# Evidence for a model of conformational change by the Plasmodium falciparum circumsporozoite protein during sporozoite development in the mosquito host through the use of camelid single-domain antibodies

Rob Geens, Line de Vocht, Manuela C Aguirre-Botero, Cécile Vincke, Ema Romão, Stefan Magez, Serge Muyldermans, Rogerio Amino, Yann G.-J. Sterckx

► To cite this version:

Rob Geens, Line de Vocht, Manuela C Aguirre-Botero, Cécile Vincke, Ema Romão, et al.. Evidence for a model of conformational change by the Plasmodium falciparum circumsporozoite protein during sporozoite development in the mosquito host through the use of camelid single-domain antibodies. Infection and Immunity, 2025, 93 (6), pp.e0008125. 10.1128/iai.00081-25. hal-05122495

HAL Id: hal-05122495

https://hal.science/hal-05122495v1

Submitted on 20 Jun 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Evidence for a model of conformational change by the *Plasmodium falciparum* circumsporozoite protein during sporozoite development in the mosquito host through the use of camelid single-domain antibodies

Rob Geens,¹ Line De Vocht,¹ Manuela C. Aguirre-Botero,² Cécile Vincke,³,⁴ Ema Romão,³ Stefan Magez,³ Serge Muyldermans,³ Rogerio Amino,² Yann G.-J. Sterckx¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 17.

**ABSTRACT** *Plasmodium* sporozoites (SPZs) are formed in the *Anopheles* mosquito midgut from where they travel to the salivary glands and subsequently to the mammalian liver after deposition into the skin. The SPZ's main surface antigen, the circumsporozoite protein (CSP), plays a pivotal role in SPZ biology and constitutes the immunodominant target for host antibodies. In this study, we raised single-domain antibodies (sdAbs) against CSP from *P. falciparum* (PfCSP) by immunizing two alpacas with recombinant versions of the antigen. We found that all identified sdAbs specifically target PfCSP's globular αTSR domain without cross-reacting with *P. berghei* CSP. Further characterization revealed that most sdAbs recognize native PfCSP on the SPZ surface, although they do not have any inhibitory effect on hepatocyte binding and invasion. Structural studies showed that all binders target the previously identified α-epitope, confirming the non-protective nature of this epitope. Comparison of sdAb binding to midgut and salivary gland SPZs revealed a shift in the exposure and accessibility of the α-epitope. Hence, our findings provide further evidence that CSP undergoes structural changes during SPZ development in the mosquito host.

**KEYWORDS** malaria, *Plasmodium falciparum*, sporozoite, circumsporozoite protein, camelid single-domain antibodies

Malaria is a devastating disease that presents an immediate health threat for almost half the world's population, with an estimated 263 million cases and 597,000 deaths in 2023 (1). The endemic 'hot spot' is Sub-Saharan Africa, which accounts for 94 and 95% of all malaria cases and deaths, respectively. The disease is caused by parasites of the *Plasmodium* genus and vectored by female *Anopheles* mosquitoes. The parasite sexually reproduces in the mosquito midgut and generates thousands of sporozoites (SPZs), which travel to the salivary glands where they await injection into the skin of the mammalian host. From their inoculation site, the SPZs travel to the liver where they differentiate into tens of thousands of merozoites. The latter re-emerge in the circulation to replicate within erythrocytes and cause the notorious malaria pathology (2). Most human malaria cases concern infections by *P. falciparum*, which causes the deadliest malaria. Due to the fast emergence of resistance against antimalarial drugs and insecticides (3, 4), intensive research has been performed during the past decades in the quest to design an effective vaccine (5). Recently, the first malaria vaccines have been approved (Mosquirix, also known as RTS,S/AS01, and R21/Matrix-M), which are subunit vaccines based on the *P. falciparum* circumsporozoite protein (PfCSP) (6, 7).

**Editor** Jeroen P. J. Saeij, University of California Davis, Davis, California, USA

Address correspondence to Yann G.-J. Sterckx, yann.sterckx@uantwerpen.be.

Line De Vocht and Manuela C. Aguirre-Botero contributed equally to this article.

The authors declare no conflict of interest.

See the funding table on p. 17.

**Received** 13 February 2025

**Accepted** 26 March 2025

**Published** 28 April 2025

Copyright © 2025 Geens et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
The choice for CSP as the basis for subunit vaccines is well considered, as it is an immunodominant SPZ surface antigen (8). Furthermore, CSP plays a central role during the SPZ life stage both in insect and mammalian hosts (9), where it has been reported to: (i) be essential for SPZ development in the mosquito (10, 11); (ii) form a shield around the SPZ to protect its plasma membrane against pore-forming proteins during its migration through the skin (12); (iii) be essential for the recognition and invasion of host hepatocytes via its interaction with highly sulfated heparan sulphate proteoglycans (HS-HSPGs) (13, 14); and (iv) function as extra- and intracellular effector biomolecules to dampen host inflammatory responses (15, 16). PfCSP contains an intrinsically disordered N-terminal domain (PfCSP<sub>N</sub>) (17), a flexible linker region composed of NANP and NVDP repeats (PfCSP<sub>rep</sub>) and is flanked by N- and C-terminal junctions (PfCSP<sub>NTJ</sub> and PfCSP<sub>CTJ</sub>, respectively) and a globular C-terminal domain (PfCSP<sub>C</sub>) adopting a so-called αTSR fold (18) that is connected to the SPZ plasma membrane via a GPI anchor (19). Most antibodies (Abs) elicited by natural exposure or immunization target PfCSP<sub>rep</sub> and are regarded as potent inhibitors of SPZ migration and invasion (12, 20–22). Importantly, despite the immunodominance of PfCSP<sub>rep</sub>, Abs targeting other CSP regions have been reported as well and may or may not be protective (23). Abs targeting PfCSP<sub>N</sub> are rare and generally bind within or close to the biologically important Region I. However, their protective efficacy is still heavily debated (24–27). Abs recognizing PfCSP<sub>NTJ</sub> have also been discovered and are currently considered the most potent inhibitory Ab type with regard to parasite development (28, 29). Finally, Abs targeting PfCSP<sub>C</sub> are relatively rare and so far found to have poor SPZ inhibitory efficacy (30–32). However, it remains unclear whether the targeted epitopes are truly non-protective or whether the Abs are unable to reach their target because of the dense CSP packing on the SPZ surface.

The limited accessibility of CSP<sub>C</sub> epitopes may be explained by the model proposed by Coppi and colleagues (33). Here, CSP is suggested to adopt two conformational states that are timely regulated to address the SPZ's needs at different stages: the αTSR-exposed (or adhesive) conformation for midgut SPZs (MG SPZs) and the αTSR-shielded (or non-adhesive) conformation for salivary gland SPZs (SG SPZs). Since the αTSR domain is presumed to be involved in high-affinity cell adhesion processes but does not selectively target hepatocytes, it is proposed to be masked by CSP<sub>N</sub> during the SPZ's journey from the mosquito midgut to the mammalian liver. Hence, the different CSP conformations are believed to be linked to SPZ migration/invasion, in which the adhesive conformation only occurs at the start (development in the mosquito midgut) and end (hepatocyte invasion) of the SPZ life stage. Regarding the latter, the transition to the adhesive form is induced by interactions between CSP<sub>N</sub> and HS-HSPGs presented on liver cells (13, 14), which eventually result in the proteolytic cleavage of CSP<sub>N</sub> (34). This is presumed to ensure that CSP<sub>C</sub> is only exposed just before hepatocyte invasion, thereby limiting possible interference by anti-CSP<sub>C</sub> Abs. Although the Coppi model is based on *P. berghei* SPZs expressing wild-type or mutant PbCSP on their surface, another study has shown that similar mechanisms are involved during a *P. falciparum* infection (25).

In this paper, we describe the functional characterization of 12 camelid single-domain
antibodies (sdAbs, also known as nanobodies) targeting PfCSP<sub>C</sub>. sdAbs correspond to
the variable domain (VHH) of heavy-chain only Abs (HCAbs) found in camelids (Ila-
mas, alpacas, camels, and dromedaries) (35, 36). Two immune libraries were generated
after the immunization of two alpacas with distinct PfCSP constructs, resulting in the
identification of 12 specific sdAbs. Most binders have no effect on hepatocyte binding
and invasion by SG SPZs. A structural characterization of the sdAb-PfCSP<sub>C</sub> complexes
through macromolecular X-ray crystallography (MX) and/or AlphaFold-Multimer shows
that most sdAbs target the non-protective α-epitope (30–32). Interestingly, prominent
sdAb binding signals could be observed on MG SPZs, thereby revealing that the
α-epitope displays greater accessibility on MG SPZs compared to SG SPZs. Hence, our
findings support that CSP is subject to conformational change during SPZ development
in the mosquito.
# MATERIALS AND METHODS

## Recombinant production and purification of PfCSP~FL~ and PfCSP~C~

The *PfCSP*~FL~, *PfCSP*~C~, and *PbCSP*~FL~ genes were synthesized, codon-optimized for bacterial expression, and cloned into the pET-21b(+) vector through the Ndel and Xhol sites via a commercial partner (GenScript). The *PfCSP*~FL~ and *PfCSP*~C~ constructs are based on *P. falciparum* NF54 CSP (Uniprot ID: P19597) and correspond to full-length CSP (F20-S384, devoid of signal peptide and GPI anchor) and its C-terminal domain (E310-S384), respectively. *PbCSP*~FL~ corresponds to full-length *P. berghei* ANKA CSP (NCBI ID XP_022712148.1, G21-S327, devoid of signal peptide and GPI anchor). All constructs contain a C-terminal TEV cleavage site (ENLYFQSGG) and a hexahistidine (His~6~-tag. Chemocompetent *Escherichia coli* SHuffle T7 Express cells (NEB) were heat-transformed for gene expression, and transformants were selected on LB-agar-Gluc-Amp plates (LB-agar plates supplemented with 2% D-glucose and 100 µg/mL ampicillin) incubated overnight at 37°C. Pre-cultures of 10–25 mL were started by inoculation of 2xTY-Gluc-Amp medium (2xTY medium at pH 8.2 supplemented with 0.2% D-glucose and 100 µg/mL ampicillin) with a single colony or cell paste from a glycerol stock and grown overnight, shaking at 37°C. Main cultures of 0.5–1 L were inoculated with a 250-fold dilution of pre-culture and grown, shaking at 37°C to an OD~600~ of 0.6–0.9, at which gene expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the temperature was decreased to 20°C for overnight incubation. Cells were harvested 20 h post-induction by centrifugation (3,220 ×g for 30 min at 4°C). Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 2 µM leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0), flash frozen in liquid nitrogen, and stored at –20°C. A biotinylated *PfCSP*~FL~ construct (*PfCSP*~FL-bio~) was designed by inserting an AVI-tag (GLNDIFEAQKIEWHEGS) in between the TEV cleavage site and the His~6~-tag for site-specific biotinylation with *E. coli* biotin ligase (BirA). Chemocompetent *E. coli* SHuffle T7 cells (NEB) were co-transformed with *PfCSP*~FL-bio~ and BirA encoding (pBirAcm, Avidity) plasmids for in vivo production and biotinylation in 2xTY-Gluc-Amp-Chl medium (2xTY-Gluc-Amp medium supplemented with 35 µg/mL chloramphenicol) according to the procedures described above. The medium was supplemented with 50 µM D-biotin 30 min before induction.

The recombinant proteins were purified through immobilized metal affinity and size exclusion chromatography (IMAC and SEC, respectively). The purification protocol for *PfCSP*~FL~/*PfCSP*~FL-bio~/*PbCSP*~FL~ contains an additional heparin affinity chromatography (HAC) step to ensure their functional and biological relevance (18). Thawed cells were lysed by ultrasonication (60 cycles of 5 s pulses at 20% amplitude output and 5 s breaks on Sonics VCX-130) on ice, after which the soluble fraction was obtained by centrifugation (20,000 ×g for 1 h at 4°C). The cell extract (supernatant) was filtered (0.45 µm filter) prior to loading it onto a 5 mL HisTRAP HP column (Cytiva), pre-equilibrated in IMAC buffer A (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 2 µM leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0). Contaminants were removed by washing the column with 10 column volumes (CVs) IMAC buffer A, and target protein was eluted by linearly increasing the imidazole concentration with IMAC buffer B (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, 2 µM leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0) from 0 to 100%. Relevant IMAC elution fractions containing the target protein were pooled and dialyzed overnight at 4°C against SEC running buffer (50 mM Tris-HCl, 500 mM NaCl, 2 µM leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0) to reduce the imidazole concentration. The dialyzed sample was concentrated using a Vivaspin Turbo 15 PES (10 or 5 k) concentrator device (Sartorius) and subjected to SEC on a pre-equilibrated HiLoad 16/600 Superdex 200 column (Cytiva) or a HiLoad 16/60 Superdex 75 column (Cytiva) for *PfCSP*~FL~/*PfCSP*~FL-bio~/*PbCSP*~FL~ and *PfCSP*~C~, respectively. For *PfCSP*~C~, relevant SEC elution fractions containing the target protein were pooled and stored at 4°C. For *PfCSP*~FL~/*PfCSP*~FL-bio~/*PbCSP*~FL~, relevant SEC elution fractions containing the target protein were pooled and dialyzed overnight at 4°C against HAC buffer A (50 mM Tris-HCl, 2 µM
leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0) to reduce the salt concentration. The dialyzed sample was loaded onto a 1 mL HiTRAP Heparin HP column (Cytiva) pre-equilibrated with HAC buffer A. Remaining contaminants and non-functional PfCSP<sub>FL</sub>/ PfCSP<sub>FL-bio</sub>/PbCSP<sub>FL</sub> were removed by washing the column with 5 CVs HAC buffer A, and functional target protein was eluted by linearly increasing the salt concentration with HAC buffer B (50 mM Tris-HCl, 1 M NaCl, 2 µM leupeptin, 125 µM AEBSF, 100 µM EDTA, pH 8.0) from 0 to 100%. Fractions containing the target protein were pooled and stored at 4°C. Purity was analyzed by SDS-PAGE and anti-His western blotting, and biotinylation of PfCSP<sub>FL-bio</sub> was verified by streptavidin western blotting.

## Generation of anti-PfCSP camelid single-domain antibodies

*Ethical statement.* All animal experiments were carried out according to the directive 2010/63/EU of the European Parliament for the protection of animals used for scientific purposes and approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (clearance number 14-220-19 for the alpaca immunization).

*Construction of anti-PfCSP immune libraries.* Two alpacas named Attitude and Cosimo were immunized with PfCSP<sub>FL</sub> and PfCSP<sub>C</sub>, respectively, over a course of 6 weeks. Every week, 100 µg recombinant protein prepared in a volume of 500 µL was mixed with an equal volume of GERBU adjuvant (GERBU Biotechnik GmbH, Germany) prior to injection. A total of 100 mL of blood was collected from each alpaca 4 days after the last immunization. The two immune libraries (one for each alpaca) were constructed as described previously (37).

*Phage display and panning of the anti-PfCSP libraries.* Enrichment and identification of PfCSP-specific sdAbs from the immune libraries were performed according to published procedures (38). The two libraries (100 µL) were grown, shaking in 100 mL 2xTY-Gluc-Amp medium (1% D-glucose) at 37°C to an OD<sub>600</sub> of 0.6-0.8. After that, they were infected with 10<sup>12</sup> M13K07 helper phages (Invitrogen) for 30 min at room temperature. Cells were harvested by centrifugation (1,500 ×g for 10 min at 4°C), inoculated in 300 mL 2xTY-Amp-Kan medium (2xTY medium supplemented with 100 µg/mL ampicillin and 70 µg/mL kanamycin), and grown, shaking overnight at 37°C. Finally, cultures were centrifuged (11,000 ×g for 30 min at 4°C), and the virion-containing supernatant was collected. The phage particles were precipitated by mixing four parts with one part of PEG/NaCl solution (20% (w/v) polyethylene glycol 6000, 2.5 M NaCl). After an incubation of 30 min on ice, the precipitated virus particles were harvested by centrifugation (2,200 ×g for 30 min at 4°C) and resuspended in PBS to a final volume of 1 mL. The concentration of phage particles was determined by measuring the OD<sub>260</sub> (an OD<sub>260</sub> of 1 equals 3 × 10<sup>10</sup> particles per mL). A total of 10<sup>11</sup> phages (diluted in blocking buffer, see below) were used for solid-phase panning of the two libraries (Attitude and Cosimo) against both recombinant antigens used for immunization (PfCSP<sub>FL</sub> and PfCSP<sub>C</sub>) and for in-solution panning of the Attitude library against PfCSP<sub>FL-bio</sub>. For solid-phase panning, the antigens (100 µg/mL in 0.1 M NaHCO<sub>3</sub> at pH 8.2) were coated overnight at 4°C in the wells of a 96-well Nunc Maxisorb microtiter plate (Thermo Scientific). Protein binding sites in antigen-coated and non-coated wells were saturated for 2 h at room temperature with 200 µL 2% milk in PBS or Pierce Protein-Free T20 (PBS) blocking buffer (Thermo Scientific) as blocking reagents (alternated to avoid non-specific binders to buffer components), after which 100 µL of the phage suspension was added and incubated for 1 h. Phage particles were eluted by incubation in 100 µL of 100 mM triethylamine (TEA at pH 11.0) for 10 min, followed by neutralization with 100 µL 1 M Tris-HCl (pH 8.0). In between each step, the wells were washed 10-20 times with PBST (0.1% Tween 20 in PBS). For in-solution panning, PfCSP<sub>FL-bio</sub> (25 mM) was mixed with the phages in 250 mL blocking buffer and incubated for 1 h. The following blocking buffers were alternated to avoid enrichment of non-specific binders: 1% BSA in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5), 0.1% casein in TBST, and Pierce Protein-Free T20 (PBS) blocking buffer. Per panning round, 20 µL Pierce streptavidin magnetic beads (Thermo Scientific) was added to 1 mL TBST and subsequently blocked
for 1 h in 1 mL blocking buffer, after which the beads were incubated with the panning
mixture for 1 h, all while rotating at room temperature. The beads were subsequently
washed seven times in 1 mL blocking buffer, followed by three times in 1 mL PBST,
each wash performed while rotating the beads for 5 min. Bound phages were eluted
and neutralized as described above. The magnetic beads were manipulated using a
magnetic stand. Eluted phage particles were amplified by: (i) infecting 2 mL of fresh,
exponentially growing *E. coli* TG1 cells (in LB medium) for 30 min at 37°C; (ii) diluting
the culture with 8 mL 2xTY-Gluc-Amp medium and continuing incubating for 1 h at 37°C
with shaking; (iii) super-infecting the cells with 10⁷ M13K07 helper phages for 30 min at
room temperature; and (iv) pelleting the bacteria by centrifugation (800 ×g for 10 min
at 4°C) to inoculate 150 mL 2xTY-Amp-Kan medium, growing them overnight by shaking
at 37°C. Panning of the immune libraries was performed for three (Cosimo) and four
(Attitude) consecutive rounds on recombinant antigens. Enrichment of antigen-specific
phages was investigated by infecting 90 µL exponentially growing *E. coli* TG1 cells (in LB
medium) with 10 µL of a 10-fold dilution series (10⁻¹–10⁻⁷ in PBS) of the eluted phages
(from the antigen-coated and non-coated wells) in a round bottom 96-well plate for
30 min at 37°C, after which 10 µL per transduced cell culture was applied in a line
on a square LB-agar-Gluc-Amp plate. The remaining cell cultures were plated on round
LB-agar-Gluc-Amp plates to grow single colonies. All plates were incubated overnight at
37°C and subsequently stored at 4°C until further use.

*Identification of PfCSP-specific sdAbs.* Screening and selection of colonies carrying sdAb
fragments specifically binding the target antigens were performed through an enzyme-
linked immunosorbent assay (ELISA) on crude bacterial extracts (BEs). From the last two
rounds of panning of the sdAb library on recombinant target antigens, 47 individual
colonies were randomly selected per target antigen and grown in 100 µL of 2xTY-Gluc-
Glyc-Amp medium (2xTY medium supplemented with 2% D-glucose, 10% glycerol, and
100 µg/mL ampicillin) in 96-well round bottom culture plates. After overnight growing
at 37°C, 10 µL of each clone was used to inoculate 1 mL of 2xTY-Gluc-Amp medium
(0.1% D-glucose) in 96 deep-well plates. After growing at 37°C for 4–5 h with aeration,
the cultures were induced with 1 mM IPTG and incubated for another 4 h, after which
cells were harvested by centrifugation (3,220 ×g for 20 min at 4°C). The BEs containing
the sdAbs were released through a freeze–thaw cycle, after which the cell pellets were
resuspended in 100 µL PBS. Subsequently, the plates were centrifuged (3,000 ×g for
20 min at 4°C), and the BEs (supernatants) were collected into a new 96-well plate.
In parallel, Nunc Maxisorb microtiter plates were coated (2.5 µg/mL antigen solution),
blocked, and washed as described above. Non-coated wells were used as a negative
control for each colony. Fivefold dilutions of each BE were loaded (100 µL) onto blocked
coated and non-coated wells and incubated for 1 h at room temperature. The ELISA
was detected by 1 h incubations at room temperature with 100 µL mouse anti-hemag-
glutinin mAb (anti-HA, BioLegend 16B12) and goat anti-mouse pAb conjugated with
alkaline phosphatase (AP; Bethyl Laboratories A90-116AP), which were 2,000-fold diluted
in PBS. The ELISA was developed with AP substrate (2 mg/mL 4-nitrophenyl phosphate
disodium salt hexahydrate in 100 mM Tris–HCl, 10 mM NaCl, 50 mM MgCl₂, pH 9.5), and
absorption was measured at 405 nm. A true binder was considered when the signal ratio
between coated/uncoated wells was ≥2.

*Recombinant production and purification of anti-PfCSP sdAbs.* Chemocompetent *E. coli* WK6 cells were heat-transformed with the pMECS-GG-sdAb plasmids as described above. Transformants were selected by plating the cell culture on LB-agar-Gluc-Amp plates. Finally, single colonies were picked to inoculate 10 mL 2xTY-Amp medium (2xTY medium supplemented with 100 µg/mL ampicillin) and grown overnight by shaking at 37°C. The next day, glycerol stocks were prepared by mixing 900 µL culture with 900 µL sterile 50% (v/v) glycerol and stored at –80°C. Pre-cultures of 10–25 mL were started by inoculating LB-Amp or 2xTY-Amp medium with a single colony from a fresh transformation or cell paste from an existing glycerol stock and grown overnight by shaking at 37°C. Main cultures of 0.25–1 L were started in baffled culture flasks by inoculating TB-Gluc-Amp
medium (1.2% tryptone, 2.4% yeast extract, 17 mM KH₂PO₄, 72 mM K₂HPO₄, 0.4% glycerol, supplemented with 100 µg/mL ampicillin and 0.2% D-glucose) with a 200-fold dilution of the pre-culture and grown by shaking at 37°C to an OD₆₀₀ of 0.6–0.9, at which the gene expression was induced with 1 mM IPTG, and the temperature was decreased to 28°C for overnight incubation. Cells were harvested by centrifugation (3,220 ×g for 30 min or 11,000 ×g for 10 min at 4°C), after which the supernatant was discarded. Cell pellets were subjected to an osmotic shock by: (i) resuspension in 15 mL TES buffer (200 mM Tris–HCl, 500 mM sucrose, 0.5 mM EDTA, pH 8.0) per pellet of 1 L culture and shaking at 4°C for 6 h; and (ii) addition of 30 mL TES/4 buffer (50 mM Tris–HCl, 125 mM sucrose, 0.125 mM EDTA, pH 8.0) per pellet of 1 L culture and shaking overnight at 4°C. The periplasmic extracts (PEs) were collected by centrifugation (3,220 ×g for 30 min at 4°C) and stored in 50 mL Falcon tubes at 4°C. This process was repeated once to collect two PEs per sdAb.

sdAbs were purified from the PEs by a two-step protocol consisting of IMAC and SEC. Approximately 1–2 mL HisPur Ni-NTA resin (Thermo Scientific) per sdAb was equilibrated in PBS, divided over the PE-containing tubes, and incubated for 1 h at room temperature while gently shaking or rotating. The extracts were poured into empty PD10 columns with filters (GE Healthcare), after which the resin was washed with 20–30 bed volumes (BVs) PBS. The sdAbs were eluted by adding 1 BV elution buffer (0.5 M imidazole in PBS) with an incubation period of 10 min prior to collecting 1 mL fractions while adding more elution buffer. Elution fractions containing the sdAbs were identified via UV spectrophotometry, pooled, and subjected to SEC on HiLoad 16/60 Superdex 75 or HiLoad 16/60 Superdex 200 columns pre-equilibrated in PBS or TBS (20 mM Tris–HCl, 150 mM NaCl, pH 7.5). Relevant SEC elution fractions were pooled, aliquoted, and stored at –20°C.

## ELISA

Nunc Maxisorb microtiter plates were coated (1 µg/mL antigen solution), blocked (5% milk in PBS), and washed as described above. Non-coated wells were used as a negative control for each antigen. Diluted sdAbs (100 µL at 1 µg/mL in blocking buffer) were loaded in triplicates onto blocked wells and incubated for 1 h at room temperature, followed by incubations with 2,000-fold dilutions (in blocking buffer) of mouse anti-HA mAb and goat anti-mouse pAb conjugated with HRP (Sigma A4416). The ELISA was developed with 1-Step Ultra TMB-ELISA substrate solution (100 µL; Thermo Scientific) for 15–30 min, after which development was stopped with 1 M H₂SO₄ (50 µL), and absorption was measured at 450 nm. For the serum ELISA, Pierce Protein-Free T20 (PBS) blocking buffer was used as the blocking agent, and threefold dilution series (10- to 7,290-fold) of the Attitude and Cosimo sera were prepared in PBS and probed for binding to the antigens. Detection was performed with 2,000-fold dilutions (in blocking buffer) of rabbit anti-camelid VHH mAb (GenScript 96A3F5) and goat anti-rabbit pAb conjugated with AP (Bethyl Laboratories A120-101AP) and developed with AP substrate after which absorption was measured at 405 nm.

## Heparin binding assays

All proteins were extensively dialyzed against HAC buffer A prior to preparing 100 µL samples of the sdAb:PfCSP<sub>FL</sub> complexes at a molar ratio of 1.2:1 and incubated for 1 h at room temperature. Approximately 100 µL Heparin Sepharose HP (GE Healthcare) per complex was equilibrated in HAC buffer A and incubated with the protein samples for 1 h. Subsequently, the samples were transferred to Pierce spin cups with cellulose acetate 0.45 µm filters (Thermo Scientific) and centrifuged (1,200 ×g for 1 min; Eppendorf Centrifuge 5417R). After that, the resin was washed twice with 50 µL HAC buffer A and eluted in two rounds with 50 µL HAC buffer B. All samples were collected and analyzed by SDS-PAGE.
## Sporozoite binding and invasion assays

*Ethical statement.* All animal experiments were performed in compliance with the French and European regulations regarding the protection and care of laboratory mice and approved by the ethics committee with references MESR 01324, APAFIS #32422-2021071317049057 v2, and APAFIS #32989-2021091516594748 v1.

*Procurement of midgut and salivary gland SPZs.* Transgenic *P. berghei* ANKA SPZs expressing full-length CSP from *P. falciparum* 3D7 (PfCSP-PbSPZs) were used. A green fluorescent protein (GFP) was expressed in the parasite cytoplasm to serve as a reporter (39). Naive *Anopheles stephensi* (strain SDA 500) mosquitoes were reared according to standard procedures. One day prior to infection, mosquitoes were deprived of sucrose to increase the bite rate, after which they were infected by allowing them to feed on two-three infected RjOrl:Swiss mice per 200 female mosquitoes for 30 min. Subsequently, mosquitoes were maintained in a humid chamber at 21°C for 1 week, after which they were allowed to feed on naive RjOrl:Swiss mice. MG and SG SPZs were isolated 13-22 days after infection in 10-30 µL 1× Dulbecco's PBS and kept on ice.

*SPZ-sdAb binding studies.* About 20,000-30,000 PfCSP-PbSPZs per sdAb were fixed in 2% formaldehyde for 20 min at room temperature. Subsequently, the SPZs were washed once with PBS, after which they were centrifuged (500 ×g for 10 min at 4°C). The supernatant was then removed, and the pelleted SPZs were resuspended in 1 mL PBS with 2% BSA and incubated on ice for 1 h. If necessary, the SPZs were further diluted in PBS with 1% BSA to obtain about 8,000 SPZs in 20 µL per reaction tube. Thereafter, 10 µL SPZs were mixed with 10 µL 20 µg/ml sdAb, and the reaction mixtures were incubated for 1 h on ice or 30 min at room temperature. Then, a 500-fold dilution of mouse anti-HA mAb conjugated with Alexa Fluor 647 tag (BioLegend, clone 16B12) was added and incubated for 30 min on ice or 15 min at room temperature. Finally, the samples were diluted with 200 µL PBS, and data were collected on a CytoFLEX S flow cytometer (Beckman Coulter). For fluorescence microscopy, 20 µL of the final samples was transferred to a µ-Slide 18 well coverslip and centrifuged for 5 min at 500 ×g prior to image acquisition with an inverted Axio Observer Z1 microscope. A non-relevant sdAb targeting β-lactamase from *Bacillus cereus* 596H (sdAb-Bcl10) (40) was used as a control.

*SPZ hepatocyte traversal and cell wounding assays.* Invasion assays were performed as previously reported (41). HepG2 cells were seeded on flat-bottom 96-well plates (40,000 cells per well) 1 day before the SPZ challenge in DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS, 1× of non-essential amino acids (Sigma), and 2% of penicillin-streptomycin-neomycin antibiotic mixture (Sigma) at 37°C with 5% CO₂. PfCSP-PbSPZ-sdAb samples were prepared as described above, after which 50 µL/well was transferred to the HepG2 cells. Tetramethylrhodamine dextran, lysine-fixable, 10,000 MW (DxRed at 1 mg/mL; Molecular Probes) was added to detect cell wounding. The plates were centrifuged (500 ×g for 5 min at 10°C) and subsequently incubated for 2 h at 37°C with 5% CO₂ and 10% O₂. Non-targeting sdAb and cytochalasin D (CytoD; 0.5 µM) were included as controls for SPZ invasion. Unchallenged HepG2 cells and manually scratched HepG2 cells were used as additional controls for cell wounding. The supernatants were transferred to another plate, and the HepG2 cells were washed twice with 50 µL/well PBS. The wash fractions were pooled with the former supernatant and corresponded to the non-adherent SPZ fraction. Subsequently, 50 µL/well trypsin was added to the HepG2 cells and incubated for 10 min at 37°C, and trypsinization was stopped by adding 150 µL/well PBS with 2% FCS. After homogenizing the solutions in the wells, they were filtered to collect the extracellular (adherent) and intracellular (invasion) SPZ fractions. Samples of 50 µL from both fractions were analyzed on a CytoFLEX S flow cytometer. DxRed+ and GFP+ HepG2 cells are considered as wounded and invaded cells, respectively; GFP+ and GFP-SPZs are viable and dead parasites, respectively. At 44 h, intracellular development was assessed based on the number of GFP+ cells. Statistical significance was determined with a one-way analysis of variance with Holm-Šídák correction for multiple comparisons using GraphPad Prism 10 (see Table S1 in the Supplemental material).
## Crystallization, data collection and processing, and structure determination

The sdAb:PfCSP~C~ complexes were prepared at a molar ratio of 1.2:1 and incubated for 1 h at room temperature. Subsequently, the complexes were concentrated to 200 µL using Vivaspin 6 (3 k) concentrator devices (Sartorius) and purified by SEC using an ENrich SEC 70 10 × 300 column (Bio-Rad), pre-equilibrated in 20 mM Tris-HCl, 150 mM NaCl, and pH 7.5. Relevant elution fractions containing the complex were pooled and concentrated to 5–10 mg/mL using Vivaspin 500 (3 k) concentrator devices (Sartorius). Crystallization conditions were screened by the sitting drop method using the Mosquito Xtal3 robotic system (TTP Labtech) mixing 100 nL protein droplets with 100 nL mother liquor in 96 well plates. Commercial screens of Jena Bioscience (JBScreen Basic, JBScreen Classic, JBScreen Wizard, and JBScreen PEG salt) and Molecular Dimensions (ProPlex, PACT Premier, and JCSG Plus) were used for screening. The crystal plates were incubated at 20°C, and crystal growth was monitored manually using a Nikon SMZ 745T stereo microscope. The affinity tags of all proteins were retained for crystallization. Diffraction-quality crystals of sdAb1:PfCSP~C~ and sdAb9:PfCSP~C~ were obtained after 3 to 4 days in JBScreen Wizard (Jena Bioscience) condition no. F12 (30% PEG 8000, 100 mM imidazole pH 8.0, 200 mM NaCl) and JBScreen Classic 1–4 (Jena Bioscience) condition no. D12 (35% PEG 4000), respectively.

The sdAb1:PfCSP~C~ and sdAb9:PfCSP~C~ crystals were cryocooled in liquid nitrogen with the addition of 25 (v/v) glycerol and 30% (v/v) glycerol to the mother liquor as a cryoprotectant, respectively. Data sets were collected at the ESRF Synchrotron (Grenoble, France) on the ID30A-3 beamline. Both data sets were processed with XDSME (42, 43). The quality of the collected data sets was verified by close inspection of the XDS output files and through phenix.xtriage in the PHENIX package (44). Twinning tests were also performed by phenix.xtriage. Analysis of the unit cell contents was performed with the program MATTHEWS_COEF, which is part of the CCP4 package (45). The structures of sdAb1:PfCSP~C~ and sdAb9:PfCSP~C~ were determined by molecular replacement with PHASER-MR (46). The following search models were employed for molecular replacement: (i) one copy of the structure of PfCSP~C~ (chain A, PDB ID 3VDJ); and (ii) one copy of an AlphaFold2 (47, 48) model of the sdAb (of which the CDRs were removed due to poor pLDDT scores). For both data sets, the searches provided single solutions (top TFZ = 22.6 and top LLG = 995.328 for sdAb1:PfCSP~C~, top TFZ = 20.3 and top LLG = 549.409 for sdAb9:PfCSP~C~). For both structures, refinement cycles using the maximum likelihood target function cycles of phenix.refine (44) were alternated with manual building using Coot (49). The crystallographic data for the sdAb1:PfCSP~C~ and sdAb9:PfCSP~C~ structures are summarized in Table S2 in the Supplemental material and have been deposited in the PDB (PDB IDs: 9HZJ and 9HZK, respectively). Molecular graphics and analyses were performed with UCSF ChimeraX (50).

## AlphaFold-based structure prediction

The structural models of sdAb:PfCSP~C~ protein complexes were predicted using AlphaFold-Multimer (51). Twenty-five models were predicted per run, and the best models underwent a final relaxation step. The models were evaluated based on the following parameters: AlphaFold-Multimer model confidence (a weighted combination of the predicted template modeling (pTM) and interface predicted template modeling (ipTM) scores [51]: 0.8*pTM + 0.2*pTM), the predicted aligned error (PAE) matrix (47), the local and global predicted local distance difference test (pLDDT) scores (47), the predicted DockQ (pDockQ) values (52), and the normalized discrete optimised protein energy (zDOPE) scores (53). Molecular graphics and analyses were performed with UCSF ChimeraX (50).
RESULTS

Camelid immunization with two different PfCSP constructs yields 12 sdAbs specifically targeting PfCSP's αTSR domain

Two anti-PfCSP VHH libraries were generated through the immunization of two alpacas (Attitude and Cosimo) with distinct recombinant antigens (Fig. 1A): while Attitude was immunized with full-length PfCSP (PfCSP~FL~, devoid of its signal peptide and GPI anchor), Cosimo only received PfCSP~C~ (corresponding to the αTSR domain). Peripheral blood samples were collected 4 days after the last immunization. The induced immune responses against the antigens were assessed via serum ELISA, indicating that the sera of both animals contained PfCSP-specific HCAbs (Fig. 1B). Interestingly, significant binding signals are observed for PfCSP~C~ with both sera, indicating that a substantial HCAb subset in the Attitude serum targets conformational epitopes within the globular αTSR domain despite the presentation of the linear B-cell epitopes in PfCSP's repeat region.

The two VHH libraries contained 8 × 10⁷ (Attitude) and 2 × 10⁹ (Cosimo) individual
transformants, with an insert percentage of ~100%. PfCSP-specific sdAbs were enriched
by phage display using both solid-state and in-solution panning. The same seven VHH
families were identified after both panning strategies (see Fig. S1 in the Supplemental
material). Interestingly, all positive clones recognize both PfCSP~FL~ and PfCSP~C~, demon-
strating that all sdAbs target epitopes within the αTSR domain. Eventually, 12 sdAbs
(sdAb1-10 from Attitude; sdAb15 and sdAb18 from Cosimo) were selected for further

<figure><img src="image_2.png" /><figcaption>**FIG 1** Generation of PfCSP-specific sdAbs. (A) Schematic overview of the wild-type (WT) PfCSP from *P. falciparum* NF54 and the recombinant protein constructs derived from it shown in the left panel. The start of each domain and the segment corresponding to the globular αTSR domain are indicated on top and below the WT schematic, respectively. A legend of the conserved regions, tandem repeats, and purification tags (TEV, tobacco etch virus protease cleavage site; His6, hexahistidine tag) is shown at the bottom. An overview of the alpaca immunization strategy used to generate two immune VHH libraries is provided in the right panel. (B) Serum ELISA. Identification of HCAbs in the sera of Attitude (left panel) and Cosimo (right panel) targeting recombinant PfCSP~FL~ and PfCSP~C~. (C) Cross-reactivity ELISA. The sdAbs raised against PfCSP were probed for binding to PfCSP~FL~ and PbCSP~FL~ (blue and orange bars, respectively). (D) The mean and standard deviation of triplicates for each condition are shown.</figcaption></figure>
characterization. Because CSP~C~ contains several regions that are relatively well conserved among *Plasmodium* species (e.g., regions III and II+), cross-reactivity of the sdAbs against recombinant full-length CSP from *P. berghei* (*PbCSP*~*FL*~) was assessed by ELISA (Fig. 1C). While clear binding can be observed to *PfCSP*~*FL*~, none of the sdAbs recognize *PbCSP*~*FL*~, indicating that key residues essential for the interaction are absent in *PbCSP*~*FL*~.

## The anti-PfCSP~C~ sdAbs do not interfere with heparin binding

CSP is essential to initiate the liver stage of the infection via its specific interaction with liver cell HS-HSPGs. Although PfCSP~N~ is presumed to be the main contributor to this interaction, a possible supporting role has been suggested for PfCSP~C~ (54). Therefore, the ability of the sdAbs to inhibit the interaction between PfCSP and heparin was investigated. Heparin is a structural analogue of HS-HSPGs that is frequently used in malaria research (18, 54–56). sdAb-PfCSP~FL~ complexes were prepared with a slight excess of sdAb to ensure complete antigen saturation and subsequently probed for binding to heparin in a spin cup filter setup (Fig. 2). After several washing steps, heparin-bound proteins were eluted with a high-salt buffer (1 M NaCl). The results were visualized by subjecting all collected fractions (flow through, FT; wash, W; elution, E) to SDS-PAGE. PfCSP~FL~ and one of the sdAbs were used as positive and negative controls, respectively. Sample analysis shows that the positive control only displays a protein band between the 55 and 70 kDa markers (expected for PfCSP~FL~) in the elution fractions, while the negative control only exhibits a protein band just above the 15 kDa marker (expected for

<figure><img src="image_3.png" /><figcaption>**FIG 2** Heparin binding experiments with sdAb-PfCSP~FL~ samples. The experimental setup is schematically illustrated in the top left panel. All collected fractions were analyzed by SDS-PAGE. Unbound PfCSP~FL~ and sdAb were included as positive and negative controls, respectively.</figcaption></figure>
a sdAb) in the flow through and wash fractions. A small and barely visible sdAb band is present in the first elution fraction as well, which is most probably due to the abundance in which the protein was used rather than a specific interaction with heparin. Hence, the controls indicate that PfCSP<sub>FL</sub> is indeed able to interact with heparin, and that the possibility of the formation of a heparin-sdAb complex can be discarded. Analysis of the sdAb-PfCSP<sub>FL</sub> samples shows that the sdAbs were present in all collected fractions, while PfCSP<sub>FL</sub> can only be observed in the elution fractions. This demonstrates that the sdAbs are indeed able to bind PfCSP<sub>FL</sub> but do not have the ability to inhibit its interaction with heparin. Hence, none of the sdAbs impair the PfCSP-heparin interaction.

## The anti-PfCSP<sub>C</sub> sdAbs bind salivary gland SPZs but do not inhibit productive SPZ invasion

The ability of the sdAbs to recognize their target antigen on the surface of live SPZs was assessed using transgenic *P. berghei* SPZ expressing PfCSP (PfCSP-PbSPZs) (39). The results show that the recognition of SG SPZs heavily depends on the assay conditions. While the sdAbs displayed no to very poor interaction with SG SPZs at 10°C, better binding signals could be obtained for a selection of anti-PfCSP sdAbs (1, 4, and 9) at ambient temperature (Fig. 3, panels A to C). This discrepancy is most likely explained by the temperature's influence on binding affinity and membrane fluidity, thereby affecting the sdAb°PfCSP interaction at the parasite surface. Nonetheless, the data indicate that some sdAbs can reach PfCSP<sub>C</sub> on SG SPZs.

Next, the anti-PfCSP sdAbs 1, 2, and 9 were tested for their effect on the viability and invasion of PfCSP°PbSPZs *in vitro*. SPZs were pretreated with the sdAbs, after which their abilities to adhere to and invade HepG2 cells were assessed. Since SPZs rely on the actin-myosin motor for their migration and invasion of host cells (57), Cytochalasin D (CytoD, an inhibitor of actin polymerization) was used as a negative control for cell invasion. None of the tested anti-PfCSP sdAbs have an inhibitory effect on SPZ adhesion or invasion (Fig. 3D), nor do they affect SPZ viability (Fig. 3E). In addition, the sdAbs have no effect on SPZ cell traversal (Fig. 3F). Quantification of parasite-infected HepG2 cells 2 h post-initiation validates that there is indeed no difference between the SPZs treated with the control sdAb and the anti-PfCSP sdAbs (Fig. 3G). Finally, parasite liver stage development was monitored for 48 h, demonstrating that there is no sdAb-mediated inhibition (Fig. 3H). In conclusion, despite being able to reach their epitope on SG SPZs, the anti-PfCSP sdAbs do not confer protection against host cell infection and parasite development.

## The anti-PfCSP<sub>C</sub> sdAbs display prominent binding to midgut sporozoites

Because CSP has been reported to adopt different conformations depending on the SPZ developmental stage within the mosquito (33), all sdAbs were also assayed for binding to MG SPZs. At 10°C, sdAbs 1, 6, 8, 9, and 10 display a clear interaction with MG SPZs, whereas the other sdAbs show weaker binding (sdAbs 3, 4, and 5) or no binding at all (sdAbs 7, 15, and 18) (Fig. 3I). Not surprisingly, the detection signals were larger at ambient temperature (recorded for sdAbs 1, 4, and 9) (Fig. 3, panels B and C). Clearly, these experiments demonstrate that these anti-PfCSP sdAbs bind MG SPZ more efficiently compared to SG SPZs. To investigate the influence of the SPZ maturation state on sdAb recognition, a similar experiment was performed with 15- and 22-day-old MG and SG SPZs (Fig. 3J). This reveals that binding depends on the SPZ developmental stage rather than SPZ maturation. Indeed, fluorescence imaging of fixed PfCSP-PbSPZs shows that sdAb1 more readily interacts with MG SPZs compared to SG SPZs, independent of their maturation state (Fig. 3E). Together, these results indicate that, while the epitopes targeted by these sdAbs are easily accessible on MG SPZs, they may be (partially) shielded or less accessible on SG SPZs.
<figure><img src="image_4.png" /><figcaption>**FIG 3** In vitro interaction studies between the anti-PfCSP sdAbs and transgenic PfCSP-PbSPZs. (A) Interaction screening of the sdAbs to 20-day-old SG SPZs on ice. PBS was used as a negative control. (B) Interaction studies of selected sdAbs to 15-day-old MG and SG SPZs at ambient temperature. sdAb-Bcl10 was included as a negative control. (C) Fluorescence imaging of MG and SG SPZs after incubation with sdAb1 at ambient temperature. Scale bars (white lines): 5 µm. (D–G) Effect of the sdAbs on SPZ invasion of HepG2 cells evaluated 2 h after parasite addition. Here, we measured the recovery of SPZs present in the suspension, attached to, or inside the cells (D), percentage of viable SPZs compared to the control (E), percentage of wounded cells (F), and percentage of infected cells (G). sdAb-Bcl10 and CytoD were included as negative and positive controls for invasion inhibition, respectively. (H) Effect of the sdAbs on the SPZ intracellular development. The percentage of parasite-infected HepG2 cells 24 and 48 h post-incubation with SPZs pre-incubated with the different sdAbs is shown. sdAb-Bcl10 was included as a negative control. (I) Interaction screening of the sdAbs to 13-day-old MG SPZs on ice. PBS was included as a (Continued on next page)</figcaption></figure>
Fig 3 (Continued)

negative control. (J) sdAb binding to MG and SG SPZs with different maturation times (15-day-old vs. 22-day-old) at ambient temperature. sdAb-Bcl10 was included as a negative control. (A, B and I, J) Binding is expressed as median fluorescence intensities (MFI). (D–H) The mean values with standard deviation of three independent measurements are shown. Statistical significance was determined by one-way analysis of variance with Holm–Šídák correction for multiple comparisons; \*P ≤ 0.05, \*\*P ≤ 0.01, \*\*\*P \_j 0.0001, ns = not significant.

Most sdAbs target the α-epitope

To unravel the structural basis of PfCSP~C~ recognition by the identified anti-PfCSP sdAbs, the high-resolution structures of sdAb–PfCSP~C~ complexes were either experimentally determined through macromolecular X-ray crystallography (MX) or predicted using AlphaFold-Multimer.

Crystallization trials were initiated for PfCSP~C~ complexed with sdAb1, 4, 6, and 9. Diffraction quality crystals were obtained for PfCSP~C~–sdAb1 and PfCSP~C~–sdAb9 complexes, and data were collected to ~2.1 Å (see Table S2 in the Supplemental material). The crystal structures reveal that sdAb1 and 9 target the PfCSP~Cα~-epitope (30, 31) (Fig. 4A). This epitope is composed of the Region III residues (adopting an α-helical structure), the flexible loop between the two antiparallel β-strands of the TSR fold (referred to as the CSP-flap), and the hydrophobic pocket between the α-helix and the CSP-flap. The paratopes of sdAb1 and 9 are largely similar, which is expected, as they belong to the same VHH family (Fig. 4A). The bulk of antigen contacts are provided by residues from their CDR3; however, N57 (CDR2) and Y59 (FR, only for sdAb1) also contribute to the interaction (Fig. 4, panels B and C for sdAb1 and sdAb9, respectively). The total buried surface areas of the PfCSP~C~–sdAb1 and PfCSP~C~–sdAb9 interfaces are 626 and 747 Å², respectively, and detailed overviews of all contacts are provided in Table S3 and S4 in the Supplemental material. Noteworthy ‘interaction hot spots’ are sdAb1 amino acids L101 and F103 that are inserted into the hydrophobic pocket delineated by PfCSP residues L320, L327, L358, Y360, and I364. The sdAb1 hydrophobic residues L101 and F103 are substituted by W101 and Y103 in sdAb9. Here, W101 is inserted into the PfCSP~C~ hydrophobic pocket, but Y103 is oriented outwards. Three other important interactions are the salt bridges formed at either side of the α-epitope between sdAb1 R105 and PfCSP E357 (left side, only for sdAb1–PfCSP~C~) and sdAb D99/D113 and PfCSP K317 (right side, both complexes), which likely stabilize the hydrophobic interaction center.

In the absence of diffraction-quality crystals for the other sdAb–PfCSP~C~ complexes, we wondered whether AlphaFold-Multimer would be able to predict these structures. As a positive control, AlphaFold-Multimer was employed to predict the sdAb1–PfCSP~C~ and sdAb9–PfCSP~C~ complexes. We note that the predictions were highly accurate, displaying an almost perfect overlap with the experimentally determined crystal structures (Cα RMSDs of 0.99 and 1.08 Å for sdAb1–PfCSP~C~ and sdAb9–PfCSP~C~, respectively). Furthermore, both predicted complexes are characterized by very good to excellent values of various local and global validation metrics (pLDDT, zDOPE, pTM, PAE, pDockQ, and ipTM) that are defined and visualized in the legend of Fig. S2 in the supplemental material. Having validated the performance of AlphaFold-Multimer for sdAbs 1 and 9, it was employed to predict the complexes for the other sdAb–PfCSP~C~ complexes. Reliable models could be obtained for all sdAbs, except for sdAb2, 4, and 8. A comparison with the sdAb1–PfCSP~C~ and sdAb9–PfCSP~C~ complex structures reveals that the sdAbs retrieved in this study are highly likely to be α-epitope binders.

DISCUSSION

Significant progress has been made in reducing malaria-endemic areas primarily by
targeting the mosquito vector (58). While vector control remains a cornerstone of current
malaria control programs, our understanding of parasite biology within the mosquito
host remains limited. A notable example is the SPZ's main surface antigen: while CSP has
been shown to play a crucial role during SPZ development in the mosquito (9), many
<figure><img src="image_5.png" /><figcaption>**FIG 4** Crystal structures of sdAb1 and sdAb9 in complex with PfCSP~C~. (A) Cartoon representations of PfCSP~C~ (in light green) in complex with antibody fragments (in gray). The top left panel displays a superposition of the Fab1710-PfCSP~C~ (PDB ID 6B0S [30]) and Fab1512-PfCSP~C~ (PDB ID 7RXP [31]) crystal structures. The right panel shows the crystal structures of PfCSP~C~ bound by sdAb1 and sdAb9 (PDB IDs 9HZJ and 9HZK, respectively, this work). A sequence alignment of the CDRs from sdAb1 and sdAb9 is presented in the bottom left panel, with amino acid substitutions highlighted by gray boxes. The CDRs are indicated in different colors (CDR1, blue; CDR2, green; CDR3, orange), and the residues that are part of the paratope are marked by an asterisk (*). Stereo views of the sdAb1-PfCSP~C~ (B) and sdAb9-PfCSP~C~ (C) interactions. PfCSP~C~ is depicted in cartoon representation. For reasons of clarity, only the sdAb residues that are part of the paratope are shown and colored as in (A). All interacting residues are labeled and shown in stick representation. Hydrogen bonds and salt bridges are indicated by black dashed lines.</figcaption></figure>
molecular details remain largely unexplored. Here, we identified 12 sdAbs targeting PfCSP by phage display and panning of two immune VHH libraries to investigate the antigen's structure-function relationship. Regardless of the immune VHH library (Attitude vs. Cosimo) or the employed immunization strategy (PfCSP<sub>FL</sub> vs. PfCSP<sub>C</sub>), all sdAbs target the globular αTSR domain. This is intriguing since most conventional anti-PfCSP Abs raised in humans and mice recognize repeat region epitopes (20, 22, 59, 60), and PfCSP<sub>C</sub>-targeting Abs are considered rare (30–32). HCAbs possess longer CDR loops (particularly CDR3) to maintain a sufficiently large paratope surface area for effective antigen recognition and compensate for the absence of a light chain (36). Therefore, VHHs possess unique paratope features that are usually not found in conventional Abs, giving them a preference for binding conformational epitopes (61–63), which may provide an explanation for our findings.

High-resolution structure determination through experimental methods (MX) or machine learning-based prediction (AlphaFold-Multimer) reveals that most sdAbs recognize the PfCSP<sub>C</sub> α-epitope. For the sdAb1–PfCSP<sub>C</sub> complex, the experimentally determined crystal structure further validates results recently obtained through a combination of molecular modeling and mass spectrometry (64). The recognition of PfCSP by the sdAbs is highly specific, as no binding to PbCSP could be observed. This is consistent with the literature on anti-PfCSP<sub>C</sub> conventional Abs, which reports that cross-reactivity to PfCSP<sub>C</sub> from different PfSPZ field isolates depends on β-epitope (and not α-epitope) characteristics (31). This can be attributed to the polymorphic nature of the Th2R (Region III) and Th3R (CSP-flap) regions constituting the α-epitope (65), while the β-epitope contains the more conserved regions II+ (TSR strands 1 and 2) and CS.T3 (TSR strand 3) (31, 66). As evidenced by heparin binding assays and functional experiments with live SG SPZs, the sdAbs studied in this work do not impede SPZ adhesion, traversal, and invasion of hepatocytes, nor its intracellular development. This is consistent with previous work reporting that α-epitope targeting Abs are poorly or non-protective (30). However, the underlying molecular mechanisms for these observations remain unclear. Three hypotheses could be presented: the targeted epitopes are (i) accessible but inherently non-protective, (ii) inaccessible to bulky, conventional Abs due to the densely packed CSP-covered parasite surface, or (iii) inaccessible due to the CSP conformation on the SG SPZ surface. Arguments for the latter two hypotheses are provided by the observation that both α- and β-epitope targeting Abs inefficiently bind SG SPZs (30–32). In contrast, due to their smaller size, sdAbs are known for their ability to easily penetrate densely coated antigen-crowded parasite surfaces and bind epitopes, which are inaccessible to conventional Abs (67, 68). Hence, while the second hypothesis cannot be excluded based on the data presented here, the sdAbs are likely to be able to infiltrate the densely packed CSP-covered parasite surface. However, the results clearly indicate that staining of SG SPZs using these sdAbs is comparable to that obtained through the use of mAbs (69).

Finally, the most intriguing result presented in this work is perhaps the observation
that, in comparison to SG SPZ, the α-epitope targeting sdAbs readily recognize MG
SPZs. The observed difference in binding signals suggests that the accessibility of the
α-epitope changes during SPZ development in the mosquito, which in turn supports a
model for conformational change by PfCSP at the parasite surface (33). This could be
an intrinsic property of the surface antigen and/or could be induced by a yet unknown
mosquito factor. We propose that our findings further refine this model (Fig. 5): the
α-epitope is exposed and accessible on the surface of MG SPZs (CSP adhesive form) but
shielded and inaccessible to large molecules (e.g., Abs) on SG SPZs (CSP non-adhesive
form). These conformations are closely linked to SPZ migration/invasion, as CSP takes
its adhesive form during SPZ development in the oocyst and hepatocyte invasion, while
the non-adhesive conformation is adopted during SPZ migration from the mosquito
midgut to the mammalian liver. Further research on the exact timing of the transition
from the adhesive to the non-adhesive conformation in the mosquito will enable further
<figure><img src="image_6.png" /><figcaption>**FIG 5** CSP is subject to conformational changes during SPZ development in the mosquito and its journey to the mammalian liver. CSP's α-epitope is exposed during SPZ development in the mosquito midgut (adhesive conformation). Upon release from the oocyst, the SPZs transition to a migratory state and travel to the salivary glands. This transition is marked by a conformational change of CSP, rendering the α-epitope inaccessible to Abs and fragments thereof (non-adhesive form). sdAbs are expected to display better penetration properties due to their smaller size. Following deposition into the mammalian skin, the SPZs migrate to the liver, where they are primed for hepatocyte invasion through the interaction between CSP and highly sulfated heparan sulphate proteoglycans (HS-HSPGs). This interaction triggers the proteolytic cleavage of CSP by a cysteine protease, causing it to revert to its adhesive conformation and expose its α-epitope. Consequently, the SPZ switches to its invasion state, allowing it to invade a final hepatocyte and settle within a parasitophorous vacuole (PV) for its further development.</figcaption></figure>

fine-tuning of the model and provide novel insights into parasite biology inside the
mosquito host.

ACKNOWLEDGMENTS

The authors wish to thank the staff of the ID30A-3 beam line at ESRF Synchrotron (Philippe CARPENTIER) for the outstanding beam line support. The authors acknowledge the use of the CalcUA and VSC supercomputing facilities and wish to thank the staff for their outstanding support.

R.G. was supported by a DOCPRO4-NIEUWZAP (code 40043) grant awarded to Y.G.-J.S. by the University of Antwerp 'Bijzonder Onderzoeksfonds (BOF)'. L.D.V. is a doctoral fellow supported by the FWO-Vlaanderen (11P4B24N). This work was supported by the University of Antwerp 'Bijzonder Onderzoeksfonds' (41391 awarded to Y.G.-J.S.). The research was supported by the Agence Nationale de la Recherche (ANR, French National Research Agency)/Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project number SporoSTOP ANR-19-CE15-0027 (RA), and the French Government's Investissement d'Avenir Program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" project number ANR-10-LABX-62-IBEID (RA). M.C.A.-B. was funded by the Pasteur—Paris University International PhD Program.
## AUTHOR AFFILIATIONS

¹Laboratory of Medical Biochemistry (LMB) and the Infla-Med Centre of Excellence, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium

²Institut Pasteur, Université Paris Cité, Malaria Infection & Immunity, Paris, Île-de-France, France

³Lab of Cellular and Molecular Immunology (CMIM), Brussels Center for Immunology (BCIM), Department of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium

⁴Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium

## PRESENT ADDRESS

Rob Geens, TrypanosomaUnit, Department of Biomedical Sciences, Institute of Tropical Medicine Antwerp (ITM), Antwerp, Belgium

## AUTHOR ORCIDs

Yann G.-J. Sterckx http://orcid.org/0000-0002-7420-0983

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Universiteit Antwerpen (UAntwerp)</td><td>BOF DOCPRO4-NIEUWZAP (code 40043)</td><td>Yann G-J Sterckx</td></tr><tr><td>Fonds Wetenschappelijk Onderzoek</td><td>11P4B24N</td><td>Line De Vocht</td></tr><tr><td>Universiteit Antwerpen</td><td>BOF (41391)</td><td>Yann G-J Sterckx</td></tr><tr><td>Agence Nationale de la Recherche</td><td>SporoSTOP ANR-19-CE15-0027</td><td>Rogerio Amino</td></tr><tr><td>Laboratoires d'excellence "Integrative Biology of Emerging Infectious Diseases"</td><td>ANR-10-LABX-62-IBEID</td><td>Rogerio Amino</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Line De Vocht, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review and editing | Manuela C. Aguirre-Botero, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – review and editing | Cécile Vincke, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – review and editing | Stefan Magez, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review and editing | Serge Muyldermans, Investigation, Methodology, Resources, Supervision, Validation, Writing – review and editing | Yann G.-J. Sterckx, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing.

## DATA AVAILABILITY

The sdAb1-PfCSP<sub>C</sub> and sdAb9-PfCSP<sub>C</sub> crystal structures have been deposited in the PDB (accession codes 9HZJ and 9HZK). All other data are contained within this article or in the Supplemental material.

## ADDITIONAL FILES

The following material is available online.
Supplemental Material

Supplemental material (IAI00081-25-S0001). Table S1 to S4; Fig. S1 and S2.

REFERENCES

1. WHO. 2024. World malaria report 2024: addressing inequity in the global malaria response

2. Cowman AF, Healer J, Marapana D, Marsh K. 2016. Malaria: biology and disease. Cell 167:610-624. https://doi.org/10.1016/j.cell.2016.07.055

3. Kozlov M. 2021. Resistance to front-line malaria drugs confirmed in Africa. Nature New Biol 597:604–604. https://doi.org/10.1038/d41586-021-02592-6

4. NormileD. 2021. Threat of drug-resistant malaria looms in Africa. Science 373:1427-1427. https://doi.org/10.1126/science.acx9160

5. Draper SJ, Sack BK, King CR, Nielsen CM, Rayner JC, Higgins MK, Long CA, Seder RA. 2018. Malaria vaccines: recent advances and new horizons. Cell Host Microbe 24:43–56. https://doi.org/10.1016/j.chom.2018.06.008

6. LaurensMB. 2020. RTS,S/AS01vaccine(Mosquirix):an overview. Hum Vaccin Immunother 16:480–489. https://doi.org/10.1080/21645515.2019.1669415

7. Datoo MS, Dicko A, Tinto H, Ouédraogo J-B, Hamaluba M, Olotu A, Beaumont E, Ramos Lopez F, Natama HM, Weston S, et al. 2024. Safety and efficacy of malaria vaccine candidate R21/Matrix-M in African children: a multicentre, double-blind, randomised, phase 3 trial. The Lancet 403:533–544. https://doi.org/10.1016/S0140-6736(23)02511-4

8. Arun Kumar K, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC, Zavala F, Nussenzweig V. 2006. The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444:937–940. https://doi.org/10.1038/nature05361

9. Aly ASI, Vaughan AM, Kappe SHI. 2009. Malaria parasite development in the mosquito and infection of the mammalian host. Annu Rev Microbiol 63:195-221. https://doi.org/10.1146/annurev.micro.091208.073403

10. Graumans W, Jacobs E, Bousema T, Sinnis P. 2020. When is a plasmo-
dium-infected mosquito an infectious mosquito? Trends Parasitol
36:705-716. https://doi.org/10.1016/j.pt.2020.05.011

11. Kojin BB, Adelman ZN. 2019. The Sporozoite's journey through the mosquito: a critical examination of host and parasite factors required for salivary gland invasion. Front Ecol Evol 7:284. https://doi.org/10.3389/fev o.2019.00284

12. Aliprandini E, Tavares J, Panatieri RH, Thiberge S, Yamamoto MM, Silvie O, Ishino T, Yuda M, Dartevelle S, Traincard F, Boscardin SB, Amino R. 2018. Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin. Nat Microbiol 3:1224–1233. https://doi.org/ 10.1038/s41564-018-0254-z

13. Pinzon-Ortiz C, Friedman J, Esko J, Sinnis P. 2001. The binding of the circumsporozoite protein to cell surface heparan sulfate proteoglycans is required for *Plasmodium* sporozoite attachment to target cells. J Biol Chem 276:26784-26791. https://doi.org/10.1074/jbc.M104038200

14. Coppi A, Tewari R, Bishop JR, Bennett BL, Lawrence R, Esko JD, Billker O, Sinnis P. 2007. Heparan sulfate proteoglycans provide a signal to *Plasmodium* sporozoites to stop migrating and productively invade host cells. Cell Host Microbe 2:316–327. https://doi.org/10.1016/j.chom.2007.10.002

15. Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, Walker JR, Winzeler EA, Fujii H, Fontoura BMA, Nussenzweig V. 2007. *Plasmodium* circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131:492–504. https://doi.org/10.1016/j.cell.2007.09.013

16. Usynin I, Klotz C, Frevert U. 2007. Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells. Cell Microbiol 9:2610-2628.
https://doi.org/10.1111/j.1462-5822.2007.00982.x

17. Geens R, Stanisich J, Beyens O, D'Hondt S, Thiberge J-M, Ryckebosch A, De Groot A, Magez S, Vertommen D, Amino R, De Winter H, Volkov AN, Tompa P, Sterckx YG-J. 2024. Biophysical characterization of the *Plasmodium falciparum* circumsporozoite protein's N-terminal domain. Protein Sci 33:e4852. https://doi.org/10.1002/pro.4852

18. Doud MB, Koksal AC, Mi L-Z, Song G, Lu C, Springer TA. 2012. Unexpec-
ted fold in the circumsporozoite protein target of malaria vaccines. Proc
Natl Acad Sci U S A 109:7817–7822. https://doi.org/10.1073/pnas.1205737109

19. Nagar R, Garcia Castillo SS, Pinzon-Ortiz M, Patray S, Coppi A, Kanatani S, Moritz RL, Swearingen KE, Ferguson MAJ, Sinnis P. 2024. The major

surface protein of malaria sporozoites is GPI-anchored to the plasma
membrane. J Biol Chem 300:107557. https://doi.org/10.1016/j.jbc.2024.1
07557

20. Triller G, Scally SW, Costa G, Pissarev M, Kreschel C, Bosch A, Marois E, Sack BK, Murugan R, Salman AM, Janse CJ, Khan SM, Kappe SHI, Adegnika AA, Mordmüller B, Levashina EA, Julien J-P, Wardemann H. 2017. Natural parasite exposure induces protective human anti-malarial antibodies. Immunity 47:1197–1209. https://doi.org/10.1016/j.immuni.2 017.11007

21. Imkeller K, Scally SW, Bosch A, Martí GP, Costa G, Triller G, Murugan R, Renna V, Jumaa H, Kremsner PG, Sim BKL, Hoffman SL, Mordmüller B, Levashina EA, Julien J-P, Wardemann H. 2018. Antihomotypic affinity maturation improves human B cell responses against a repetitive epitope. Science 360:1358–1362. https://doi.org/10.1126/science.aar530 4

22. Murugan R, Scally SW, Costa G, Mustafa G, Thai E, Decker T, Bosch A, Prieto K, Levashina EA, Julien J-P, Wardemann H. 2020. Evolution of protective human antibodies against *Plasmodium falciparum* circumsporozoite protein repeat motifs. Nat Med 26:1135-1145. https://doi.org/10.1038/s41591-020-0881-9

23. Julien J-P, Wardemann H. 2019. Antibodies against *Plasmodium falciparum* malaria at the molecular level. Nat Rev Immunol 19:761-775. https://doi.org/10.1038/s41577-019-0209-5

24. Espinosa DA, Gutierrez GM, Rojas-López M, Noe AR, Shi L, Tse S-W, Sinnis P, Zavala F. 2015. Proteolytic cleavage of the *Plasmodium falciparum* circumsporozoite protein is a target of protective antibodies. J Infect Dis 212:1111-1119. https://doi.org/10.1093/infdis/jiv154

25. Herrera R, Anderson C, Kumar K, Molina-Cruz A, Nguyen V, Burkhardt M, Reiter K, Shimp R Jr, Howard RF, Srinivasan P, Nold MJ, Ragheb D, Shi L, DeCotiis M, Aebig J, Lambert L, Rausch KM, Muratova O, Jin A, Reed SG, Sinnis P, Barillas-Mury C, Duffy PE, MacDonald NJ, Narum DL. 2015. Reversible conformational change in the *Plasmodium falciparum* circumsporozoite protein masks its adhesion domains. Infect Immun 83:3771-3780. https://doi.org/10.1128/IAI.02676-14

26. Thai E, Costa G, Weyrich A, Murugan R, Oyen D, Flores-Garcia Y, Prieto K, Bosch A, Valleriani A, Wu NC, Pholcharee T, Scally SW, Wilson IA, Wardemann H, Julien J-P, Levashina EA. 2020. A high-affinity antibody against the CSP N-terminal domain lacks *Plasmodium falciparum* inhibitory activity. J Exp Med 217:e20200061. https://doi.org/10.1084/jem.20200061

27. Tan J, Cho H, Pholcharee T, Pereira LS, Doumbo S, Doumtabe D, Flynn BJ, Schön A, Kanatani S, Aylor SO, et al. 2021. Functional human IgA targets a conserved site on malaria sporozoites. Sci Transl Med 13:eabg2344. http://doi.org/10.1126/scitranslmed.abg2344

28. Kisalu NK, Idris AH, Weidle C, Flores-Garcia Y, Flynn BJ, Sack BK, Murphy S, Schön A, Freire E, Francia JR, et al. 2018. A human monoclonal antibody prevents malaria infection by targeting A new site of vulnerability on the parasite. Nat Med 24:408–416. https://doi.org/10.10 38/nm.4512

29. Tan J, Sack BK, Oyen D, Zenklusen I, Piccoli L, Barbieri S, Foglierini M, Fregni CS, Marcandalli J, Jongo S, Abdulla S, Perez L, Corradin G, Varani L, Sallusto F, Sim BKL, Hoffman SL, Kappe SHI, Daubenberger C, Wilson IA, Lanzavecchia A. 2018. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med 24:401–407. https://doi.org/10.1038/nm.4513

30. Scally SW, Murugan R, Bosch A, Triller G, Costa G, Mordmüller B, Kremsner PG, Sim BKL, Hoffman SL, Levashina EA, Wardemann H, Julien J-P. 2018. Rare PfCSP C-terminal antibodies induced by live sporozoite vaccination are ineffective against malaria infection. J Exp Med 215:63–75. https://doi.org/10.1084/jem.20170869

31. Beutler N, Pholcharee T, Oyen D, Flores-Garcia Y, MacGill RS, Garcia E, Calla J, Parren M, Yang L, Volkmuth W, Locke E, Regules JA, Dutta S, Emerling D, Early AM, Neafsey DE, Winzeler EA, King CR, Zavala F, Burton DR, Wilson IA, Rogers TF. 2022. A novel CSP C-terminal epitope targeted by an antibody with protective activity against *Plasmodium falciparum*. PLOS Pathog 18:e1010409. https://doi.org/10.1371/journal.ppat.1010409
32. Oludada OE, Costa G, Burn Aschner C, Obraztsova AS, Prieto K, Canetta C, Hoffman SL, Kremsner PG, Mordmüller B, Murugan R, Julien J-P, Levashina EA, Wardemann H. 2023. Molecular and functional properties of human *Plasmodium falciparum* CSP C-terminus antibodies. EMBO Mol Med 15:e17454. https://doi.org/10.15252/emmm.202317454

33. Coppi A, Natarajan R, Pradel G, Bennett BL, James ER, Roggero MA, Corradin G, Persson C, Tewari R, Sinnis P. 2011. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J Exp Med 208:341–356. https://doi.org/10.1084/jem.20101488

34. Coppi A, Pinzon-Ortiz C, Hutter C, Sinnis P. 2005. The *Plasmodium* circumsporozoite protein is proteolytically processed during cell invasion. J Exp Med 201:27–33. https://doi.org/10.1084/jem.20040989

35. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hammers C, Songa EB, Bendahman N, Hammers R. 1993. Naturally occurring antibodies devoid of light chains. Nature 363:446–448. https://doi.org/10.1038/363446a0

36. Muyldermans S. 2013. Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797. https://doi.org/10.1146/annurev-biochem-063011-092449

37. Romão. 2018. Construction of high-quality camel immune antibody libraries, p 169–187. In Hust M, Lim TS (ed), Phage display, vol 1701 of methods in molecular biology. Springer, New York, New York, NY.

38. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs, p 145–176. 2012. In Antibody Engineering, vol 907 of Methods in Molecular Biology. Humana Press, Totowa, NJ.

39. Wang LT, Pereira LS, Flores-Garcia Y, O'Connor J. 2020. A potent antimalarial human monoclonal antibody targets circumsporozoite protein minor repeats and neutralizes sporozoites in the liver. Immunity 53:733–744. https://doi.org/10.1016/j.immuni.2020.08.014

40. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LGJ, Muyldermans S, Wyns L, Matagne A. 2002. Single-domain antibody fragments with high conformational stability. Protein Sci 11:500–515. https://doi.org/10.1110/ps.34602

41. Aguirre-Botero MC, Pacios O, Celli S. 2024. Late killing of *Plasmodium berghei* in the liver by an anti-circumsporozoite protein monoclonal antibody. https://doi.org/10.1101/2024.04.30.591864

42. Kabsch W. 2010. XDS. Acta Crystallogr D Biol Crystallogr 66:125–132. https://doi.org/10.1107/S0907444909047337

43. Legrand P. 2017. XDSME:XDSMadeEasier. Available from: https://github.com/legrandp/xdsme

44. Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, Hintze B, Hung LW, Jain S, McCoy AJ, Moriarty NW, Oeffner RD, Poon BK, Prisant MG, Read RJ, Richardson JS, Richardson DC, Sammito MD, Sobolev OV, Stockwell DH, Terwilliger TC, Urzhumtsev AG, Videau LL, Williams CJ, Adams PD. 2019. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877. https://doi.org/10.1107/S2059798319011471

45. Agirre J, Atanasova M, Bagdonas H, Ballard CB, Baslé A, Beilsten-Edmans J, Borges RJ, Brown DG, Burgos-Mármol JJ, Berrisford JM, et al. 2023. The CCP4 suite: integrative software for macromolecular crystallography. Acta Crystallogr D Struct Biol 79:449–461. https://doi.org/10.1107/S2059798323003595

46. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. 2007. Phaser crystallographic software. J Appl Crystallogr 40:658–674. https://doi.org/10.1107/S0021889807021206

47. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589. https://doi.org/10.1038/s41586-021-03819-2

48. Tunyasuvunakool K, Adler J, Wu Z, Green T, Zielinski M, Žídek A, Bridgland A, Cowie A, Meyer C, Laydon A, et al. 2021. Highly accurate protein structure prediction for the human proteome. Nature 596:590–596. https://doi.org/10.1038/s41586-021-03828-1

49. Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. https://doi.org/10.1107/S0907444910007493

50. Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE. 2023. UCSF ChimeraX: tools for structure building and analysis. Protein Sci 32:e4792. https://doi.org/10.1002/pro.4792

51. Evans R, O'Neill M, Pritzel A, Antropova N, Senior A, Green T, Žídek A, Bates R, Blackwell S, Yim J, Ronneberger O, Bodenstein S, Zielinski M, Bridgland A, Potapenko A, Cowie A, Tunyasuvunakool K, Jain R, Clancy E, Kohli P, Jumper J, Hassabis D. 2022. Protein complex prediction with AlphaFold-multimer. Bioinformatics. https://doi.org/10.1101/2021.10.04.463034

52. Bryant P, Pozzati G, Zhu W, Shenoy A, Kundrotas P, Elofsson A. 2022. Predicting the structure of large protein complexes using AlphaFold and monte carlo tree search. Nat Commun 13:6028. https://doi.org/10.1038/s41467-023-33729-4

53. Shen M-Y, Sali A. 2006. Statistical potential for assessment and prediction of protein structures. Protein Sci 15:2507–2524. https://doi.org/10.1110/ps.062416606

54. Zhao J, Bhanot P, Hu J, Wang Q. 2016. A comprehensive analysis of *Plasmodium* circumsporozoite protein binding to hepatocytes. PLoS ONE 11:e0161607. https://doi.org/10.1371/journal.pone.0161607

55. Rathore D, McCutchan TF. 2000. Role of cysteines in *Plasmodium falciparum* circumsporozoite protein: interactions with heparin can rejuvenate inactive protein mutants. Proc Natl Acad Sci U S A 97:8530–8535. https://doi.org/10.1073/pnas.140224597

56. Rathore D, McCutchan TF. 2000. The cytotoxic T-lymphocyte epitope of the *Plasmodium falciparum* circumsporozoite protein also modulates the efficiency of receptor-ligand interaction with hepatocytes. Infect Immun 68:740–743. https://doi.org/10.1128/IAI.68.2.740-743.2000

57. Frénaud K, Dubremetz J-F, Lebrun M, Soldati-Favre D. 2017. Gliding motility powers invasion and egress in Apicomplexa. Nat Rev Microbiol 15:645–660. https://doi.org/10.1038/nrmicro.2017.86

58. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle K, Moyes CL, Henry A, Eckhoff PA, et al. 2015. The effect of malaria control on *Plasmodium falciparum* in Africa between 2000 and 2015. Nature 526:207–211. https://doi.org/10.1038/nature15535

59. Martin GM, Torres JL, Pholcharee T, Oyen D, Flores-Garcia Y, Gibson G, Moskovitz R, Beutler N, Jung DD, Copps J, Lee W-H, Gonzalez-Paez G, Emerling D, MacGill RS, Locke E, King CR, Zavala F, Wilson IA, Ward AB. 2023. Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection. Nat Commun 14:4546. https://doi.org/10.1038/s41467-023-40151-x

60. Fisher CR, Sutton HJ, Kaczmarski JA, McNamara HA, Clifton B, Mitchell J, Cai Y, Dups JN, D'Arcy NJ, Singh M, Chuah A, Peat TS, Jackson CJ, Cockburn IA. 2017. T-dependent B cell responses to *Plasmodium* induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein. PLOS Pathog 13:e1006469. https://doi.org/10.1371/journal.ppat.1006469

61. Yu T, Zheng F, He W, Muyldermans S, Wen Y. 2024. Single domain antibody: development and application in biotechnology and biopharma. Immunol Rev 328:98–112. https://doi.org/10.1111/imr.13381

62. Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Hölzer W, De Genst E, Wyns L, Muyldermans S. 1998. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17:3512–3520. https://doi.org/10.1093/emboj/17.13.3512

63. De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne J, Muyldermans S, Wyns L. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103:4586–4591. https://doi.org/10.1073/pnas.0505379103

64. Opuni KFM, Ruß M, Geens R, Vocht LD, Wielendaele PV, Debuy C, Sterckx YG-J, Glocker MO. 2024. Mass spectrometry-complemented molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the *Plasmodium falciparum* circumsporozoite protein's C-terminal domain. Comput Struct Biotechnol J 23:3300–3314. https://doi.org/10.1016/j.csbj.2024.08.023

65. Jalloh A, Jalloh M, Matsuoka H. 2009. T-cell epitope polymorphisms of the *Plasmodium falciparum* circumsporozoite protein among field isolates from Sierra Leone: age-dependent haplotype distribution? Malar J 8:120. https://doi.org/10.1186/1475-2875-8-120

66. Reece WHH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, Plebanski M, Akinwunmi P, Everaere S, Watkins KR, Voss G, Torniephor N, Alloueche A, Greenwood BM, Kester KE, McAdam KPWJ, Cohen J, Hill AVS. 2004. A CD4+ T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural *Plasmodium falciparum* infection and disease. Nat Med 10:406–410. http://doi.org/10.1038/nm1009

67. Stijlemans B, Conrath K, Cortez-Retamozo V, Van Xong H, Wyns L, Senter P, Revets H, De Baetselier P, Muyldermans S, Magez S. 2004. Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm. J Biol Chem 279:1256–1261. https://doi.org/10.1074/jbc.M307341200
68. Hempelmann A, Hartleb L, van Straaten M, Hashemi H, Zeelen JP, Bongers K, Papavasiliou FN, Engstler M, Stebbins CE, Jones NG. 2021. Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome. Cell Rep 37:109923. https://doi.org/10.1016/j.celrep.2021.109923

69. Wang LT, Pereira LS, Kiyuka PK, Schön A, Kisalu NK, Vistein R, Dillon M, Bonilla BG, Molina-Cruz A, Barillas-Mury C, Tan J, Idris AH, Francica JR,

Seder RA. 2021. Protective effects of combining monoclonal antibodies and vaccines against the *Plasmodium falciparum* circumsporozoite protein. PLOS Pathog 17:e1010133. https://doi.org/10.1371/journal.ppat.1010133