Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2

HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.


INTRODUCTION
immunogen with nanomolar EC50 values in ELISA (Fig 2B). Surface plasmon resonance (SPR) showed that Fabs of two mAbs, 2/6.14 and 2/1.12, bind with affinities of 5 to 10 nM ( Supplementary Fig. 1). The antibodies also immunoprecipitated a shorter D3 (502-617) with all putative N-glycosylation sites mutated out (Fig 2C, top  panel). Immunoprecipitation was comparable to the His-tag antibody, showing that the epitopes of the 5 mAbs resided within residues 502-617 and were unaffected by N-glycosylation. One antibody (1/5.13) that reacted with the C-terminal His tag and is comparable in specificity and sensitivity to commercial His tag antibodies is described in Methods.
Antibody reactivity with gametes and blockade of fertilization in vitro and in vivo Two individual assays with gametes, immunofluorescent staining with mAbs and ookinete conversion, were carried out in vitro. The change of environment from the bloodstream to the mosquito midgut triggers the development of gametocytes in infected erythrocytes into mature, highly motile "male" microgametes and more sessile "female" macrogametes and gamete emergence from erythrocytes. These events can be mimicked in vitro by reducing the temperature or pH and adding xanthurenic acid to the medium (ookinete medium). Each male gametocyte gives rise to eight microgametes, which look like a number of waving cilia as they emerge from the infected erythrocyte; this process is thus termed "exflagellation".
Reactivity of mAbs to native HAP2 in P. berghei microgametes was determined using cultured, infected erythrocytes undergoing exflagellation. After cultures were fixed, gametes adsorbed to slides were incubated with mouse D3 mAb and rabbit anti-alpha-tubulin followed by staining with fluorochrome-conjugated secondary antibodies specific for mouse and rabbit IgG and DAPI. Anti-tubulin and DAPI were used to identify microgametes by their highly elongated microtubule cytoskeletons and nuclei. All 5 D3 mAbs specifically stained P. berghei microgametes (Fig 3A).
We next tested the ability of the D3 mAbs to inhibit P. berghei microgametes from fertilizing macrogametes and forming ookinetes in vitro. Mouse blood infected with P. berghei gametocytes was mixed with D3 mAbs or control mouse IgG diluted in ookinete medium. After 24 h, ookinetes and unfertilized female gametocytes were identified by their morphology and staining with a fluorescent antibody, counted, and ookinete conversion rates were calculated. Only a single microgamete is required to fertilize a macrogamete, and each ookinete observed comprises a successful fertilization event. In control IgG, conversion rates were ~80% (Fig.  3B). Strikingly, mAb 2/6.14 at 500 µg/ml completely inhibited ookinete conversion and at 250 µg/ml inhibited by 96% (p<0.001). In contrast, none of the other mAbs inhibited by >50%, although mAb 2/1.40 showed significant inhibition at 500 µg/ml but not at 250 µg/ml (Fig 3B).
We then tested mAb 2/6.14 for its ability to block transmission in vivo in mosquitoes. Female Anopheles stephensi mosquitoes were allowed to feed through membranes on blood from P. berghei infected mice mixed with mAb 2/6.14 or control IgG. After 14 days, mosquito midguts were dissected and oocysts/midgut were counted, i.e., oocyst intensity was determined. Oocyst intensity was significantly reduced by mAb 2/6.14, particularly at 250 and 100 µg/ml (Fig. 3C). We also measured the prevalence of infection among the fed mosquitoes, i.e. the % of mosquitoes with at least one oocyst. Prevalence was significantly reduced by mAb 2/6.14 at both 250 and 100 µg/ml ( Supplementary Fig. S3). Summing the results over all mosquitos in the three independent experiments showed significant inhibition of intensity at all three mAb 2/6.14 concentrations, significant inhibition of prevalence at 250 and 100 µg/ml of mAb 2/6.14, dose-dependent reduction of both measures over the three antibody concentrations, and a reduction of intensity by 85% at 250 µg/ml (Fig. 3D). Overall, the results show that all five antibodies react with microgametes, that mAb 2/6.14 exhibits potent ability to block P. berghei fertilization in vitro and in vivo, and thus that purified HAP2 D3 has potential as a transmission-blocking vaccine.
Structural characterization of P. berghei HAP2 and complexes of D3 with Fabs To obtain structural insights relevant to rational development of a transmission-blocking vaccine, we obtained crystal structures of the P. berghei HAP2 D3 502-617 fragment with its three N-linked sites mutated out in complex with Fab fragments. Diffraction data was collected and refined to resolutions of 2.8 and 2.1 Å with Rfree of 29% and 23% for complexes with the transmission-blocking Fab 2/6.14 and cross-reactive Fab 2/1.12, respectively (Table S1). Each structure has two independent D3-Fab complexes in the asymmetric unit, giving us four examples of D3. The two Fab fragments bind to opposite faces of D3 (Fig. 4A).
D3 contains seven β-strands labeled A to E that are arranged into two β-sheets containing the ABE and DCFG β-strands ( Fig. 4A and B). These β-sheets associate through hydrophobic faces to form a β-sandwich. The way in which the sequence folds into this arrangement classifies it as a fibronectin type III (Fn3) domain. The D3 β-strands connect to one another through loops at each end of the domain. The A-B, C-D, and F-G loops link adjacent β-strands within a sheet and the B-C, D-E, and E-F loops link the two sheets.
Comparisons among the four examples of D3 show markedly different conformations for the loops at the C-terminal end of D3, which abut the polypeptide segment that connects to the plasma membrane (Fig. 4B). Quantitation of flexibility in these loops, A-B, C-D and E-F, shows high RMSD values or lack of comparison because of residues missing in density (dashes, Fig. 4E). In contrast, the loops at the N-terminal end that abut D1 show very similar conformations in all independent crystallographic examples. These loops, B-C, D-E, and F-G, show low RMSD values (Fig. 4E), consistent with the presence of several backbone-backbone or sidechainbackbone hydrogen bonds that stabilize each loop. Three sidechains that support the hydrogen bond networks in these loops, Asn-531 and Asn-532 in the B-C loop and Asp-598 in the FG-loop (overlined in Fig. 4E), are invariant in the 6 Plasmodia species.
The two antibodies bind largely to opposite faces of D3, with 2/1.12 binding primarily to the ABE sheet and 2/6.14 binding primarily to the DCFG sheet (Fab contacts are colored in green and red, respectively in Although the 2/1.12 Fab primarily engages the ABE β-sheet, it extends over the edge of this sheet to also contact the D strand, where it overlaps with the 2/6.14 epitope ( Fig. 4A and E). Fab 2/1.12 has the most contact with four residues in β-strands A, B and E of D3 (Fig. 4F). The sidechains of Thr-508 from D3 β-strand A and His-528 from strand B form hydrogen bonds with the sidechain of Tyr-32 from light chain complementaritydetermining region 1 (L-CDR1). In adjacent interactions, Tyr-577 in β-strand E forms a sidechain hydrogen bond to the backbone of Gly-91 in L-CDR3 while the Tyr-577 backbone hydrogen bonds to the sidechain of Asn-104 in H-CDR3. Another close contact and backbone-backbone hydrogen bond between D3 E β-strand residue Ala-575 and Tyr-102 from H-CDR3 further strengthens interaction ( Fig 4F). All four of these major epitope residues are invariant among the species examined here (Fig. 4E), correlating with excellent cross-reactivity of mAb 2/1.12.
Although mAb 2/6.14 contacts residues in each β-strand of the D3 DCFG β-sheet, the major contacts are formed by Arg-565 in strand D and six residues in the F-G loop (Fig 4E and G). The F-G loop forms a large bulge on the D3 surface that fits into a cavity in the Fab centered between the three H chain CDR loops (Fig. 4D). The Arg-565 sidechain forms bidentate hydrogen bonds to the backbone of Tyr-91 in L-CDR3 and a cation-pi interaction with Tyr-32 in L-CDR1. All three Fab H chain CDR loops surround the D3 F-G loop with each CDR forming at least one of a total of four mainchain-mainchain hydrogen bonds. A network of two sidechainsidechain and two sidechain-backbone hydrogen bonds forms around FG loop residue Glu-603 and H-CDR2 residue Asn-56. Hydrogen bonds between Lys-597 and Ser-57 of H-CDR2 and salt bridge between Lys-602 and Asp-31 of H-CDR1 further extend the interaction with the F-G loop (Fig 4G).
D3 contains six cysteine residues, all of which are conserved among Plasmodium species; however, only four of these form disulfide bonds ( Fig. 4E and Fig. 5A). Cys-513 in the A-B loop forms a short-range disulfide to Cys-523 in the B strand that likely stabilizes the residues between Cys-513 and the beginning of the B strand, which are highly polar and mostly disordered. Cys-546 in the C strand and Cys-592 in the F strand form a longrange disulfide bond to bridge these two adjacent β-strands in the middle of the DCFG β-sheet. Two D3 cysteines are free. Free cysteines buried in hydrophobic regions are occasionally present in extracellular proteins. Cys-542 is well buried in the hydrophobic core. Cys-604 is exposed in D3-Fab complexes but in the full ectodomain may be buried by residues at the interface between D1 and D3, including residues N-terminal to D3 residue 502, which were not present in the crystallization construct ( Fig. 5A and B).
HAP2 D3 in P. berghei is only 9 and 18% identical in sequence to C. reinhardtii and A. thaliana HAP2 D3, respectively; however, their three-dimensional structures are highly conserved (Fig. 5B). Their seven βstrands superimpose with very low root mean square deviations (Fig 5C). The long-range disulfide bond that connects the C and F strands is also conserved (Fig 5C). Both C. reinhardtii and P. berghei but not A. thaliana have long insertions in their A-B loops and a disulfide bond that may help to stabilize these long loops, which orient quite differently (Fig. 5C).
There is little variation in the orientation and length of the BC, DE and FG loops at the N-terminal end of D3. These loops are stabilized by an Asn in the BC loop, an Asp in the FG loop, and the hydrogen bonds they make. The Asn, residue 531 in P. berghei (asterisked in Fig. 5C), forms hydrogen bonds to both the BC and DE loops near the D1 junction ( Fig. 5D and E). Invariance in the other two species, despite the evolutionary distance between the unicellular algae, the plant, and protozoan parasite, is of particular interest because Asn-531 is in an N-glycosylation NX(S/T) sequon in P. berghei but its equivalents in the other species are not ( Fig 5C). The two other species are known to N-glycosylated but Plasmodium species are not. In D3 constructs mutated to remove N-linked sites, the N was left unchanged because it was much more conserved than the (S/T) position in other Plasmodium species. The B, D, and E strands that connect in these loops all contribute residues important in the mAb 2/1.12 epitope (Fig. 5E).
To better understand the P. berghei HAP2 ectodomain and its binding to 2/6.14 Fab, we obtained negative stain EM images and subjected ~2,000 particles to iterative alignment and classification. The P. berghei HAP2 ectodomain is rod-like with three or four oval densities arranged linearly along the rod (Fig. 4H panel 1 and Supplementary Fig. S4). The 2/6.14 Fab -ectodomain complex is L-shaped ( Fig. 4H panel 2). Similar results were obtained for complexes with ectodomains with residues 43-617 and 61-611 ( Supplementary Fig. S4). The two globules with stronger density correspond to the Fab, which has two domains per globule. The globules with weaker density correspond to the HAP2 ectodomain. Fab 2/6.14 binds essentially perpendicularly to one end of the HAP2 rod, in agreement with the crystal structure showing that the Fab binds to the side of D3 (Fig. 4A). When the putative Fab-D3 portion of the class average was isolated with a mask (Fig.4H panel 3), it correlated well (0.959) with the projection of the crystal structure of the 2/6.14 Fab-D3 complex ( Fig. 4H panel 4), as shown with the ribbon cartoon in the same orientation ( Fig. 4H panel 6). When the mask was omitted and the D3 Fab complex was cross-correlated with the entire ectodomain Fab complex, the same result and orientation was obtained, but the cross-correlation score was lower because the crystal structure lacks HAP2 D1 and D2.
Differences in antibody reactivity with D3 and the ectodomain Because of their differences in blocking transmission, we wondered whether mAbs differed in reactivity with the HAP2 ectodomain. Yields of monomeric ectodomain were lower than for D3 as described in Methods; nonetheless, gel filtration yielded a sharp peak (Fig. 6A). The peak primarily contained a band at 75 kDa, in agreement with the expected size of the ectodomain; furthermore, its C-terminus was present as shown by detection of the C-terminal His-tag by Western blotting (Fig. 6B). Treatment with Endo D reduced the size of the ectodomain to ~71kDa (Fig. 6B).
Presence of D3 epitopes in the ectodomain was tested by immunoprecipitation. Immunoprecipitation of the ectodomain, and D3 as a control, was detected using Western blotting of the His tag (Fig. 6C). mAb 2/6.14 was fully reactive with the ectodomain as shown by immunoprecipitation comparable to that with the His-tag antibody. In contrast and surprisingly, the other four mAbs were only partially reactive with the ectodomain, although they reacted with D3 comparably to mAb 2/6.14.
These differences were followed up by further comparisons that focused on mAbs 2/6.14 and 2/1.12. Calculations of affinity and dissociation constants assume fully active material; if only a fraction is active, then the apparent affinity is lower. In SPR, only the concentration of the analyte enters into affinity calculations. Therefore, we compared affinities measured for the two Fabs using them either as analyte or immobilized on the chip ( Fig. 6E and Supplementary Fig. S1). For Fab 2/1.12, the ratio of KD values for D3 (analyte/immobile) was 5.2/5.9 = 0.88±0.09 (Fig. 6E). In contrast, these values for the ectodomain were 106/11.7 = 9.1±6.2. Thus, the ectodomain appears to be only 0.88/9.1 = 9±6% active in binding to Fab 2/1.12. Comparable KD value ratios for Fab 2/6.14 were 6.0/10.2 = 0.59±0.23 for D3 and 22.9/50.5 = 0.45±0.04 for the ectodomain. Thus, the ectodomain appears to be 0.59/0.45 = 130±52% active in binding mAb 2/6.14, within error of the expected value of 100%, while Fab 2/1.12 binding to the ectodomain was far below the expected value.
We also assessed epitope content of the ectodomain by mixing it with equimolar concentrations of Fab and separating the complexes from components in gel filtration. Gel filtration of the mixtures was compared to that of the same concentrations of unmixed components (Fig. 6D). With Fab 2/6.14, much of the ectodomain was shifted to higher molecular weight and the Fab fragment was substantially depleted. With Fab 2/1.12 in contrast, only a small portion of the ectodomain was shifted to a higher molecular weight shoulder and there was little or no depletion of the Fab.
We further measured affinity with bio-layer interferometry (BLI) and complex formation with an independent preparation of the ectodomain. mAb IgG were immobilized on anti-mouse Fc capture sensors. HAP2 D3 bound well to both mAbs 2/6.14 and 2/1.12. In contrast, HAP2 ectodomain bound to mAb 2/6.14 but not to mAb 2/1.12 ( Supplementary Fig. S5). In conclusion, mAb 2/6.14 binds well to both D3 and the ectodomain, whereas mAb 2/1.12 and three other antibodies bind well to the D3 immunogen but bind poorly to or do not recognize the ectodomain.

DISCUSSION
We have generated a mAb to HAP2 D3 that blocks Plasmodium ookinete formation in vitro and in vivo and have determined a crystal structure of D3 that reveals the epitope to which the neutralizing mAb binds. Furthermore, we found that a subset of D3 mAbs were not fully reactive with the HAP2 ectodomain and that these antibodies were ineffective in preventing ookinete conversion. These findings have important implications for choice of future HAP2 immunogens. Previously, in the absence of structural knowledge, a large number of P. berghei HAP2 cDNA clones beginning and ending in different positions were tested in E. coli. Only one sequence, residues 355-609, was found to be expressed well; fortunately, it yielded an immunogen that elicited polyclonal antisera that inhibited gamete fertilization in vitro and reduced transmission in vivo (9). This sequence contained about half of D2, a small portion of D1, and all of D3 except for the last four residues of strand G. Recently, polyclonal antibodies to the putative fusion loops of D2 of Plasmodium berghei HAP2 also showed efficacy in blocking fertilization (8). The same laboratory and procedures were used for these studies, so the results are directly comparable, except for the use of a polyclonal antibody in the previous and monoclonal antibody in the current study. Surprisingly, mAb 2/6.14 to D3 is at least 5-fold more effective than that affinitypurified polyclonal antibody, as 100 µg/ml reduced oocyst intensity by 78%, whereas 500 µg/ml affinity-purified polyclonal antibodies to the fusion loop reduced intensity by 61%. Recently, residues 231-459 of P. vivax HAP2 were also tested. It contains ~30% of D1 and ~60% of D2 and, as expected from lack of a complete domain, had to be recovered from inclusion bodies in insect cells (10). This material elicited antibodies that inhibited transmission, but the results cannot be directly compared. The present study is the first time that a defined domain from HAP2 has been used in immunization, that a monoclonal antibody has been found to be effective in blocking transmission, and that a Plasmodium species HAP2 structure has been reported. Thereby, this study advances the rational development of transmission-blocking malaria vaccines.
HAP2 is highly conserved, both within and between Plasmodium species. In P. falciparum, 199 isolates in PlasmoDB (www.plasmodb.org, release 54) evaluated have identical D3 amino acid sequences and only a few polymorphisms are present in D1 and D2 of HAP2 ectodomain in 2 or more isolates (R99E, n=2; N184S, n=14; D185Y/E, n=11/7 and D455N, n=47). The high sequence identity of gamete antigens contrasts to malaria vaccine antigens expressed by sporozoites such as TRAP and the C-terminal region of CSP used in the RTS,S vaccine and those expressed by blood stage parasites such as MSP1 and AMA1, which have high levels of polymorphisms and have proven to be challenging for vaccine antigen design (22)(23)(24)(25). Compared to CSP and TRAP, HAP2 is also highly conserved among species; for example, P. berghei HAP2 is 60-70% identical in D3 to the five species we examined that are capable of infecting humans.
We structurally characterized HAP2 D3 in complex with two Fab fragments that bound to opposite faces of D3, on the β-sheets that form its β-sandwich domain. Conservation or variation among residues in these interfaces among Plasmodium species provides insights into which residues are important in their epitopes. The four residues most important for mAb 2/1.12 binding were identical among all six species examined, explaining the wide cross-reactivity of this mAb. Although the D3 F-G loop was central in the 2/6.14 epitope, many of its contacts were between backbone atoms. The sidechain with the most contacts was Arg-565 in the D strand. All three interactions by the Arg-565 sidechain would be abolished by the Asn substitution in P. vivax and P. falciparum (Fig 4E), which had the lowest affinity.
Thus far, structural information on HAP2 has been on its post-fusion, trimeric form or on isolated domains (2)(3)(4)(5). The rod-like, linear conformation of the HAP2 ectodomain and its monomeric state has not previously been seen for HAP2. Its resemblance to the linear conformation of pre-fusion states of structurally homologous class II viral fusogens suggests that this linear conformation of the HAP2 ectodomain corresponds to its pre-fusion state. The pre-fusion state is preferred over the post-fusion state as the target for transmission blocking antibodies, because antibodies that bind to the pre-fusion state not only can act earlier but can also sterically block the interfaces required for trimer formation and the fold-back of D3 onto D1 and D2 (Fig. 1).
In addition to immunological mechanisms for blocking gamete fertilization in the mosquito blood meal (11), mAb 2/6.14 might also block conformational changes required for conversion of the monomeric pre-fusion state of HAP2 to the trimeric post-fusion state (Fig 1), especially reorientation of D3 to fold over D1 and D2 in the trimer, which is required for fusion (Fig 1). Direct blocking of fusion has been demonstrated with antibodies to D3 of viral type II fusion proteins that are structurally homologous to HAP2 (13)(14)(15).
To test this idea, we superimposed D3 from P. berghei HAP2 on D3 from the trimeric fusion conformation of HAP2 in other species (Fig. 5B). The C. reinhardtii and A. thaliana D3 structures superimpose well, despite only 8 and 17 % identity with P. berghei HAP2 D3, respectively. This identity is too low for immunological crossreactivity. Superposition showed that the F-G loop recognized by mAb 2/6.14 is buried in D3 interfaces in the postfusion state trimer. The mAb 2/1.12 epitope also substantially overlaps with the D3 postfusion state trimer interfaces in the C strand. Moreover, both antibody epitopes overlap in the D strand with sites buried in the postfusion conformation. The incompatibility of Fab binding and conformational change to the postfusion state is shown by burial of the Fabs in superpositions on one monomer in a postfusion trimer of C. reinhardtii HAP2 (Supplemental Fig. S6). Thus, binding of either of these mAbs to D3 would block folding back of D3 in the fusion state (Fig. 1C). Why then did mAb 2/6.14 and not mAb 2/1.12 block conversion of macrogametes to ookinetes? mAb 2/6.14 is differentiated from the other 4 mAbs studied here by its ability to completely react with the monomeric HAP2 ectodomain. Although all five mAbs reacted with D3 with nanomolar EC50 values by ELISA and pulled down similar amounts of isolated D3 by immunoprecipitation, only mAb 2/6.14 fully immunoprecipitated the ectodomain. SPR measurements further showed that the affinity of mAb 2/1.12 was substantially lower for the ectodomain than for D3, but only when the ectodomain was used as analyte and not when immobilized on the chip, suggesting that only ~10% of the ectodomain was active in binding to mAb 2/1.12. Gel filtration showed that the ectodomain complexed well with mAb 2/6.14, whereas only a minor fraction of the ectodomain complexed with mAb 2/1.12. Thus, three independent methods show that only a minor fraction of the HAP2 ectodomain reacts with mAb 2/1.12, and one of these methods, immunoprecipitation, showed that another three mAbs behaved like mAb 2/1.12. While all 5 mAbs gave clear-cut staining of P. berghei microgametes, the microgametes had been fixed in 4% paraformaldehyde and incubated overnight in this solution, which denatures many proteins, prior to staining.
The scope of this study was to investigate the effectiveness of HAP2 D3 as an immunogen for transmission-blocking malaria vaccines. Nonetheless, our study also provides some useful guideposts for future use of the HAP2 ectodomain in transmission-blocking vaccines. Most eukaryotic extracellular proteins are highly dependent on N-glycosylation for stability and expression; however, lack of N-glycosylation sites in D3 appeared to have little or no effect on expression yield, which favors the hypothesis that N-glycans are not added to HAP2.
Unfortunately, efficiently expressing eukaryotic extracellular proteins with complex multi-domain structures, multiple disulfide bonds, and no N-glycosylation requires refolding from E. coli, which is highly challenging. Therefore, the best approach for successfully expressing the HAP2 ectodomain may be to use eukaryotic hosts and to mutationally remove N-glycosylation sequons, as was done here for D3 but not for the ectodomain.
In conclusion, we find that D3 of HAP2 can elicit antibodies that block transmission of malaria. Furthermore, we have obtained a mAb that blocks transmission, determined the first structure of a fragment of HAP2 in Plasmodium, and determined the structure of HAP2 of D3 in Plasmodium in complex with antibodies that either block or do not block transmission. We have confirmed the principle that HAP2 D3 can elicit transmission-blocking antibodies. On the other hand, we have also identified limitations of D3 as an immunogen because some antibodies to D3 did not react well with the HAP2 ectodomain. We also outline a possible strategy for obtaining improved expression and more native folding of the HAP2 ectodomain as an alternative immunogen for transmission-blocking immunity. This proposed approach is validated by our EM studies showing we can isolate a monomeric, pre-fusion state of the HAP2 ectodomain. Furthermore, stabilizing the pre-fusion state of HAP2, as successfully done for the respiratory syncytial virus fusion protein (26) and SARS-CoV-2 spike protein (27), may not only increase expression but also efficacy in inducing neutralizing antibodies.

MATERIALS AND METHODS
Study design. This study was designed to provide insights for developing a vaccine to block or reduce malaria transmission. This objective was addressed by first conducting producing monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2. CB6F1 mice were immunized with the glycan-shaved D3 protein.
All in vitro characterization of binding properties was carried out after a detailed antibodies screening as described in the associated figure legends. We next tested the ability of the D3 mAbs to inhibit P. berghei microgametes from fertilizing macrogametes and forming ookinetes in vitro using standard membrane feeding assay. The results show that all five antibodies react with microgametes, that mAb 2/6.14 is outstanding for its ability to block P. berghei fertilization in vitro. Therefore, we decided to test mAb 2/6.14 for its ability to block transmission in vivo in mosquitoes. For in vivo characterization of the ability of mAb 2/6.14 to reduce malaria transmission, Female Anopheles stephensi mosquitoes were randomized to group feed through membranes on blood from P. berghei infected mice mixed with mAb 2/6.14 or control IgG. After 14 days, oocyst intensity and prevalence of infection mosquito were counted to evaluate the ability of mAb 2/6.14 inhibition.
Ethical statement for animal studies. Mouse immunization was conducted in accordance with and was approved by Boston Children  (3). All constructs were codon-optimized for mammalian cells. UniProt accession Q4YCF6.1, which was used for P. berghei HAP2 ectodomain construct codon optimization, was retrospectively discovered to be deleted for S206, which locates to the bd-strand of D2.2 in the alignment to Chlamydomonas reinhardti HAP2, at the opposite end of the ectodomain from D3. All other eight HAP2 accessions recovered from the nonredundant protein database with NCBI BLAST in 2020, including at least seven distinct P. berghei strains including ANKA, are identical to one another and differ from the Q4YCF6.1 sequence only at residue S206 and the signal sequence. This error has been reported to help@uniprot.org. Our recent experience is that, among hundreds of sequences of human and mouse extracellular proteins of similar length, ~10% of UniProt but not RefSeq accessions have similar errors.
For purification, HAP2 fragments in 1 L of culture supernatant were adjusted to 1 mM NiCl2 in D3 buffer (20 mM Tris, pH 8 and 300 mM NaCl) or ectodomain buffer (20 mM Tris, pH 8.5, 500 mM NaCl) and applied to 10 ml Ni-NTA-Sepharose (Qiagen) columns. After washing with 15 mM imidazole in D3 or ectodomain buffer, the protein was eluted with 300 mM imidazole in the same buffers. Fragments were then subjected to gel filtration chromatography using Superdex 75 (GE life Sciences) in D3 or ectodomain buffer, concentrated, and frozen at -80°C. Purification of P. berghei 477-621 D3 construct expressed stably in S2 cells was similar, except after the Ni-NTA step it was concentrated (1 mg/ml in 0.2 ml) and shaved with endoglycosidase D (10 µl, 100 unit) (New England BioLabs) for 16 hrs at 4°C prior to gel filtration. Final yield was 22 mg/L culture supernatant. Yields for transiently expressed D3 constructs were ~ 5 to 8 mg/L culture supernatant. Ectodomain yields were ~0.2-0.3 mg/L culture supernatant.
Monoclonal antibodies. CB6F1 mice (Charles River, Wilmington, MA) were immunized intraperitoneally with 20 µg of N-glycan shaved P. berghei HAP2 D3 (residues 477-621) in PBS and complete Freund's adjuvant (Sigma) and 3 weeks later with the same material in incomplete Freund's adjuvant (Sigma). Mice were boosted both intravenously and intraperitoneally 2 weeks later with 20 µg of the same antigen in PBS. Three days later, splenocytes were fused with the murine myeloma P3X63Ag8.653 (CRL 1580, ATCC, Rockville, MD) as described (28). Hybridomas supernatants were screened by ELISA in microtiter wells coated with the immunogen. Hybridomas that produced IgG mAbs were subcloned. Five produced antibodies specific for P. berghei HAP2 D3 and are characterized in Results. mAb 1/5.13 was found to be specific to the His-tag. It reacts with proteins with C-terminal His tags, but not with N-terminal His tags. It is fully active in ELISA, Western blot and immunoprecipitation and has sensitivity comparable to the THE TM His Tag Antibody (Genscript) in ELISA and Western Blot ( Supplementary  Fig.S7).
For antibody purification, hybridoma cells were adapted to and expanded in serum free medium containing 1:2 vol/vol Cell™ MAB medium (Life Technologies) and HyClone™ CDM4MAB medium (GE Life Sciences). IgG was purified using protein G affinity chromatography (Invitrogen).
Heavy and light chain V region cDNA sequences of mAbs 2/6.14 and 2/1.12 were determined by Syd Labs (Natick, MA). The mAbs each have γ1 heavy and κ light chains and unique V regions as shown by BLAST searches. A somatic mutation in the 2/6.14 VL domain that introduced an N-glycosylation site into the framework region was reversed with an N74S mutation (mature protein numbering) in recombinantly expressed Fab and IgG. Fab or intact H chains used g-Blocks encoding the murine k chain secretion signal peptide, VH and γ1 CH1 domain, with or without hinge, CH2 and CH3 domains, followed by a Gly-Ser linker and 6xHis tag, that were assembled in EcoRV-linearized pVRC8400 vector (29) using NEBuilder ® HiFi DNA reagents and protocol (New England BioLabs). κ light chains used g-Blocks encoding the murine κ chain secretion signal peptide, VL domain, and CL domain that were similarly assembled with SapI-linearized pD2529-CAG vector (Atum, Newark, CA).
Fabs and IgGs were expressed in Expi293F cells co-transfected with H and L chain constructs in 2:1 ratios. Fab fragments were purified from culture supernatant by Ni-NTA affinity purification followed by Superdex 200 gel filtration chromatography. IgG was purified by protein G affinity chromatography. 2/6.14 IgG purified from Expi293F and hybridoma cell supernatants bound to P.berghei HAP2 D3 in ELISA with comparable EC50 values.
Enzyme-linked immunosorbent assay (ELISA). 96-well ELISA plates (Costar) were coated overnight at 4°C with 50 µl of purified, His-tagged HAP2 D3 at 5 µg/ml in 50 mM sodium carbonate buffer, pH 9.5 and blocked with 3% BSA for 90 min at 37 o C. 50 µl of diluted hybridoma supernatants or purified mAbs in triplicate were incubated for 2 hrs at 37°C. After 3 washes, 50 µl of 1:10,000 diluted HRP-conjugated goat-anti-mouse IgG (H+L) (Abcam) was added. After 1 h at room temperature and 4 washes, peroxidase substrate (Life Technologies) was added and after 10 min plates were read at 405 nM on an Emax plate reader (Molecular Devices). As a control, hybridoma medium or mAb dilution buffer (1% BSA in PBS) was substituted in the antibody binding step. For measuring binding of His-tagged D3 to immobilized mAbs, ELISA plates were coated with 5 µg/ml mAb, and HRP-anti-His (Penta-His Ab, Qiagen) was used in the detection step. Titration curve fitting and EC50 measurements used the sigmoidal, 4 parameter logistic equation in GraphPad Prism 7.
Surface plasmon resonance (SPR). Purified HAP D3 (residues 502-617 with N516T, S533N and N539Q mutations) or ectodomain (residues 43-617) fragments or Fabs 2/1.12 or 2/6.14 were either used as analytes or amine immobilized on a CM5 chip in a Biacore 3000 (GE Healthcare) according to the manufacturer's instructions. For immobilization, protein was diluted to 5 μg/ml in 0.15 M NaCl, 20 mM Hepes pH 7.4, and injected at 10 μL/min. The surface was regenerated with a 10-to 60-s pulse of 18 mM HCl at the end of each cycle to restore resonance units to baseline. Kinetics and affinity analysis were performed with SPR evaluation software version 4.0.1 (GE Healthcare). A 1:1 Langmuir binding model, with or without a conformational change model, was applied for experimental data fitting, and kinetic parameters were fit globally to sensorgrams at different analyte concentrations.
Bio-layer interferometry (BLI). Bio-layer interferometry (30) experiments were performed on a ForteBio Octet RED384 instrument using anti-mouse Fc capture (AMC, 18-5090) sensors. The reaction was measured on a 384 well plate (working volume of 25 µL) in 20 mM Tris-HCl, pH7.5, 150mM NaCl, 0.01%BSA, 0.02% Tween 20 (Assay buffer). Biosensors were hydrated in assay buffer for 10 min before starting the measurements. Each biosensor was sequentially moved through 5 wells with different components: (1) Assay buffer for 1 minute in baseline equilibration step; (2) 5ug/ml (33.3 nM) 2/6.14 or 2/1.12 IgG for 3 minutes for immobilization of the antibodies onto the biosensor; (3) Assay buffer for 3 minutes for another baseline equilibration; (4) indicated concentrations of PbHAP2 ectodomain (aa:43-617 ) or PbD3(aa: 477-621) for 10 minutes for the association phase measurement; and (5) Assay buffer for 10 minutes for the dissociation phase measurement. Each biosensor has a corresponding assay buffer reference sensor that went through the same 5 steps. Kinetics and affinity analysis were performed with Octet RED384 Data Analysis 11.0. A 1:1 Langmuir binding model was applied for experimental data fitting, and kinetic parameters were fit globally to different analyte concentrations for each IgG and HAP2 combination, with k on and k off as shared fitting parameters and maximum response (Rmax) as individual fitting parameter.
Immunoprecipitation and Western blot. Culture supernatants or purified HAP2 D3 and ectodomain fragments diluted in TBS (25 mM Tris, pH 8, 300 mM NaCl) containing 0.5% BSA were incubated with mAbs or control non-binding IgG overnight at 4°C. Immunocomplexes were pulled down with protein G beads by incubation at 4°C for 2 hrs with rotation. Beads were washed 3 times with 1 ml TBS and bound proteins eluted in 1x Laemmli sample buffer containing 5% β-mercaptoethanol and subjected to reducing SDS-polyacrylamide gel electrophoresis. Blots were probed with polyclonal rabbit anti-His (0.4 µg/ml, Cell Signaling), followed by incubation with HRP-conjugated goat-anti-rabbit (GE Healthcare) and chemiluminescence imaging using LAS-4000 system (Fuji Film). Quantitation of protein bands used ImageJ software.
In vitro ookinete conversion was assayed as described (8,32). Briefly, infected mouse blood containing P. berghei (strain ANKA 2.34) female and male gametocytes (20 ul) was mixed with ookinete medium (100 ul) containing HAP2 D3 antibodies or control IgG (500 and 250 ug/ml final concentration) and incubated at 19°C. After 24 h, cultures were incubated with Cy3-conjugated Pbs28 mAb 13.1, which stains both ookinetes and unfertilized macrogametes (33d5904), for 20 min on ice. Larger, elongated, banana-shaped ookinetes were distinguished from smaller, round macrogametes and counted by fluorescence microscopy. Conversion rates were calculated as % ookinetes /(macrogametes + ookinetes). Inhibition of ookinete conversion was expressed as the percentage reduction in ookinete conversion with respect to the negative control IgG at the same concentration.
In vivo transmission-blocking activity of HAP2 antibodies was assayed using standard membrane feeding assay (SMFA) as described (32). Briefly, heparinized P. berghei infected blood containing gametocytes was mixed with HAP2 antibodies or negative control IgG. Female Anopheles stephensi (SDA 500 strain) were starved for 24 hours and then fed on the mixtures using membrane feeders (>50 mosquitoes per each blood-antibody feed). 24 hours later, unfed mosquitoes were removed. Mosquitoes were maintained on 8% (w/v) fructose, 0.05% (w/v) p-aminobenzoic acid at 19-22°C and 50-80% relative humidity. Day 14 post-feeding, mosquito midguts were dissected, and oocyst numbers per midgut in each mosquito was determined by phase contrast microscopy. Reductions in oocyst intensity (number of oocysts/midgut) and prevalence (number of infected over total mosquitoes fed) in the presence of an HAP2 antibody were calculated with respect to the negative control IgG present at the same concentration in the feeds.
Crystallization and structure determination. The P. berghei D3 (502-617) construct with N516T, S533N and N539Q mutations to abolish N-glycosylation was used for crystallization. D3 and Fab were mixed in 1:1.3 molar ratios and complexes were isolated by gel filtration. Complexes were crystallized at 20°C by hangingdrop vapor diffusion with equal volumes of complex and well solution. The Fab 2/1.12 complex (4.5 mg/ml) was crystallized with 0.2 M ammonium sulfate, 25% PEG 3350, 0.1 M Bis-Tris pH5.5. The 2/6.14 Fab complex (7.5 mg/ml) was crystallized with 0.3 M proline, 22% PEG 1500, 0.1M HEPES pH7.5; crystals were dehydrated by soaking in solutions that had the starting concentrations of components in the protein and reservoir solutions while raising the concentration of PEG 1500 to 31% in 3% steps. Crystals of Fab 2/1.12-D3 and Fab 2/6.14-D3 were cryo-protected with reservoir solution containing 15% glycerol or 15% ethylene glycerol, respectively, in 2 steps of 5 and 10% increase and plunged in liquid nitrogen. Data were collected at 100K on GM/CA beamline 23-IDB at the Advanced Photon Source (Argonne National Laboratory) and processed with XDS (34). Structures were refined with PHENIX, built with Coot (35) and validated with MolProbity (36). Figures were made with PyMol.
The 2/1.12 Fab D3 complex structure was solved by molecular replacement in the Phenix suite (37) using a Fab search model (PDB ID 2A6D). The 2/6.14 Fab-D3 complex structure was solved by molecular replacement using D3 from the 2/1.12 Fab complex, the H chain of PDB ID 1IGC, and the L chain of PDB ID 5AZ2. Each crystal structure has two complexes in the asymmetric unit.
During model building and refinement of the 2/6.14 Fab complex, the Fab constant domains of one complex had good density, but the variable domains and D3 had broad but continuous density that was difficult to trace. In contrast, all domains of the other complex were easily traced. Furthermore, refinement remained stuck. Alternative space groups including those with lower symmetry or use of twin rules provided no improvement. We then realized that in the troublesome D3-Fab complex in the asymmetric unit, two alternative conformations were present for D3, VH, and VL, whereas CH1 and CL had a single conformation. The transition between dual and single conformations occurred at the elbows between VH and CH1 and between VL and CL; i.e. the two conformations differed in elbow angle. Further refinement, which largely treated each of the dual conformations of D3, VH1, and VL as rigid bodies based on their structure in the single conformation of the other D3-Fab complex, resulted in drop to the final Rwork and Rfree of 25.3 and 29.2%, respectively.
The alternate conformations allow the VL domain in chain B and the D3 domain in chain C to pack against alternate conformation symmetry mates in different complexes along adjacent edges of the unit cell. Symmetry mates with the B conformation severely clash, while those with the distinct conformations A and B have good lattice contacts. In contrast, symmetry mates with conformation A are too far apart to provide stabilizing lattice contacts. Thus, clashes in one conformation together with a lack of stabilizing lattice contacts in the other conformation may have driven the formation of a crystal lattice with dual conformations of VL, VH, and D3 in one of two Fab-D3 complexes in the asymmetric unit.
Negative stain electron microscopy. Each P. berghei HAP2 ectodomain construct (residues 43-617 or 61-611), with or without Fab at a molar ratio of 1:1.3, was subjected to Superdex 200 gel filtration in 20 mM Tris-HCl, pH8, 500 mM NaCl. Peak fractions were adsorbed to glow-discharged carbon-coated copper grids, washed with deionized water, and stained with freshly prepared 0.75% uranyl formate. Images were acquired with an FEI Tecnai-T12 transmission electron microscope at 120 kV and a nominal magnification of 52,000×. About two thousand particles were picked interactively and subjected to 2D alignment, classification and averaging using RELION-2.1 (38). Selected EM class averages were masked and cross-correlated using EMAN2 (39) with 2D projections generated at 2° intervals from the 2/6.14 Fab-D3 complex crystal structure filtered to 25 Å. Fig. S1. Surface plasmon resonance (SPR) analysis of binding Interactions of Fab 2/6.14 and Fab 2/1.12 with PbHAP2 D3 and monomeric ectodomain. Fig. S2. Titration of binding of HAP2 D3 from Plasmodium species to immobilized mAbs. Fig. S3. Transmission blocking activity of mAb 2/6.14 in standard membrane feeding assay. Fig. S4. EM class averages. Fig. S5. Bio-layer interferometry (BLI) analysis of binding Interactions of IgG 2/6.14 and IgG 2/1.12 with PbHAP2 D3 and monomeric ectodomain. Fig. S6. Analysis the mAb 2/6.14 and 2/1.12 binding in the context of postfusion C. reinhardtii HAP2 trimer. Fig. S7. Reactivity of mAb 1/5.13 to fusion proteins with His tags. Table S1. Statistics of X-ray diffraction and structure refinement of PbHAP2 domain 3 (D3) complexed with 2/6.14 Fab or 2/1.12 Fab   Structural and sequence conservation of D3 and its 2/1.12 and 2/6.14 epitopes. Top line: b-strands shown as arrows. Second line: Root mean square deviation (RMSD) of Cα atom position (Å) among the two independent Fab 2/1.12 -D3 complexes and the Fab 2/6.14-D3 complex with a single conformation, calculated after structure alignment by RaptorX (41). A dash shows positions where residues were defined in only 0 or 1 of the three 3 structures. Third line: Filled circles show Fab contacts (green for 2/1.12 Fab and red for Fab 2/6.14); residues that mediate major interactions (>10 Å 2 of buried solvent accessible surface area or H-bonds) or minor interactions (<10 Å 2 buried solvent accessible surface) are shown with large or small circles, respectively. Remaining lines show D3 sequence in P. berghei, P. knowlesi, P. vivax, P. malariae, P. ovale and P. falciparum with identities to P. berghei shown as dots. P. berghei residues in red are wild-type and were mutated to remove N-linked sites and residues in italics were not visualized in any of the three structures. P. berghei residues with sidechains mentioned in the text that stabilize hydrogen bonds in the B-C and F-G loops are overlined. Cysteines are highlighted in yellow and connected when disulfide-linked (F and G). Details of D3 interactions with the 2/1.12 Fab (F) and 2/6.14 Fab (G). D3 is silver, Fab H and L chains are light blue and wheat, respectively, and residues with major interactions with Fabs as defined in (E) have carbons colored green (F) or red (G). Dashes show hydrogen bond and pi-cation interactions. (H) The most populated negative stain EM class averages of the HAP2 ectodomain (residues 43-617) alone (panel 1, 1128 particles) and in complex with the 2/6.14 Fab (panel 2, 379 particles). Panel 3 shows the best correlating projection directly from the complex crystal structure. Panel 4 shows a ribbon diagram of the crystal structure in the same orientation. D3 colored cyan, Fab H and L chains colored light blue and wheat, respectively, and N-terminal of D3 shows as a red sphere, C terminal of D3 colored as black. Structure-based sequence alignment from the superimposition shown in (A). b-strands and root mean square deviation (RMSD) (Å) of Cα atom positions are shown above the sequences. Green and red filled circles above the P. berghei sequence show Fab 2/1.12 and Fab 2/6.14 contacts, respectively as defined in Fig. 4E legend. In the P. berghei sequence, the three residues in red mark residues that were mutated to remove N-linked sites. Orange and blue filled large and small circles above Arabidopsis thaliana and Chlamydomonas reinhardtii sequences mark residues buried in trimer contacts with >10 Å² burial or a hydrogen bond or < 10 Å², respectively. Solvent accessible surface area burial was calculated with PISA (42). (D) Asn-531, present in an N-glycosylation sequon in the P. berghei sequence, forms stabilizing hydrogen bonds to the backbones of the B-C and D-E loops, a function that is conserved in HAP2 in other phyla. The color code is the same as in panel A, except P. berghei is in silver. (E) Asn-531 locates near mAb epitopes. Asn-531 and sidechains or backbones to which it hydrogen bonds are shown in stick. Residues with major or minor contacts with Fabs as defined in Fig. 4E legend are shown with both  iii Figure S2. (Related to Figure 2). Titration of binding of HAP2 D3 from Plasmodium species to immobilized mAbs. Elisa plates on which mAb 2/6.14 (A), 2/1.12 (B) or 2/1.40 (C) at 5 ug/ml were immobilized were then incubated with purified, His-tagged D3 from Plasmodium spp. at varying concentrations. Binding was detected by incubation with HRP-conjugated anti-His. Nonlinear titration curve fitting was performed using GraphPad Prism 7 software. Data shown are mean ± SD of triplicate measurements in one representative experiment.