We selected donor A, a 69-year-old Dutch expatriate who resided in Central Africa for approximately 30 years and whose serum was shown to strongly reduce Pf transmission, to isolate sexual stage-specific mAbs. We previously demonstrated the serum of this donor, donor A, largely retained its TRA when depleted of antibodies directed against the main transmission-blocking epitopes of Pfs48/45 and Pfs230 (Stone et al., 2018), suggesting the presence of antibodies targeting other epitopes on these two proteins or directed at other proteins also involved in transmission.

PBMCs from the donor were thawed and a total of 1496 IgG+ memory B cells were sorted in 384-well plates (Figure 1) (Figure 1—figure supplement 1A). After activation, single-cell culture supernatants potentially containing secreted IgGs were screened in a high-throughput 384-well ELISA for their reactivity against a crude Pf gamete lysate (Figure 1—figure supplement 1B). A subset of supernatants was also screened against gametocyte lysate (Figure 1—figure supplement 1C). In total, supernatants from 84 wells reacted with gamete and/or gametocyte lysate proteins, representing 5.6% of the total memory B cells. Of the 21 supernatants that were screened against both gamete and gametocyte lysates, six recognized both, while nine appeared to recognize exclusively gamete proteins, and six exclusively gametocyte proteins.

To isolate the corresponding mAbs, single B cell lysates from the 84 ELISA-positive wells were subjected to single B cell reverse transcriptase PCR (RT-PCR) for amplification of immunoglobulin variable genes. We obtained and cloned heavy and light chain sequences for 11 out of 84 wells. For three wells we obtained a kappa light chain sequence and for five wells a lambda light chain sequence. For three wells we obtained both a lambda and kappa light chain sequence suggesting that either both chains were present in a single B cell or that two B cells were present in the well. For all 14 wells we retrieved a single heavy chain sequence. Following amplification and cloning, 14 mAbs were expressed as full human IgG1s (Supplementary files 1 and 2).

The 14 mAbs were first tested for binding to Pf sexual stage surface antigens in a surface immunofluorescence assay (SIFA) using wild-type female gametes (Figure 2A). The mAbs were also tested for binding to Pfs48/45 knockout female gametes, which lack surface-bound Pfs48/45 and Pfs230 (Eksi et al., 2006; Stone et al., 2018) Seven mAbs exhibited binding to approximately half or more of gametes when tested at a concentration of 100 µg/mL. Among these, four mAbs, B1C5K, B1C5L, B2C10L, and B2E9L, recognized wild-type gametes with high scores (>68%) even at concentrations as low as 1 µg/mL. The binding of the B1C5K, B1C5L, B2C10L, and B2E9L mAbs strongly decreased when using gametes that lacked surface-expressed Pfs48/45 and Pfs230, indicating that these four mAbs likely targeted one of these two antigens. Three other mAbs, B2D10L, B1C8L, and B1E7K, displayed a similar recognition profile, albeit with notably smaller percentages of labeled wild-type gametes, particularly at the lower concentrations tested. This suggested a potential low-affinity recognition of either Pfs48/45 or Pfs230 for these latter three mAbs.

Six of the remaining seven mAbs, B1C8K, B1D3L, B1D3K, B1F9K, B1C3L, and B2F7L, exhibited very weak or no binding to gametes. For B1C8K, this showed that the light chain (kappa) did not correspond to the antibody that was originally selected in the screening process as the lambda version (B1C8L) exhibited strong binding. As for the other mAbs, the results indicated that they may be specific for proteins not expressed or only poorly expressed at the gamete surface.

Finally, one mAb, B1E11K, exhibited a distinctive gamete surface binding profile, recognizing only a fraction (approximately a third to a fifth) of the wild-type and Pfs48/45 knockout gametes across all tested concentrations, suggesting potential binding to non-Pfs48/45 and Pfs230 proteins.

We were interested in investigating potential TRA for all identified mAbs. To do this, a standard membrane feeding assay standard membrane feeding assay (SMFA) was conducted in the presence of the isolated mAbs, revealing a range of TRAs (Figure 2B; Figure 2—source data 1). Overall, seven mAbs were confirmed to strongly reduce transmission (TRA>80%) when tested at 500 µg/ml: B1C5K, B1C5L, B2C10L, B2E9L, B1C8L, B1D3L, and B1F9K. Of those, two mAbs, B1C8L and B2C10L, retained >50% TRA at a lower concentration (100 µg/ml). Notably, despite not showing gamete surface recognition, B1F9K and B1D3L displayed TRA – although only at high concentrations. Conversely, three mAbs recognizing the gamete surface, B2D10L, B1E7K, and B1E11K, showed no activity in SMFA (Standard membrane feeding assay).

To gain deeper insight into the specificity of the various isolated mAbs, regardless of their TRA, we conducted further characterization via western blot analyses using gametocyte extracts (Figure 2C). The B1C5K and B1C5L mAbs recognized a protein with a molecular weight matching that of Pfs48/45. This result was consistent with the findings from the gamete surface binding experiment in which these mAbs recognized wild-type gametes but not Pfs48/45 knockout gametes, providing further validation of the specificity of the B1C5K and B1C5L mAbs in targeting Pfs48/45. The B2C10L mAb displayed pattern of recognition that corresponded to Pfs230, similar to the anti-Pfs230 control mAb RUPA-96 (Ivanochko et al., 2023). Once again, these results were in agreement with the findings from the gamete surface binding experiment confirming the specificity of the B2C10L mAb in targeting Pfs230. The B1E11K mAb also appeared to bind to Pfs230 on the western blot. However, in contrast to the RUPA-96 mAb, B1E11K only recognized the higher molecular band corresponding to Pfs230, suggesting exclusive recognition of the unprocessed form of this protein (Brooks and Williamson, 2000). Interestingly, besides Pfs230, B1E11K also recognized several unidentified proteins ranging from 70 kDa and 250 kDa with various intensities. These findings were consistent with the gamete binding assay that showed recognition of gametes lacking Pfs48/45 and Pfs230, suggesting potential recognition of proteins other than Pfs48/45 and Pfs230. Finally, all the other mAbs showed no clear binding to any protein from the gametocyte extract on the western blot. In the case of those demonstrating binding to gamete surfaces, B2E9L, B2D10L, B1C8L, B1E7K, this may be attributed to their recognition of conformational epitopes that are lost during western blotting preparation, or possibly of specific recognition of proteins expressed in gametes but not gametocytes.

The recognition of Pfs48/45 by the B1C5K and B1C5L mAbs was subsequently confirmed through an ELISA using full-length recombinant Pfs48/45 (Figure 2D). Notably, none of the other mAbs of the panel displayed binding to this Pfs48/45 recombinant protein construct. To further pinpoint the domain targeted by B1C5K and B1C5L, an ELISA was performed using constructs corresponding to domains 1–2, 2–3, and 3 revealing that these two mAbs targeted domain 2 of Pfs48/45 (Figure 2E). This is in agreement with prior work indicating mAbs to domain 2 of Pfs48/45 are generally mAbs with low potency (Fabra-García et al., 2023).

The combined findings above strongly pointed to Pfs230 as the target of mAb B2C10L. We thus tested this mAb in ELISA for binding to Pfs230CMB, a construct containing Pfs230 domain 1 and part of the pro-domain (Farrance et al., 2011). No reactivity was observed (Figure 2F) suggesting that B2C10L may recognize other Pfs230 domains than the one tested, or recognize epitopes not properly displayed in the construct used.

In summary, our target-agnostic mAb isolation approach successfully identified mAbs against Pf sexual stage proteins, some of which exhibited TRA and some of which target Pfs48/45 or Pfs230. However, given that the mAbs isolated in this study showed substantially lower TRA than mAbs identified previously (Kundu et al., 2018; Coelho et al., 2021), we elected not to investigate them further. Instead, we were intrigued by the binding properties of B1E11K, which showed cross-reactivity with various Pf proteins, including Pfs230. Such cross-reactivity has been shown as a hallmark of the human antibody response to Pf and explored at the serum level but to our knowledge has never been studied at the mAb level (Hou et al., 2020; Raghavan et al., 2023). Thus, we rationalized a more detailed molecular characterization of this mAb may provide insights into this relatively unexplored phenomenon.

First, to ensure the ability of B1E11K to recognize different proteins in western blotting experiments was not due to polyreactivity, the mAb was tested in ELISA against a panel of human proteins, single-stranded DNA (ssDNA) and lipopolysaccharide (LPS). The 4E10 mAb, a well-known anti-HIV gp41 polyreactive mAb (Cardoso et al., 2005), was used as a positive control. The B1E11K mAb did not bind any of the antigens on the panel at any significant level, even at a high 50 µg/ml concentration, and therefore polyreactivity was ruled out (Figure 3—figure supplement 1A).

To identify antigens recognized by B1E11K, immunoprecipitation experiments were conducted using gametocyte extract. Proteins of different molecular weights were specifically detected in the B1E11K immunoprecipitate but not when using the anti-HIV control mAb (Figure 3—figure supplement 1B). Mass spectrometry analysis of the corresponding gel slices revealed recognition of Pfs230, confirming the western blot results.

The specificity of B1E11K was further tested using a protein microarray featuring recombinant proteins corresponding to putative antigens expressed at the sexual stage as well as proteins expressed at different stages of the Pf life cycle (Stone et al., 2018). The results showed that B1E11K exhibited high level reactivity (>8-fold higher than the negative control, minimum signal intensity rank 15th of 943 array targets) against several antigens, some expressed at the sexual stage (i.e. Pf11.1), others at the asexual stage (i.e. LSA3, RESA, RESA3) (Figure 3A; Figure 3—source data 1). Analysis of the primary amino acid sequence of the antigens recognized in the array suggested homology in several cases, based on the presence of glutamate-rich regions (Figure 3—figure supplement 2). To analyze the numerous repeated motifs contained in these proteins, we used the RADAR (Rapid Automatic Detection and Alignment of Repeats) software (Heger and Holm, 2000). Although B1E11K recognition of Pfs230 fragments on the array was lower than our cutoff for further analysis (3.4-fold higher than the negative control, maximum signal intensity rank 30th of 943 array targets), its sequence was also analyzed using RADAR due to its recognition by B1E11K in the immunoprecipitation experiments (Figure 3—figure supplements 3 and 4). The analysis showed Pfs230 and several of the proteins recognized by B1E11K on the array contained diverse patterns of glutamate-rich repeats of different lengths and compositions. Among these proteins, Pfs230, Pf11.1, RESA, RESA3, and LSA3 presented the most similar glutamate repeats, following an 'EE-XX-EE' pattern (Figure 3—figure supplements 3 and 4). Pfs230 contains adjacent EE-VG-EE repeats which are located in the domain of the protein which is cleaved upon gametocyte egress from erythrocytes (Williamson et al., 1996). RESA and RESA3 contain 20 and 9 EE-NV-EE overlapping repeats at the C-terminus of the protein, respectively. LSA3 contains two overlapping EE-NV-EE repeats. Finally, 221 non-adjacent EE-LV-EE repeats span the whole Pf11.1 megadalton protein.

To verify the recognition of the aforementioned proteins, a western blot was performed with recombinant forms of RESA, RESA3, LSA3, and of a Pf11.1 domain (de Jong, 2023; Figure 3B). Domain 1 of Pfs230, which does not contain the EE-VG-EE repeats, was also included. The results confirmed the binding of B1E11K to all the proteins tested except for Pfs230D1, as expected. Overall, the data showed that the B1E11K mAb recognizes various Pf proteins from different stages, all containing glutamate-rich repeats.

To validate the B1E11K mAb specifically targets glutamate-rich repeats, we synthesized five biotinylated peptides derived from the various repeats found in the proteins identified above to test for binding in sandwich ELISA experiments (Figure 3C). Overall, the B1E11K mAb bound to all peptides (Figure 3D). However, it exhibited a higher specificity for the RESA-derived peptides with an EC₅₀ at least 100 times greater compared to EC₅₀ values obtained with the other glutamate-rich peptides. Altogether, this suggests the main antigenic targets of B1E11K are RESA and RESA3, which contain the EENV repeats.

Since B1E11K bound to RESA-based peptides the strongest, we synthesized shorter RESA peptides for a more precise determination of the B1E11K minimal sequence epitope (Figure 4A). When tested on the RESA peptide panel, B1E11K mAb binding to RESA P2 (16AA) and RESA 14AA, 12AA, and 10AA was similar, all exhibiting close EC₅₀ values (Figure 4B; Figure 4—figure supplement 1). No binding was observed for the 8AA RESA peptide, suggesting the 10AA peptide contained the minimal epitope. We hypothesized the similar EC₅₀ values may be a result of avidity effects and thus, we performed the same experiment with recombinant B1E11K Fab (Figure 4C). Although B1E11K Fab bound both RESA P2 (16AA) and RESA 14AA peptides with comparable strength of binding, both RESA 12AA and RESA 10AA peptides displayed comparatively poorer EC₅₀ values with the RESA 10AA peptide displaying the lowest detectable binding strength. The binding affinity and kinetics of the interaction was also determined through biolayer interferometry (BLI). We performed experiments using the minimal sequence required for binding determined through ELISA (RESA 10AA peptide and the RESA P2 peptide [16AA]). When immobilizing the peptide to the sensors, an approximately six-fold difference in affinity between the 10AA peptide (K_D = 484 nM) (Figure 4D) and the P2 peptide (16AA) (K_D = 74 nM) (Figure 4E) was observed.

Given the repetitive nature of the antigenic targets of B1E11K and differences in binding events captured in our BLI experiments, we hypothesized that more than one B1E11K Fab could potentially bind to the longer, RESA P2 (16AA) peptide. Thus, we performed isothermal titration calorimetry (ITC) using the same two RESA peptides as in the BLI experiments to determine the binding stoichiometries. We observed when titrating the B1E11K Fab into RESA 10AA, a binding stoichiometry of N=1.0 ± 0.2 (Figure 4F; Table 1). When using the RESA P2 (16AA) peptide, a stoichiometry of N=2.1 ± 0.1 was observed (Figure 4G; Table 1). The determined binding affinity from our ITC experiments (Table 1) differed from our BLI experiments (Figure 4D and E), which can occur when measuring antibody-peptide interactions (Kratochvil et al., 2021). Regardless, our data all trend toward the same finding in which a stronger binding affinity is observed toward the longer RESA P2 (16AA) peptide.

To further corroborate our binding stoichiometry findings, we performed size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine the molecular weight of the 2:1 Fab:peptide complex (Figure 4H). We incubated a molar excess Fab:peptide (6:1) sample to saturate all B1E11K Fab binding sites present on the RESA peptide to obtain a solution containing the putative complex and excess monomeric Fab. The resulting chromatogram revealed two species eluted from the column. The molecular weight of the heavier species was in line with what would be expected from a 2:1 Fab:peptide complex (92 kDa) in which the mass determined fell within the range of experimental error. A negative-stain electron microscopy (nsEM) map reconstruction of the 2:1 Fab:peptide complex recovered from the SEC-MALS experiment (Figure 4H) permitted the fitting of two Fab molecules, further supporting the 2:1 binding model (Figure 4I).

To obtain a full structural understanding of the observed repeat cross-reactivity and selectivity for RESA exhibited by B1E11K, we solved a 2.6 Å crystal structure of the B1E11K:RESA P2 (16AA) peptide complex (Figure 5A; Table 2; Supplementary file 3). The electron density at the binding interface is unambiguous and included density for the entirety of the repeat region of the peptide (Figure 5—figure supplement 1). Looking at the binding interface between the two Fabs and peptide reveals the structural basis for cross-reactivity (Table 1; Figure 5—figure supplement 2; Supplementary file 4). The paratope of B1E11K is highly enriched in arginine and histidine residues giving rise to a highly electropositive groove (Figure 5B–D). These residues form a plethora of salt-bridging interactions with the glutamate residue side chains of RESA repeats (Figure 5E). These interactions are supplemented by hydrogen bonding interactions of backbone serine and glycine residues of the B1E11K paratope as well as a hydrogen bond involving W33 found in the heavy chain of Fab B. Multiple hydrogen bonding interactions are made with B1E11K through the side chains of the asparagine residues of RESA repeats (EENV) (Figure 5F) that would not exist in the context of binding to the repeats of Pf11.1 (EELV or EEVIP or EEFIP or EEVVP) or Pfs230 (EEVG) (Figure 3C), as these residues lack side chains that can form hydrogen bonds. This likely leads to the observed higher specificity of B1E11K for RESA repeats demonstrated in our ELISA experiments (Figure 3D).

Additionally, the crystal structure of the antibody-antigen complex revealed the presence of homotypic antibody-antibody contacts through two interfaces surrounding the repeat peptide binding groove (Figure 5G; Figure 5—figure supplement 3; Supplementary file 5). The first interface features a salt-bridging network involving D60 of the Fab B kappa chain forming two salt bridges with R52 and H52A of the Fab A HCDR2 (Figure 5H). Additionally, R54 of the Fab A HCDR2 forms two hydrogen bonds and a salt bridge with the Fab B kappa chain side chains of S55 and E56. Finally, Y58 of the Fab B HCDR2 forms a hydrogen bond with the side chain of S53 of the Fab A HCDR2. The second interface is less extensive featuring two hydrogen bonds between Y32 and S31 of the Fab B HCDR1 and the Fab A KCDR1 T29 and R27, respectively (Figure 5I).

Analysis of the B1E11K sequences with IgBLAST (Ye et al., 2013) reveals that the B1E11K heavy and light chain have high similarity to the IGHV3-7 and IGKV3-20 germline sequences (Figure 5—figure supplement 4). This analysis also indicates multiple residues involved in the homotypic interaction interface have undergone somatic hypermutation. Residues of the CDR2 heavy chain of Fab A, R52 and H52A, and kappa chain CDR1 residues of Fab B, T29 and R27, form various electrostatic and van der Waals interactions which are mutated from the inferred germline sequences (Figure 5F and G; Figure 5—figure supplements 3 and 4). In summary, our biophysical and structural characterization revealed the basis of cross-reactivity and specificity to RESA repeats (EENV) through a binding interface highly electrostatic in nature, featuring affinity-matured homotypic interactions between adjacent antibody molecules when in its antigen-bound state.

Donor A (Stone et al., 2018) had lived in Central Africa for approximately 30 years and reported multiple malaria infections during that period. At the time of sampling PBMCs, in 1994, donor A had recently returned to the Netherlands and visited the hospital with a clinical malaria infection. After providing informed consent, PBMCs were collected, but gametocyte prevalence and density were not recorded.

Plates were prepared the day ahead of sorting and incubated at 37°C. Three-hundred-eighty-four-well cell culture plates (Corning #CLS3570-50EA) were prepared with the appropriate memory B cell stimulation media 1 day before cell sorting, to allow the feeder cells sedimentation at the bottom of the wells. Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco #12440061) was complemented with 1% penicillin-streptomycin (Thermo Fisher Scientific #10378016) and supplemented with 20% FBS (Gibco #16170-078). A cytokine cocktail was added to stimulate MBCs activation, with IL21 (Preprotech #200-21) at 100 ng/mL and IL2 (Preprotech #200-02) at 51 ng/mL. Fibroblasts expressing CD40L 'L cells' (Garrone et al., 1995) were irradiated at 50 Gy and 5000 were added in each well as feeder cells.

Cryopreserved PBMCs were thawed by a brief incubation in a 37°C warm bath and stained with the following Miltenyi REA antibodies before sorting by flow cytometry: anti-CD27 APC (#130-113-636), anti-CD3 VioBlue (#130-114-519), anti-CD19 PE-Vio (#130-113-647), anti-CD20 PE-Vio (#130-111-340), anti-IgM PE (#130-113-476), anti-IgD PE (#130-110-643), and anti-IgA PE (#130-113-476). An Aqua LIVE/DEAD stain was also used (Thermo Fisher Scientific #L34957). Following staining, MBCs were sorted into the 384-well cell culture plates. After an 11-day culture period, supernatants were harvested using a pipetting robot (Eppendorf #5073) and transfered to storage plates (Greiner #788860-906). MBCs were lysed and their mRNA purified using the mRNA TurboCapture kit for 384 wells (QIAGEN #72271). Lysates were stored at –80°C. Lysates from selected wells were further transferred into 96-well RT-PCR plates (Bio-Rad #HSP9641) to perform RT-PCR.

Gamete or gametocyte lysate were prepared as described (Fabra-García et al., 2023). 384-well plates (Thermo Fisher Scientific #460372) were coated with 7500 lysed gametes/gametocytes per well. Plates were incubated at 4°C overnight, then wells were washed three times with PBS-Tween 0.05%, prior to 1 hr blocking (with a 1% BSA-1% PBS-Tween 0.05% solution). Cell culture supernatants (diluted twofold in blocking solution) were dispensed into the wells for a 1 hr incubation step. Following washing, anti-human IgG AP antibody (Thermo Fisher Scientific #A18814) diluted at 1/2000 was added and incubated for 1 hr. Plates were then washed and 15 µl of CDP-substrate (Thermo Fisher Scientific #T2146) was added. The reaction was measured using Biotek Synergy 2 reader. Positivity threshold was determined as the average background OD + (3× SD (background OD)). 0.3 µg/mL TB31F (anti-Pfs48/45 mAb) and 1.0 µg/mL 2544 (anti-Pfs25 mAb) were used as positive controls, while 30.0 µg/mL 399 (anti-CSP mAb) was used as negative control.

Nested multiplexed PCRs were performed on single MBCs from selected wells following the protocol outlined by Tiller et al., 2008. PCR products were sent for sequencing (Genewiz) and sequences analyzed for Ig gene features using the IMGT (ImMunoGeneTics) database (Lefranc et al., 2015). Ig gene family-specific primers were used for cloning, as described by Tiller et al., 2008. Purified PCR products were cloned into vectors encoding for either IgG1 lambda, kappa, or heavy constant regions. For transient mAb expression and secretion, HEK293F cells were co-transfected with plasmids coding the antibody heavy chain and the corresponding light chain using 293Fectin (#12347500). A protein A-Sepharose column (Sigma #ge17-1279-03) was used for mAb purification. Elution of mAbs was conducted with 4.5 mL of glycine 0.1 M (pH 2.5) and 500 µl of Tris 1 M (pH 9). The purified mAbs were subsequently subjected to buffer exchange and concentration with AmiconUltra (Merck #36100101).

The DNA sequences of VK and VH of the B1E11K Fab domain were cloned upstream of human Igκ and Igγ1-CH1 domains respectively and inserted into a custom pcDNA3.4 expression vector. The plasmids were co-transfected into FreeStyle 293 F cells that were cultured in FreeStyle 293 Expression Media (Gibco #12338018) using Fectopro (Polyplus, 101000014). The recombinant Fab was purified via KappaSelect affinity chromatography (Cytiva #17545812) and cation exchange chromatography (MonoS, Cytiva 17516801).

Gamete SIFA was performed with Pf NF54 wild-type and Pfs48/45 knockout (Dijk et al., 2001) strains. The Pfs48/45 knockout lacks both surface-bound Pfs48/45 and Pfs230 (Stone et al., 2018; Eksi et al., 2006; Kumar, 1987). Wild-type or Pfs48/45 knockout gametes were obtained following gametocyte activation in FBS for 1 hr at room temperature. Gametes were washed with PBS and incubated with mAbs diluted in PBS containing 0.5% PBS and 0.05% NaN₃ (SIFA buffer) for 1 hr at 4°C in sterile V bottom plates (VWR#736-0223). After incubation, wells were washed three times with SIFA buffer and secondary antibody Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) (Invitrogen#A11001) diluted 1/200 added for a 1 hr incubation step on ice. Following a washing step, gametes were suspended in 4% paraformaldehyde and transferred into 384-well clear bottom black plates. Four images per well were taken using the ImageXpress Pico Cell Imaging System (Molecular Devices).

For western blots with gametocyte extract, Pf NF54 gametocyte extract was prepared as described above. The extract was mixed with NuPAGE LDS sample buffer (Thermo Fisher Scientific # NP0008) and heated for 15 min at 56°C. The equivalent of 1 million lysed gametocytes was loaded per lane. A NuPAGE 4–12% Bis-Tris 2D-well gel (Thermo Fisher Scientific #NP0326BOX) was used for proteins separation. Using the Trans-Blot Turbo system (Bio-Rad #1704150) samples were then transferred to a 0.22 µm nitrocellulose membrane (Bio-Rad #1620150). The blots were cut into strips, blocked with 5% skimmed milk in PBS and incubated with 5 µg/mL of the mAb to be tested. Strips were incubated with the secondary anti-human IgG-HRP antibody (Pierce #31412), diluted 1/5000 in PBS-T. Clarity Western ECL substrate (Bio-Rad #1705060) was used for development and strips were imaged with the ImageQuant LAS4000 equipment (GE HealthCare).

For western blot with recombinant proteins, we used Pfs230CMB (amino acids 444–730) expressed in a plant-based transient expression system (Farrance et al., 2011), and RESA3 (amino acids 570–1090), RESA (amino acids 66–585), LSA3 (amino acids 805–1558), and Pf11.1 (amino acids 3657–3734) that were expressed wheat germ cell free extract (https://repository.ubn.ru.nl/bitstream/handle/2066/289602/289602.pdf?sequence=1). All antigens contained a C-terminal His-tag. RESA and LSA3 also had an N-terminal GST-tag. The equivalent of 150 ng of protein was loaded per lane. An SDS 4–20% gel (Bio-Rad # 4561094) was used for protein separation under non-reducing conditions. Further steps were performed following the protocol described above.

ELISAs with full-length Pfs48/45 and fragments thereof, and Pfs230CMB were performed as previously described (Fabra-García et al., 2023; Farrance et al., 2011). In short, Nunc MaxiSorp 96-wells plates (Thermo Fisher) were coated with 0.5 µg/mL recombinant Pfs48/45 or Pfs230CMB proteins, blocked with 5% skimmed milk in PBS+0.1% Tween-20 and washed. Plates were then incubated with 10 µg/mL or 30 µg/mL mAb in 1% skimmed milk in PBS. After washing, plates were incubated with 1:60,000 Goat Anti-Human IgG/HRP-conjugated antibody (Pierce, #31412) in 1% skimmed milk in PBS+0.1% Tween-20. After washing, plates were developed with 3,3′,5,5′-tetramethylbenzidine and the reaction was stopped with H₂SO₄. Absorbance was measured at 450 nm. mAbs were considered positive when the absorbance was higher than the mean absorbance plus three standard deviations of seven negative mAbs.

The coating antigens were diluted to 1 µg/mL. Antigens used were: ssDNA (Sigma #D8899-5MG), disialoganglioside GD1α (Sigma #G2392-1MG), LPS (Sigma #L2630-10MG), transferrin (Sigma #T3309-100MG), apotransferrin (Sigma #T1147-100MG), hemocyanin (Sigma #H7017-20MG), insulin (Sigma #I2643-25MG), cardiolipin (Sigma #C0563-10MG), albumin and histone (Sigma #H9250-100MG). Secondary antibody used was a phosphatase-coupled goat anti-human IgG (Jackson ImmunoResearch #109 056 098). Optical densities were read at 405 nm, 1 hr after the addition of pNPP.

SMFA experiments were performed using Pf NF54 wild-type gametocytes with oocyst count readout, following a protocol set up by Ponnudurai et al., 1989. Briefly, blood meals containing cultured gametocytes mixed with antibodies were fed to A. stephensi mosquitoes (Nijmegen colony). For each condition, 20 fully fed mosquitoes were analyzed. Reported antibody concentrations are concentrations in the total blood meal volume. mAbs that showed >80% TRA, i.e., reduction in oocysts compared to a negative control, were tested in a second independent SMFA experiment. TRA from one or two independent SMFA experiments was calculated using a negative binomial regression model as previously described (de Jong et al., 2022b). SMFA data analyses were done in R (version 4.1.2).

Microarray design and protocol have been extensively detailed in Stone et al., 2018, and de Jong et al., 2022a. Briefly, the selection of proteins to be printed on the array was made on the basis of a systematic analyses of proteomic data by Meerstein-Kessel et al., 2018. In total, 943 protein targets representing 528 unique gene IDs were expressed for the array using an in vitro transcription translation system; these were printed onto nitrocellulose-coated slides at the University of California, Irvine, as described previously (de Jong et al., 2022a). Microarray slides were rehydrated in blocking buffer (GVS #10485356) while B1E11K was diluted 1:100 in a 20% Escherichia coli lysate/blocking buffer solution and incubated for 30 min. Blocking buffer was discarded and diluted B1E11K added to the array slides for incubation overnight at 4°C with continual rocking. Following three washes with TBS-Tween-20 0.05%, slides were probed with a fluorophore-conjugated secondary antibody (Southern Biotech, Goat Anti-Human IgG-TXRD) at a concentration of 0.5 µg/mL (1:2000) in 2% E. coli lysate/blocking buffer solution. After three washes, slides were removed from their casettes and rinsed in ddH₂O air dried in a centrifuge and scanned using a GenePix 4300A High-resolution Microarray Scanner (Molecular Devices). Data treatment and analysis were performed using R (Staples, 2023). Correction for local array target spot background was done using the ‘backgroundCorrect’ function of the limma package (Ritchie et al., 2015). Background corrected values were log2 transformed and normalized to systematic effects by subtraction of the median signal intensity of the negative IVTT controls (internally within four subarrays per sample). The final normalized data are a log2 MFI ratio of target to control reactivity: a value of 0 represents equality with the vehicle control, and a value of 1 indicates a signal twice as high.

Briefly, we used tosyl-activated beads (Invitrogen #14203) to covalently link B1E11K and incubated these beads with a gametocyte lysate to enable antigen capture. Immunoprecipitated antigens were eluted, and the elution fraction was run on an SDS-PAGE gel and silver-stained. A negative control immunoprecipitation experiment was performed using an anti-HIV gp120 mAb. As shown in sup Figure 3—figure supplement 1B, two bands with a molecular weight greater than 250 kDa, and a third one with a molecular weight around 55 kDa were specifically detected in the B1E11K immunoprecipitate, in comparison to the control antibody. The three bands were cut and sent for mass spectrometry analysis. Data were analyzed by querying the entire proteome of Pf (NF54 isolate) in the UniProt database.

Peptides were produced by P. Verdié team, IBMM – SynBio3, Montpellier, France. Lyophilized peptides were solubilized in PBS.

ELISA protocol was similar to the protocol described above. Briefly, 96-well ELISA plates were coated overnight at 4°C with 0.5 µg/mL of streptavidin (Thermo Fisher Scientific # 434301). Plates were washed and then blocked for 1 hr. Following wash, peptides diluted at 0.5 µg/mL were added to the plates for a 1 hr incubation. The plates were washed and serially diluted mAbs were added. mAb fixation was detected using phosphatase-coupled goat anti-human IgG (Jackson ImmunoResearch #109 056 098) and para-nitrophenylphosphate (Interchim #UP 664791). The enzymatic reaction was measured at 405 nm using a TECAN Spark 10M plate reader. Half maximal effective concentration (EC50) was calculated from raw data (OD) after normalization using GraphPad Prism (version 9) 'log agonist versus normalized response' function.

All BLI experiments were performed using an Octet Red96e Instrument (PallForteBio), at 25°C and under 1000 rpm agitation. SAX biosensors (Sartorius #18-5117) were pre-wetted in BLI Buffer (PBS [pH 7.4]+0.01% [wt/vol] BSA +0.002% [vol/vo] Tween-20) for 10 min. Biotinylated peptides were loaded onto the biosensors until the top concentration of B1E11K Fab utilized in kinetic assays (2500 nM for RESA 10AA peptide and 625 nM for RESA P2 peptide) yielded a response value of ~1.2 nm. An association step was conducted by dipping the sensors into a titration series of ½ serially diluted B1E11K Fab for 30 s. The dissociation step was conducted by dipping the biosensors into BLI Buffer for 1200s. Background subtractions were done using measurements where experiments were performed with biosensors treated in the same conditions but replacing Fab solution with BLI Buffer. Kinetic data were processed using the manufacturer’s software (Data Analysis HT v11.1).

An Auto-ITC200 (Malvern) was used to conduct calorimetric experiments. The RESA P2 peptide, RESA 10AA peptide, and recombinant B1E11K Fab were buffer-exchanged into Tris-buffered saline (20 mM Tris pH 7.0, and 150 mM NaCl). The B1E11K Fab was concentrated at 90–110 μM for experiments utilizing the RESA P2 peptide and 60–70 μM for those utilizing the RESA 10AA peptide. The RESA P2 peptide and RESA 10AA peptide were concentrated at 5–6 μM and 7–10 μM respectively. Fab (syringe) was titrated into the cell (peptide) at 25°C using a protocol involving 19 injections each at a volume of 2.0 μl. The curves were fitted to a 2:1 or 1:1 binding model using the MicroCal ITC Origin 7.0 Analysis Software.

SEC-MALS experiments were performed at 4°C using a Superdex 200 Increase 10/300 GL (Cytiva #GE17-5175-01) column. RESA P2 peptide was incubated with B1E11K Fab at a 1:6 molar ratio for 30 min prior to loading onto the Superdex 200 column. The column was set up onto an Agilent Technologies 1260 Infinity II HPLC coupled with a MiniDawn Treos MALS detector (Wyatt), Quasielastic light scattering (QELS) detector (Wyatt), and Optilab T-reX refractive index (RI) detector (Wyatt). Data processing was performed using the ASTRA software (Wyatt).

Fractions of the first peak of the SEC-MALS experiments containing the 2:1 B1E11K:RESA P2 complex were used to make nsEM grids. 50 µg/mL of the complex was deposited onto homemade carbon film-coated grids (previously glow-discharged in air for 15 s) and stained with 2% uranyl formate. Data was collected onto a Hitachi HT7800 microscope paired with an EMSIS Xarosa 20 Megapixel CMOS camera. Micrographs were taken with the microscope operating at 120 kV at ×80,000 magnification with a pixel size of 1.83 Å/px. Image processing, particle picking, extractions, 2D classifications, and 3D reconstructions were done in cryoSPARC v2 (Punjani et al., 2017).

The RESA P2 peptide was incubated with B1E11K Fab at a 1:5 molar ratio for 30 min prior to loading onto a Superdex 200 column Increase 10/300 GL column. Fractions containing the complex were pooled and concentrated at 8.6 mg/mL. A seed stock prepared from a previous crystallization trial was used for seeding. The stock was prepared from condition G9 of a JCSG Top96 screen (0.2 M (NH₄)2SO₄ 25% [wt/vol] PEG4000, and 0.1 M sodium acetate [pH 4.6]). The complex, reservoir solution, and seed stock were mixed at a 3:4:1 volumetric ratio into an optimization tray derived from condition G9 of the JCSG Top96 screen. Crystals grew within 6 hr in a reservoir condition consisting of (0.1 M NH₄)2SO₄ 25% (wt/vol) PEG4000, and 0.1 M sodium acetate (pH 5.2). Crystals were cryo-protected with 15% ethylene glycol (vol/vol) before being flash-frozen in liquid nitrogen. Data collection was performed at the 23-ID-B beamline at the Argonne National Laboratory Advanced Photon Source. Datasets were initially processed using autoproc (Vonrhein et al., 2011) and further optimized using xdsgui (Kabsch, 2010). Molecular replacement was performed using PhaserMR McCoy et al., 2007 followed by multiple rounds of refinement using phenix.refine (Liebschner et al., 2019) and Coot (Emsley et al., 2010). Inter- and intra-molecular contacts were determined using PISA (Krissinel and Henrick, 2007) and manual inspection. Structural figures were generated using UCSF ChimeraX (Goddard et al., 2018; Pettersen et al., 2021).

Materials generated in this study will be made available upon reasonable request.