Author response: Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2
Year: 2021
Type: peer-review
Abstract: 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 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. Editor's evaluation This study describes the generation of monoclonal antibodies targeting the fusogen HAP2 from the malaria parasite that is required for parasite transmission to mosquitoes. Using structural approaches in combination with biophysical methods, the authors provide insights into the differences in transmission blocking potencies for different monoclonal antibodies, which may inform the design of HAP2 as a potential vaccine candidate. https://doi.org/10.7554/eLife.74707.sa0 Decision letter eLife's review process Introduction HAP2, a gamete fusogen required for fertilization, is found in eukaryotic plant, metazoan, and protozoan kingdoms (Clark, 2018). Crystal structures are known of HAP2 from the unicellular green alga, Chlamydomonas reinhardti; a model plant, Arabidopsis thaliana; and a protozoan parasite, Trypanosoma cruzi (Fédry et al., 2017; Feng et al., 2018; Fedry et al., 2018; Baquero et al., 2019). HAP2 can form a trimeric structure that is structurally homologous to the fusion state of viral class II fusion proteins. These fusogens have three extracellular domains, arranged in the order D2, D1, and D3, a disordered stem region, a single-pass transmembrane domain, and a cytoplasmic domain, and are expressed on the plasma membrane of male gametes or the envelope of viruses (Figure 1). Figure 1 Download asset Open asset Diagram of HAP2 pre-fusion monomer, trimerization and membrane fusion. The model is based on postfusion structures of HAP2 referenced in the text, their similarity to postfusion structures of viral class II proteins, models for the conversion of prefusion to postfusion class II fusion proteins (Kielian and Rey, 2006), and work showing that D1 and D2 are sufficient for trimer formation (Liao et al., 2010), which suggests that the last step may be concerted foldback of D3 over D1 and D2 and fusion of the macrogamete and microgamete plasma membranes. (A) pre-fusion monomer on the surface of male gametes, (B) extended trimeric intermediate, and (C) post-fusion trimer and fusion of gamete membranes. Domains are colored. Fusion loops at the tip of D2 are shown as '*'. Antibodies to D3, shown as Y shapes, may agglutinate microgametes, trigger antibody Fc-dependent effector functions, or neutralize HAP2 function by interfering with trimer formation and gamete membrane fusion. HAP2 is required for fertilization in Plasmodium parasites (Hirai et al., 2008; Liu et al., 2008) and is thus an attractive target for vaccines that block transmission of malaria. Gamete fertilization takes place in the mosquito midgut in a blood meal taken from an infected individual. Blocking fertilization prevents transmission to the individual from which the mosquito takes its second, final blood meal. Plasmodium gametes emerge from infected erythrocytes well before digestion of the blood meal; specific antibodies present in the blood meal will react with and can agglutinate or otherwise neutralize gametes (Angrisano et al., 2017; Blagborough and Sinden, 2009; Qiu et al., 2020; Graves et al., 1985). HAP2 is present on male gametes (microgametes) and has hydrophobic fusion loops at the tip of D2 that insert into the plasma membrane of the female macrogamete and trigger trimerization and structural rearrangements of HAP2 to the fusion state, which mediates membrane fusion and merging of the cytoplasm of the two gametes through a fusion pore (Kielian and Rey, 2006; Figure 1). To mediate fusion, D3 of HAP2 folds over an inner trimeric core composed of D1 and D2 (Figure 1). We reasoned that antibodies to D3 of HAP2 that blocked the interface formed with D1 and D2 in the fusion state might block fusion, as has been demonstrated with viral fusogens (Austin et al., 2012; Zhao et al., 2016; Li et al., 2018). Thus, we hypothesized that HAP2 D3 antibody taken up in a blood meal could inhibit fertilization and infection of the mosquito and subsequent transmission to humans (Delves et al., 2018; Figure 1). Immunization of mice with HAP2 fragments or putative HAP2 fusion loop peptides has proved the concept that antibodies to different domains of HAP2 can, to different extents, block transmission of Plasmodium berghei or P. vivax, species that infect rodents and humans, respectively (Angrisano et al., 2017; Blagborough and Sinden, 2009; Qiu et al., 2020). However, we are far from having an optimized HAP2 immunogen. Furthermore, the high-sequence divergence of HAP2 in Plasmodium species from structurally characterized HAP2 in other phyla emphasizes the importance of Plasmodium HAP2 structures for rational vaccine design. Here, to advance a vaccine that would block malaria transmission, we have expressed a D3 fragment of P. berghei HAP2, raised monoclonal antibodies, crystallized D3 in complex with Fab fragments of two antibodies, and examined Fab complexes with the complete HAP2 ectodomain (D1-D3) by negative stain electron microscopy (EM). Furthermore, we show that one of these antibodies potently blocks gamete fertilization and transmission and thus for the first time that a monoclonal antibody to HAP2 can block transmission. We define the advantages, as well as the limitations, of using the D3 domain of HAP2 as an immunogen for transmission blockade. Moreover, the two structurally characterized antibodies have cross-reactivity with HAP2s among multiple plasmodial species that can cause malaria in humans. The insights into Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition are important steps toward the development of a vaccine to block malaria transmission. Results P. berghei HAP2 D3 elicits antibodies that cross react with human malaria pathogens Apicomplexans such as Plasmodium are eukaryotes that have extracellular proteins that are disulfide-linked and glycosylated; therefore, we produced HAP2 proteins in insect and mammalian cells which are competent for such modifications. C-mannosylation and O-glycosylation in Plasmodium have been verified; however, while Plasmodium species have been suggested to have unusually short N-glycans, N-glycosylation has yet to be verified with any specific protein (Bushkin et al., 2010; Macedo de et al., 2010; Bandini et al., 2019; Swearingen et al., 2016; Swearingen et al., 2019). To elicit antibodies, we expressed in Drosophila S2 cells a P. berghei HAP2 D3 construct (residues 477–621, containing four putative N-glycosylation sites) containing a C-terminal His tag. After Ni-affinity chromatography, the material was nearly homogenous, as shown by SDS-PAGE and Coomassie blue staining (Figure 2A, lane 1). Shaving the N-glycans with endoglycosidase D (Endo D) followed by gel filtration decreased size and heterogeneity in SDS-PAGE and confirmed N-glycosylation of D3 (Figure 2A, lane 2). Figure 2 with 3 supplements see all Download asset Open asset Cross-reactivity of HAP2 D3 mAbs among Plasmodium species. (A) Reducing SDS 12.5% PAGE of purified HAP2 D3 stained with Coomassie blue. Lanes 1 and 2: P. berghei HAP2 D3 (aa 477–621) purified by Ni-NTA (lane 1) and then treated with Endo D and purified by gel filtration (lane 2). Lanes 3–8: Purified HAP2 D3 fragments from Plasmodium species treated with Endo D or from P. berghei (aa 502–617) with N-linked sites removed by mutation. (B) Titration of antibody binding to Endo D-treated P. berghei HAP2 D3 (aa 477–621) by ELISA. Sigmoidal curve fits show one representative experiment with mean ± difference from the mean of duplicates; EC50 values show mean ± SD of three experiments. (C) Cross-species reactivity of HAP2 D3 mAbs by immunoprecipitation. Purified D3 proteins shown in (A) (2 µg each) were subjected to immunoprecipitation with the indicated mAbs, anti-His 1/5.13, or control IgG (8 µg each). Immunocomplexes were analyzed by reducing 12.5% SDS-PAGE and Western blot using rabbit polyclonal antibodies to the C-terminal His tag of D3. (D) Quantitation of results from (C) and a repeat experiment. Intensities of bands were quantitated and data normalized to anti-His. Results shown are averages of the two experiments ± difference from the mean. (E) EC50 measurements of HAP2 D3 binding to immobilized mAbs by ELISA. Purified D3 proteins shown in (A), lanes 3–8, were used. Sigmoidal curve fitting of D3 titration (Figure 2—figure supplement 3) and EC50 used GraphPad Prism 7 software. EC50 shown are mean ± difference from means of two experiments, each experiment with triplicates. ND, Not determined. To produce monoclonal antibodies (mAbs), mice were immunized with the glycan-shaved D3. Antibodies elicited to the shaved protein, which contains one N-glycan residue at each N-glycosylation site, would be expected to react with D3 without being influenced by its glycosylation status. Five IgG mAbs reacted with the immunogen with nanomolar EC50 values in ELISA (Figure 2B). Surface plasmon resonance (SPR) showed that Fabs of two mAbs, 2/6.14 and 2/1.12, bind with affinities of 5–10 nM (Figure 2—figure supplement 1). The antibodies also immunoprecipitated a shorter D3 (502–617) with all putative N-glycosylation sites mutated out (Figure 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 Materials and methods (Figure 2—figure supplement 2). HAP2 D3 was also expressed and shaved with Endo D from Plasmodium species that can infect humans: P. knowlesi, P. vivax, P. malariae, P. ovale, and P. falciparum (Figure 2A). As their D3 domains share 60–70% amino acid sequence identity with P. berghei, we tested cross-reactivity with the mAbs to P. berghei D3 using immunoprecipitation (Figure 2C). We quantified immunoprecipitation (Figure 2D) and also measured cross-reactivity using ELISA (Figure 2E and Figure 2—figure supplement 3). mAbs 2/6.14 and 2/1.12 were especially efficient at recognizing D3 from multiple species (Figure 2C). mAb 2/6.14 pulled down about 70%, 30%, and 20% of P. knowlesi, P. ovale, and P. malariae D3, respectively, and mAb 2/1.12 was even more reactive, pulling down 60–100% of D3 from all species except P. falciparum D3 (Figure 2C and D). In contrast, mAb 2/1.40 showed strong reactivity to P. ovale, but not to D3 of other species, while mAbs 2/3.3 and 2/4.36 were highly specific for P. berghei (Figure 2C and D). mAbs 2/6.14 and 2/1.12 bound all species of D3 tested, as shown by ELISA EC50 values in the 1–1000 nM range (Figure 2E and Figure 2—figure supplement 3). Strong binding of P. knowlesi D3 to both 2/6.14 and 2/1.12 was observed with EC50 of <10 nM. 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 (Figure 3A). Figure 3 with 1 supplement see all Download asset Open asset Immunofluorescent staining and inhibition of P. berghei fertilizaton by mAbs. (A) Immunofluorescent staining of microgametes from P. berghei. Fixed microgametes were stained with mouse D3 mAbs or control IgG, rabbit anti-α-tubulin, secondary Alexa Fluor-488 anti-mouse IgG and Alexa Fluor-594 anti-rabbit IgG, DAPI, and imaged with epifluorescence. Scale bars = 5 µm. (B) Fertilization in vitro measured as macrogamete conversion to ookinetes (% ookinetes/(ookinetes + macrogametes)) in the presence of indicated antibodies. Results are mean ± SEM of three independent experiments, analyzed by paired t test: *** p < 0.001, **p < 0.01, ns, non-significant p > 0.05. Total number of macrogametes + ookinetes in all three experiments are from right to left at 500 µg/ml: 139, 177, 147,104, 199 and 129, and at 250 µg/ml: 222, 120, 270, 410, 275, and 306. (C) ( and D) Mosquitoes were allowed to feed on antibody or control IgG diluted in infected mouse blood placed in membrane feeders; results are shown from three independent experiments. (C) Oocyst intensities (oocysts per mosquito) are shown as filled circles with means as horizontal bars ± SEM. ****p < 0.0001, ***p = 0.0001–0.001, **p = 0.001–0.01, *p = 0.01–0.05, ns p > 0.05 by Mann-Whitney test. (D) Summary of intensity and prevalence (% of infected mosquitoes) with SEM from all three experiments. Prevalence and N values are shown in Figure 3—figure supplement 1. Inhibition (%) was calculated relative to negative control IgG at the same concentrations. The significance of inhibition is shown in parentheses for intensity (Mann-Whitney test) and prevalence (Fisher's exact test). 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 hr, 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% (Figure 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 (Figure 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, that is, oocyst intensity was determined. Oocyst intensity was significantly reduced by mAb 2/6.14, particularly at 250 and 100 µg/ml (Figure 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 (Figure 3—figure supplement 1). 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 (Figure 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 1). 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 (Figure 4A and B). Table 1 Statistics of X-ray diffraction and structure refinement of PbHAP2 domain 3 (D3) complexed with 2/6.14 Fab or 2/1.12 Fab. D3-2/6.14 FabD3-2/1.12 FabData collection statistics Space groupP222P21α, β, γ, °90, 90, 9090, 95.8, 90Unit cell (a, b, c), Å78.3, 122.6, 168.543.9, 187.1, 74.4Resolution range (Å)50.0–2.80 (2.87-2.80)*50.0–2.10 (2.15–2.10)Completeness (%)99.3 (99.5)98.4 (96.9)Number unique reflections40,741 (2,938)68,138 (4,935)Redundancy3.8 (4.0)3.6 (3.7)Rmerge (%)†5.7 (70.2)8.3 (153)I/σ(I)12.8 (2.2)8.42 (0.52)CC½ (%) ‡99.8 (90.9)99.7 (10.9)Wavelength (Å)1.03321.0332Refinement statistics Rwork (%) §25.3 (33.7)18.97 (37.3)Rfree (%)29.2 (34.8)23.27 (40.5)Bond RMSD (Å)0.0030.003Angle RMSD (°)0.5480.603Ramachandran plot ¶(Favored/allowed/outlier)91.5/7.6/0.995.93/4.07/0.1Number of atoms Protein10,3108,183 Ligand2254 Water18353B factor Protein128.672.7 Ligand112.8112.9 Water138.064.9Molprobity percentile (Clash/Geometry)98/9799/97 PDB7LR3 7LR4 * The numbers in parentheses refer to the highest resolution shell. † Rmerge = Σh Σi |Ii(h) -< I(h)> | / ΣhΣi Ii(h), where Ii(h) and< I(h)> are the ith and mean measurement of the intensity of reflection h. ‡ Pearson's correlation coefficient between average intensities of random half-datasets for each unique reflection (Karplus and Diederichs, 2012). § Rfactor = Σh||Fobs (h)|-|Fcalc (h)|| / Σh|Fobs (h)|, where Fobs (h) and F calc (h) are the observed and calculated structure factors, respectively. No I/σ(I) cutoff was applied. ¶ Calculated with MolProbity (Davis et al., 2007). Figure 4 with 2 supplements see all Download asset Open asset Structures of HAP2 D3 in complex with 2/6.14 Fab or 2/1.12 Fab. (A) Overview of D3-2/1.12 Fab and D3-2/6.14 Fab complexes (B) of D3 with residues that Fab 2/1.12 Fab 2/6.14 or both Fabs (C) of three independent in the two the one with multiple Each is in a different of loops with (D) and (E) of complexes are shown in in which the Fab and D3 are like two in an In D and atoms in D3 and Fabs within 5 Å of one are green 2/1.12 or 2/6.14 and are otherwise blue or Structural and sequence of D3 and its 2/1.12 and 2/6.14 shown as RMSD of among the two independent Fab 2/1.12 D3 complexes and the Fab complex with a single calculated structure by et al., where residues were in or 1 of the three circles show Fab for 2/1.12 Fab and for Fab residues that mediate ( > of surface or or ( < are shown with or respectively. show D3 sequence in P. berghei, P. knowlesi, P. vivax, P. malariae, P. ovale, and P. falciparum with to P. berghei shown as P. berghei residues in are and were mutated to N-linked in were not in any of the three P. berghei residues with in the that in the and loops are are in and and of D3 with the 2/1.12 Fab and 2/6.14 Fab D3 is Fab and are blue and respectively, and residues with with Fabs as in have green or show and The negative stain class averages of the HAP2 ectodomain (residues and in complex with the 2/6.14 Fab 3 the from the complex crystal 4 a of the crystal structure in the same D3 Fab and blue and respectively, and of D3 shown as a D3 contains to that are arranged into two containing the and (Figure and These through hydrophobic faces to form a The in which the sequence folds into this as a The D3 to one through loops at each of the The and loops within a and the and loops the two among the four examples of D3 show different for the loops at the C-terminal of D3, which the that to the plasma membrane (Figure Quantitation of in these and mean values or of of residues in Figure In contrast, the loops at the that D1 show in all independent These and show RMSD values (Figure with the presence of or that each that the in these and in the loop and in the loop in Figure are in the species. The two antibodies bind to opposite faces of D3, with 2/1.12 binding to the and 2/6.14 binding to the are in green and respectively in Figure The of the are shown in in which the Fab and D3 are like two in an (Figure and In each both the and domains significantly to the with D3. The with ( > of surface or are shown as circles over the in Figure and shown in in Figure and the 2/1.12 Fab the over the of this to also the D where with the 2/6.14 (Figure and Fab 2/1.12 has the with four residues in and of D3 (Figure The of from D3 and from form with the of from 1 In in a to the of in while the to the of in and between D3 residue and residue (Figure All four of these residues are among the species examined (Figure with cross-reactivity of mAb mAb 2/6.14 residues in each of the D3 the are formed by in D and residues in the loop (Figure and The loop a on the D3 surface that fits into a in the Fab between the three loops (Figure The to the of in and a with in All three Fab loops the D3 loop with each forming at least one of a of four of two and two loop residue and residue between and of and between and of the with the loop (Figure D3 contains all of which are among Plasmodium however, four of these form and in the loop a to in the that the residues between and the of the which are highly and in the and in the F form a to these two in the of the Two D3 are in hydrophobic are present in extracellular proteins. is well in the hydrophobic is in D3-Fab complexes but in the ectodomain may be by residues at the interface between D1 and D3, residues to D3 residue which were not present in the construct (Figure and B). Figure 5 Download asset Open asset Structural of D3 of HAP2 (A) P. berghei HAP2 D3 from the Fab 2/1.12 complex showing the and (B) of HAP2 D3 from the P. berghei 2/6.14 Fab complex and trimeric fusion of Chlamydomonas and Arabidopsis Structures were using et al., (C) sequence from the shown in and RMSD of are shown the and filled circles the P. berghei sequence show Fab 2/1.12 and Fab 2/6.14 respectively as in Figure In the P. berghei the three residues in residues that were mutated to N-linked and blue filled and circles Arabidopsis and Chlamydomonas residues in trimer with or a or <10 respectively. surface was calculated with and 2007). (D) present in an N-glycosylation in the P. berghei to the of the and a function that is in HAP2 in other The is the same as in except P. berghei is in (E) mAb and or to which are shown in with or with Fabs as in Figure are shown with both and or respectively, and to the Figure and the in both epitopes are in HAP2 D3 in P. berghei is and in sequence to and HAP2 D3, however, their structures are highly (Figure with RMSD (Figure The that the and F is also (Figure and P. berghei but
Source: eLife
Language: English
Topic: Malaria Research and Control
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4210618366