specific B cells was indicative of a T-dependent response. To determine whether there might also be a T-independent component to the B cell response we immunized CD28^-/- mice as well as C57BL/6 controls with P. berghei CS^Pf radiation attenuated sporozoites (RAS) and measured serum (NANP)_n specific antibody by ELISA and the B cell response using our Tetramers. CD28^-/- mice have CD4+ T cells but they are unable to provide help to B cell responses [36]. Interestingly 4 days post immunization there were comparable IgM and IgG anti-(NANP)_n responses in the CD28^-/- mice and control animals (Fig 6A), indicative of a T-independent component to immunity. However by day 27 post immunization there was no detectable IgM or IgG antibody specific for (NANP)_n in the CD28^-/- mice suggesting the T-independent response is short-lived. We further analyzed (NANP)_n specific B cell responses using our tetramers, in particular examining the number and phenotype (plasmablast vs GC B cell) of activated IgD^- Tetramer⁺ cells (Fig 6B). In agreement with our antibody data, similar numbers of antigen specific B cells were seen at 4 days post immunization in the CD28^-/- and control mice and most of these cells were plasmablasts (Fig 6C). However by 7 days post immunization the number of antigen specific cells declines in the CD28^-/- mice as the T dependent GC reaction begins to predominate. Thus CSP on the surface of sporozoites is able to induce short-lived T-independent B cell response, but subsequently T-dependent responses predominate.

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Fig 6. The B cell response to CSP has a T-independent component.

CD28^-/- and control C57BL/6 mice were immunized with P. berghei CS^Pf radiation attenuated sporozoites (RAS) or rCSP in alum. Sera were taken and the spleens analyzed for antigen specific B cells using tetramers 4, 7 and 27 days post-immunization. (A) IgM and IgG (NANP)_n ELISA responses following RAS immunization (B) Representative flow cytometry plots 7 days post RAS immunization showing the gating of different B cell populations among Tetramer⁺ cells. (C) Absolute numbers of (i) total Tetramer⁺ IgD^-ve (ii) Tetramer⁺ Plasmabalsts and (iii) Tetramer⁺ GC B cells post RAS immunization. (D) Antibody responses and (E) absolute numbers of Tetramer⁺ IgD^- B cells 4 days post immunization with rCSP. Log-transformed data pooled from 2 independent experiments for each immunization (>3 mice/group/timepoint) were analyzed using linear mixed models with day and genotype/immunization as experimental factors and the individual experiment as a random factor; only significant differences are shown.

https://doi.org/10.1371/journal.ppat.1006469.g006

We wanted to know if to induce a T-independent response it was necessary for CSP to be presented on the surface of the sporozoite or if free rCSP was sufficient. We found that indeed rCSP could induce a T-independent response as evidenced by similar IgM and IgG levels and IgD^-Tetramer⁺ responses 4 days post immunization in control and CD28^-/- mice (Fig 6D and 6E). Finally we were concerned that there may be some residual CD4⁺ T cell help in the CD28^-/- mice so we performed experiments in which we used the antibody GK1.5 to deplete CD4⁺ T cells [37]. In agreement with our previous data we found that sporozoites (live or RAS) and rCSP induced IgM responses in CD4 depleted mice, though we were unable to detect a significant IgG response (S4 Fig). We also detected primed antigen specific B cells in GK1.5 treated mice following RAS or rCSP immunized mice 4 days post-immunization, albeit at lower levels than in mice treated with isotype control antibodies (S4 Fig). Overall our data with GK1.5 depleted mice support our results in the CD28^-/- model.

A restricted repertoire of BCRs can bind to the (NANP)_n repeat

Our ability to identify and sort (NANP)_n specific B cells with our tetramers also allows us to examine the repertoire of antibodies that can bind the (NANP)_n by sequencing the BCRs of the identified cells. While the repeat structure of CSP has been hypothesized to induce a broad polyclonal response based on data that the CSP repeat can absorb most of the sporozoite binding activity of human sera from immune individuals [23,38], an alternative hypothesis is that the antigenically simple structure of the repeat epitope might only be recognized by a small number of naïve B cells. We therefore sorted (NANP)_n-specific cells 35 days post immunization of BALB/C mice with sporozoites. We performed this analysis in BALB/C mice as this is the background of mice from which the 2A10 antibody was derived. We then prepared cDNA from the cells and amplified the heavy and kappa chain sequences using degenerate primers as described previously [39,40]. Heavy and kappa chain libraries were prepared from 4 immunized mice as well as from 3 naïve mice from which we bulk sorted B cells as controls. We obtained usable sequences from 3 of the 4 mice for both the heavy chain and kappa chain. Analysis of the heavy chain revealed that in each mouse 3 or 4 V regions dominated the immune response (Fig 7A). The V regions identified (IGHV1-20; IGHV1-26; IGHV1-34 and IGHV5-9) were generally shared among the mice. As a formal measure of the diversity of our V region usage in the (NANP)_n specific cells and the bulk B cells from naïve mice we calculated the Shannon entropy for these populations. This analysis formally demonstrated that the diversity of the antigen specific B cells was significantly lower than the diversity of the repertoire in naïve mice (Fig 7B). We further found that each V region was typically associated with the same D and J sequences even in different mice. For example, IGHV1-20 was typically associated with J4, IGHV5-9 with J4 while in different mice IGHV1-34 was variously paired with J1 or J4 (Fig 7C). Similar results were obtained for the kappa chain with the response dominated by IGKV1-135; IGKV5-43/45; IGKV1-110; IGKV1-117 and IGKV14-111 (Fig 7D and 7E). The V regions were typically paired with the same J regions even in different mice (Fig 7F), for example IGKV5.43/45 was typically paired with IGKJ5 or IGKJ2 and IGKV1-110 was typically paired with IGKJ5, although IGKV1-135 was typically more promiscuous. One limitation of our high throughput sequencing approach is that the degenerate primers only amplified ~70% of the known IGHV and IGKV sequences in naïve mice, suggesting that we may not capture the full diversity of the response. However, comparison with the 5 published antibody sequences (S2 and S3 Tables) that include IGHV-1-20, IGKV5-45 and IGKV1-110 reveals that we are likely capturing the bulk of the antibody diversity. Together these data suggest that the number of B cell clones responding to CSP may be limited, potentially reducing the ability of the immune system to generate effective neutralizing antibodies.

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Fig 7. Limited diversity of (NANP)_n specific antibodies.

BCR sequences were amplified from Tetramer⁺ cells sorted from BALB/C mice 35 days after immunization with live P. berghei CS^Pf sporozoites under CQ cover as well as bulk (B220+) B cells from naïve BALB/C mice (A) IGHV gene usage from among B cells from a representative naïve mouse (grey bars) and Tetramer+ cells from immune mice (red, blue and yellow bars). (B) Shannon's diversity calculated for the diversity of IGHV region usage among bulk B cells and Tetramer⁺ cells. (C) Circos plots showing the IGHV-IGHJ pairings in a representative naïve mice and 3 immune mice. (D) IGKV gene usage from among B cells from a representative naïve mouse (grey bars) and Tetramer+ cells from immune mice (red, blue and green bars). (E) Shannon's diversity calculated for the diversity of IGKV region usage among bulk B cells and Tetramer⁺ cells. (F) Circos plots showing the IGKV-IGKJ pairings in a representative naïve mouse and 3 immune mice. Statistical analysis of Shannon’s diversity index was by Student’s T test.

https://doi.org/10.1371/journal.ppat.1006469.g007

CSP-binding antibodies undergo somatic hypermutation to improve affinity

Finally we were interested in knowing if the GC reaction we could see following sporozoite immunization was inducing higher affinity antibodies. We therefore examined our deep sequencing data to determine if CSP-specific antibodies had undergone somatic hypermutation (SHM) that would be indicative of B cells specific for CSP entering the GC. Taking advantage of the fact that our kappa chain primers capture the entire V-J sequences of the antibodies we sequenced we asked: 1) if the kappa chains shared between immune animals differed from the germline (providing evidence of SHM) and 2) if the mutations were conserved between different mice indicative of directed selection. Analysis of the reads from the kappa chains of the three immune mice showed that these had a much higher degree of mutation than bulk B cells from naïve mice, demonstrating SHM in the CSP-specific antibodies (Fig 8A). We further examined each specific common kappa chain in turn (IGVK1-110; IGKV1-135; IGVK5-43/45) comparing the sequences obtained from naïve B cells and (NANP)_n specific cells in immune mice. This analysis showed that while, as expected, sequences from naïve mice contained few mutations, the sequences from immune mice had much higher levels of SHM. Importantly mutations were found to be concentrated in the CDR loops, and were frequently shared by immunized mice providing strong circumstantial evidence for affinity maturation (Fig 8B; data for IGVK1-110 only shown).

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Fig 8. CSP-binding antibodies undergo somatic hypermutation and affinity maturation.

(A) Violin plots showing the number of mutations per kappa chain read from bulk B cells from 3 individual naïve mice and sorted (NANP)_n specific B cells from sporozoite immunized mice (B) Skyscraper plots showing the location of mutations away from germline in the IGKV1-110 gene in a naïve mouse and in sorted (NANP)_n specific cells in three sporozoite immunized mice. (C) ELISA binding to the (NANP)₉ peptide of recombinant antibodies corresponding to the 2A10 mAb, the predicted germline precursor, and hybrid antibodies containing the 2A10 heavy chain (mHC) paired with the germline light chain (gLC) and the 2010 light chain (mLC) paired with germline heavy chain (gHC). (D) Predicted mutations in the gLC were introduced to the germline precursor and their effect on binding to (NANP)₉ measured by ELISA (E) Predicted mutations in the gHC were introduced to hybrid antibodies consisting of the mLC and the gHC heavy chain and their effect on binding to (NANP)₉ measured by ELISA.

https://doi.org/10.1371/journal.ppat.1006469.g008

To directly test if CSP-binding antibodies undergo affinity maturation we expressed the predicted germline precursor to the 2A10 antibody (2A10 gAb) in HEK293T cells. We identified the predicted germline precursors of the 2A10 heavy and light chains using the program V-quest [41] (S5 and S6 Figs). This analysis identified the heavy chain as IGHV9-3; IGHD1-3; IGHJ4 and the light chain as IGKV10-94;IGKJ2, with the monoclonal antibody carrying 6 mutations in the heavy chain and 7 in the light chain. The 2A10 gAb had considerably lower binding in ELISA assays compared to the 2A10 mAb itself (Fig 8C), indicative that affinity maturation had taken place in this antibody. To determine the relative contribution of mutations in the heavy and light chain to enhancing binding we also made hybrid antibodies consisting of the mAb heavy chain and the gAb light chain and vice versa. Interestingly mutations in the light chain were almost entirely sufficient to explain the enhanced binding by the mAb compared to the gAb (Fig 8C).

To identify the specific mutations that were important we introduced the mutations individually into the gAb light chain construct. We prioritized mutations that were shared with the 27E antibody which has previously been found to be clonally related to 2A10 having been isolated from the same mouse and which shares the same germline heavy and light chains as the 2A10 mAb [20]. We found that two mutations (L114F and T117V) in the CDR3 of the light chain appeared to account for most of the gain in binding (Fig 8C). The effect of these antibodies appeared to be additive rather than synergistic as revealed by experiments in which we introduced these mutations simultaneously (Fig 8D). A further mutation close to the light chain CDR2 (H68Y) also caused a modest increase in binding. As expected mutations in the heavy chains appeared generally less important for increasing binding though M39I, N59I and T67F all gave modest increases in binding (Fig 8E). Collectively our data suggest that CSP repeat antibodies can undergo SHM in GCs resulting in affinity maturation, however the antibody response may be limited by the number of naïve B cells that can recognize and respond to this antigen.

Ethics statement

All animal procedures were approved by the Animal Experimentation Ethics Committee of the Australian National University (Protocol numbers: A2013/12 and A2016/17). All research involving animals was conducted in accordance with the National Health and Medical Research Council's (NHMRC) Australian Code for the Care and Use of Animals for Scientific Purposes and the Australian Capital Territory Animal Welfare Act 1992.

Mice, immunizations and cell depletions

BALB/C, C57BL/6 or CD28^-/- [55] mice (bred in-house at the Australian National University) were immunized IV with 5 x 10⁴ P. berghei CS^5M sporozoites expressing mCherry [56] or 5 x 10⁴ P. berghei CS^Pf sporozoites dissected by hand from the salivary glands of Anopheles stephensi mosquitoes. Mice were either infected with live sporozoites and then treated with 0.6mg choloroquine IP daily for 10 days or immunized with irradiated sporozoites (15kRad). For immunization with rCSP, 30ug rCSP was emulsified in Imject Alum according to the manufacturer’s instructions (ThermoFisher Scientific) and delivered intra-peritoneally. All mice received only a single immunization in these experiments. To deplete CD4+ T cells mice were treated with two doses of 100ug GK1.5 antibody on the 2 days prior to immunization (BioXCell); control mice received an irrelevant isotype control antibody (LTF2; BioXCell).

Flow cytometry and sorting

Single cell preparations of lymphocytes were isolated from the spleen of immunized mice and were stained for flow cytometry or sorting by standard procedures. Cells were stained with lineage markers (anti-CD3, clone 17A2; anti-GR1, clone RB6-8C5 and anti-NKp46, clone 29A1.4) antibodies to B220 (clone RA3-6B2), IgM (clone II/41), IgD (clone 11-26c2a), GL7 (clone GL7), CD38 (clone 90), CD138 (clone 281–2) and (NANP)₉ tetramers conjugated to PE or APC. Antibodies were purchased from Biolegend while tetramers were prepared in house by mixing biotinylated (NANP)₉ peptide with streptavidin conjugated PE or APC (Invitrogen) in a 4:1 molar ratio. Flow-cytometric data was collected on a BD Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo). Where necessary cells were sorted on a BD FACs Aria I or II machine.

Sequencing of (NANP)_n specific cells and BCR analysis

Single cell suspensions from the spleens of immunized mice were stained with (NANP)_n tetramers and antibodies to B cell markers as described in the supplementary experimental procedures. Antigen specific cells were sorted on a FACS ARIA I or II instrument prior to RNA extraction with the Arturus Picopure RNA isolation kit (Invitrogen) and cDNA preparation using the iScript cDNA synthesis kit (BioRad). BCR sequences were amplified using previously described heavy and kappa chain primers including adaptor sequences allowing subsequent indexing using the Nextera indexing kit (Illumina). Analysis was performed in house using R-scripts and the program MiXCR as described in supplementary experimental procedures.

Binding of antibody variants

Variants of the 2A10 antibody were expressed in HEK293 T cells (a kind gift of Carola Vinuesa, Australian National University) as described in the supplemental experimental procedures. Binding to the CSP repeat was tested by ELISA and ITC using standard techniques as described in the supplementary experimental procedures.

Statistical analysis

Statistical analysis was performed using Prism6 (GraphPad) for simple T tests and one-way ANOVAs from single experiments. Where data were pooled from multiple experiments, analysis was performed using linear mixed models in R version 3.3.3 (R foundation for Statistical Computing). Linear mixed models are a regression analysis model containing both fixed and random effects: fixed effects being the variable/treatment under examination, whilst random effects are unintended factors that may influence the variable being measured. If significance was found from running a linear mixed model, pair-wise comparisons of the least significant differences of means (LSD) was undertaken to determine at which level interactions were occurring. Statistical significance was assumed if the p-value was < 0.05 for a tested difference. (ns = not significant, * = p < 0.5, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Accession numbers

Sequence data generated in this paper is deposited at the NCBI sequence read archive (SRA) with accession number SRP092808 as part of BioProject database accession number PRJNA352758. Atomic coordinates and related experimental data for structural analyses are deposited in the Protein Data Bank (PDB) with PDB codes 5SZF and 5T0Y.