Polypeptide subdomain malaria vaccine

Abstract

The invention is in the field of vaccines, particularly in the field of polypeptide vaccines that target malaria. Malaria is caused by a parasite that is transmitted by mosquitoes. Transmission- blocking vaccines can prevent transmission from human to mosquito and thus can prevent spread of the disease. The invention provides polypeptides that show remarkable efficiency when used in a transmission-blocking vaccine strategy.

Description

[0001] Polypeptide subdomain malaria vaccine

[0002] Field of the invention

[0003] The invention is in the field of vaccines, particularly in the field of polypeptide vaccines that target malaria. Malaria is caused by a parasite that is transmitted by mosquitoes. Transmissionblocking vaccines can prevent transmission from human to mosquito and thus can prevent spread of the disease. The invention provides polypeptides that show remarkable efficiency when used in a transmission-blocking vaccine strategy.

[0004] Background a

[0005] The burden of malaria has increased in recent years, with 450,000 fatal malaria cases in 2016 rising to 608,000 in 2022. Malaria is caused by Plasmodium parasites, of which Plasmodium falciparum is the deadliest. These are transmitted by Anopheles mosquitoes. Transmission to mosquitoes relies on the uptake of sexual stage parasites, female and male gametocytes, through a bloodmeal. Inside the mosquito midgut, gametocytes activate to become female macrogametes and exflagellating male microgametes that fertilize to form zygotes. From this point onwards Plasmodium parasites continue their lifecycle by developing into ookinetes that traverse the epithelial layer and form oocysts on the outside of the mosquito midgut. Within these oocysts sporozoites are formed that find their way to the salivary glands, resulting in infectious mosquitoes.

[0006] Transmission to mosquitoes forms a bottleneck in the lifecycle of malaria parasites and is therefore an attractive target for interventions. Transmission-blocking vaccines (TBVs) target this bottleneck with the aim to reduce the number of mosquitoes that become infectious; TBVs thereby can form valuable assets for malaria elimination strategies (Alonso, P. L. et al. PLoS Med 8, e1000406, doi:10.1371 / journal.pmed.1000406 (2011)).

[0007] TBVs induce antibodies in the human host against surface antigens of gametes and / or zygotes. These antibodies are taken up via the bloodmeal together with gametocytes and human complement. Inside the midgut, where parasites egress from the red blood cells and become accessible to antibodies, the antibodies prevent further development of the parasite through neutralisation or activation of human complement that results in parasite lysis. The functional activity of antibodies is quantified by measuring the reduction in oocyst numbers compared to a negative control, and expressed as percentage transmission-reducing activity (TRA).

[0008] A clinically advanced TBV candidate is based on the polypeptide Pfs230 (SEQ ID NO: 1), which plays a role in fertilization and further development into oocysts. Pfs230 is an abundant gamete surface protein that consists of fourteen 6-Cys domains (Fig. 1A). The large size of Pfs230 hampers recombinant expression of full-length Pfs230. Therefore vaccine development has focused on expression of fragments of the protein (Duffy, P. E., Am J Trop Med Hyg, doi:10.4269 / ajtmh.21-1337 (2022)), resulting in a focus on the Pfs230 Pro-domain (Pro) and Domain 1 (D1). In immunization studies such polypeptides induced a functional response in rodents, leading to the development of Pfs230D1-EPA, a TBV candidate that progressed to phase 2 clinical studies (Sagara, I. et al. Lancet Infect Dis, doi:10.1016 / S1473-3099(23)00276-1 (2023)).

[0009] Not much is known about epitopes for functional antibodies in domains outside Pro and D1 . Studies showed that functional monoclonal antibodies (mAbs) induced by whole parasite immunization or natural exposure do in fact target Pfs230 epitopes outside ProD1 , yet the specific domain targeted by these mAbs remained unknown (Simons, L. M. et al. Vaccine 41 , 3367-3379, doi:10.1016 / j.vaccine.2023.04.042 (2023); Se Jong, R. M. et al. NPJ Vaccines 6, 101 , doi:10.1038 / s41541-021-00366-9 (2021); Axelle Amen et al., BioRxiv, doi:https: / / doi.org / 10.1101 / 2023.11 .03.565335 (2024).) Later studies identified the target of two of such functional mAbs as Domain 4 (D4) and Domain 7 (D7) (De Jong 2021 vide supra, Inklaar, M. R. et al. NPJ Vaccines 8, 186, doi:10.1038 / s41541-023-00784-x (2023)). However, recombinant fragments containing non-ProD1 fragments, such as fragments based on D4 or D7, have so far failed to induce a functional response in vivo (Tachibana, M. et al. Vaccine 37, 1799-1806, doi: 10.1016 / j.vaccine.2019.02.021 (2019)) and ProD1 thus remains the only Pfs230-based vaccine candidate with demonstrated in vivo efficacy described to date.

[0010] There is a need for improved strategies to reduce the spread of Malaria. There is a need for improved Malaria vaccines. There is a need for complementary or supplementary strategies to reduce the spread of Malaria. There is a need for improved polypeptide vaccines. There is a need for improved or complementary or supplementary compositions with transmission-reducing activity

[0011] Summary of the invention

[0012] The inventors surprisingly found that properly formed Domain 12 (D12) polypeptides induced antibodies with strong transmission-reducing activity (TRA) in membrane feeding assays with lab cultured parasites as well as membrane feeding assays with naturally circulating parasites from human donors. It was also found that human subjects with natural exposure to malaria parasites can possess antibodies that recognize this subdomain (Pfs230D12). These results confirm that polypeptides based on Pfs230D12 are suitable for development as transmission-blocking vaccine (TBV) candidate.

[0013] Thus the invention provides a polypeptide comprising a first amino acid sequence that has at least 80% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is domain 12.

[0014] Preferably the first amino acid sequence has a length of 120-200 amino acids, preferably of 145-165 amino acids, or wherein the polypeptide has a length of 160-180 amino acids, preferably 165-175 amino acids. Preferably the first amino acid sequence comprises at least 100 consecutive amino acids from Pfs230. In some embodiments first amino acid sequence has at least 90% sequence identity with SEQ ID NO: 14, preferably at least 95%, more preferably 100%.

[0015] In some embodiments the polypeptide further comprises a first recognition tag, wherein the first recognition tag is preferably C-terminal to the first amino acid sequence, wherein the first recognition tag preferably consists of the sequence EPEA. Preferably the polypeptide does not comprise a sequence represented by SEQ ID NO: 535. Preferably the first recognition tag and the first amino acid sequence are separated by a linker sequence, wherein the linker sequence preferably comprises 1-40 amino acids. Preferably the polypeptide exhibits transmission-reducing activity of at least 40%, wherein transmission-reducing activity is assessed using a standard membrane feeding assay (SMFA).

[0016] Also provided is a composition comprising a polypeptide as described above and a pharmaceutically acceptable excipient, the composition optionally comprising one or more further polypeptides comprising a first amino acid sequence that has at least 80% sequence identity with Plasmodium falciparum surface protein Pfs230 subdomain, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is a domain that is not domain 12.

[0017] Also provided is the polypeptide or the composition, for use as a medicament, wherein the medicament is preferably for the prevention of malaria. Also provided is a nucleic acid construct comprising a first nucleotide sequence encoding a polypeptide as defined above. Preferably the nucleic acid construct comprises a second nucleotide sequence that is 5’ of the first nucleotide sequence, wherein the second nucleotide sequence encodes a signal peptide such as a binding protein (BiP) signal peptide. Also provided is an expression vector comprising the nucleic acid construct as defined above. Also provided is a cell comprising the nucleic acid construct or comprising the expression vector.

[0018] Also provided is a method for providing a polypeptide as defined above, the method comprising the steps of: i) providing a cell as defined above wherein the cell comprises a nucleic acid construct comprising a first and a second sequence as described above; ii) culturing the cell under conditions conducive to polypeptide expression to produce the polypeptide; and iii) optionally purifying the produced polypeptide.

[0019] Description of embodiments

[0020] The inventors surprisingly found that properly formed Domain 12 (D12) polypeptides induced antibodies with strong transmission-reducing activity (TRA) in membrane feeding assays with lab cultured parasites and as well as membrane feeding assays with naturally circulating parasites from human donors. It was also found that human subjects with natural exposure to malaria parasites can possess antibodies that recognize this subdomain (Pfs230D12). These results confirm that polypeptides based on Pfs230D12 are suitable for development as transmission-blocking vaccine (TBV) candidate.

[0021] Accordingly the invention provides a polypeptide comprising a first amino acid sequence that has at least 80% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is domain 12. Such polypeptides can be referred to herein as polypeptides according to the invention.

[0022] In other embodiments the subdomain is domain 1. In other embodiments the subdomain is domain 2. In other embodiments the subdomain is domain 3. In other embodiments the subdomain is domain4. In other embodiments the subdomain is domain 5. In other embodiments the subdomain is domain 6. In other embodiments the subdomain is domain 7. In other embodiments the subdomain is domain 8. In other embodiments the subdomain is domain 9. In other embodiments the subdomain is domain 10. In other embodiments the subdomain is domain 1 1 . In other embodiments the subdomain is domain 13. In other embodiments the subdomain is domain 14.

[0023] The polypeptide has a length of 100-300 amino acids. In some embodiments the polypeptide has a length of 110-290, 120-280, 130-270, 140-260, 150-250, 160-240, 170-230, 180-220, 190- 210, or 195-200 amino acids. In preferred embodiments the polypeptide according to the invention has a length of 140-200 amino acids, more preferably 150-190 amino acids, even more preferably 160-180 amino acids, most preferably 165-175 amino acids such as 168, 169, or 170 amino acids, preferably 169 amino acids.

[0024] First amino acid sequence

[0025] Comprised in the polypeptide according to the invention is a first amino acid sequence that has at least 80% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230. This first amino acid sequence has a length of at least 100 amino acids. Preferably, the first amino acid sequence has a length of 100-300 amino acids, more preferably of 110-250 amino acids, even more preferably 115-200 amino acids, still more preferably 120-200 amino acids. In preferred embodiments the first amino acid sequence has a length of 120-200 amino acids, preferably of 125- 195, more preferably of 130- 190, more preferably of 135-185, more preferably of 140-180, more preferably of 145-175, still more preferably of 145-170, even more preferably of 145-165 amino acids, most preferably of 150-160 amino acids, such as 154, 155, or 156 amino acids, preferably 155 amino acids.

[0026] Plasmodium falciparum surface protein Pfs230 is a polypeptide Pfs230 (SEQ ID NO: 1) that plays a role in parasite fertilization and further development into oocysts. Pfs230 is an abundant gamete surface protein that consists of fourteen 6-Cys domains (Fig. 1A). These fourteen domains, as well as the Pro-domain at the N-terminus, are referred to as subdomains of Pfs230. The table below shows which SEQ ID NO represent which subdomain of Pfs230. Accordingly, when an amino acid sequence of subdomain 12 is referred to, this can be seen as reference to SEQ ID NO: 14. Subdomains are generally referred to by their number, preceded by a capital D. Accordingly D12 refers to subdomain 12, which refers to a polypeptide consisting of the amino acids shown in SEQ ID NO: 14. A subdomain can also be referred to as a domain when it is clear from context which subdomain is being referred to. For instance, domain 12 and subdomain 12 can be used interchangeably.

[0027] The first amino acid sequence that has at least 80% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230. Preferably it has at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with the subdomain. In preferred embodiments it has at least 85%, more preferably at least 90%, still more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, most preferably 100% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230.

[0028] Particularly, in preferred embodiments the first amino acid sequence has at least 90% sequence identity with SEQ ID NO: 14, preferably at least 95%, more preferably at least 98%, still more preferably 100%. In other embodiments the first amino acid sequence has at least 90% sequence identity with any one of SEQ ID NOs: 3-16, preferably at least 95%, more preferably at least 98%, still more preferably 100%. The first amino acid sequence preferably comprises at least 90, more preferably at least 100, still more preferably at least 110, even more preferably at least 120, even more preferably at least 130, even more preferably at least 140, most preferably at least 150 consecutive amino acids from Pfs230 (SEQ ID NO: 1). In preferred embodiments the first amino acid sequence consists of consecutive amino acids from Pfs230.

[0029] Further characteristics of the polypeptide

[0030] The polypeptide according to the invention can comprise further sequences in addition to the first amino acids sequence. For instance, polypeptides can be made more versatile by introduction of one or more recognition tags. Such tags are widely known, and can aid in purification (for instance a histidine-tag) or recognition (for instance a FLAG-tag). In this context a tag can be as small as a single residue, although a tag will generally have a length of at least 2, 3, or 4 amino acids, preferably a recognition tag has a length of at least 3 amino acids, more preferably at least 4. In general, tags are designed to be small so as to not influence the overall behavior of the polypeptide.

[0031] In preferred embodiments the polypeptide further comprises a first recognition tag, wherein the first recognition tag is preferably C-terminal to the first amino acid sequence. In other embodiments the polypeptide further comprises a first recognition tag, wherein the first recognition tag is preferably N-terminal to the first amino acid sequence.

[0032] In some embodiments the polypeptide comprises a first recognition tag, and a second recognition tag, and optionally a third recognition tag, wherein the second recognition tag is distinct from the first recognition tag, and wherein the second recognition tag is distinct from the third recognition tag, and wherein the first recognition tag is distinct from the third recognition tag. It is generally not useful to feature multiple instances of the same tag. In some embodiments, the polypeptide comprises a first recognition tag, and comprises a second recognition tag.

[0033] Suitable recognition tags are an epitope tag, an affinity-tag, or an aptamer-tag, preferably the recognition tag is a histidine-tag or a FLAG-tag or a C-tag or a Strep-tag, more preferably a C- tag or a Strep-tag.

[0034] An epitope tag is a tag that can be recognized by an antibody. An example of an epitope tag is a C-tag, which is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed. This makes epitope tags (such as C-tag) suitable for purification ofthe linked polypeptide (for instance via affinity chromatography). C-tag is represented by the four amino acids EPEA. Examples of a first amino acid sequence followed by two amino acids followed by a C-tag are SEQ ID NOs: 48-61 , which represent polypeptides wherein the first amino acid sequence consists of subdomains 1-14, respectively.

[0035] An affinity-tag is a tag that can bind a material. Affinity-tags allow polypeptides to be purified from for instance their crude biological source using an affinity technique such as by using histidine- tag (His-tag), chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, or glutathione- S-transferase (GST). Preferred affinity-tags are histidine-tags and Strep-tags. Such tags are widely known. A suitable Strep-tag is represented by SEQ ID NO: 531 . Suitable His-tags are represented by SEQ ID NOs: 528-530. Examples of a first amino acid sequence preceded by two amino acids preceded by a His-tag are SEQ ID NOs: 108-121 , which represent polypeptides wherein the first amino acid sequence consists of subdomains 1-14, respectively.

[0036] In preferred embodiments the polypeptide according to the invention comprises a first recognition tag, wherein the first recognition tag consists of the sequence EPEA. This is referred to as a C-tag. Most preferably the C-tag is C-terminal to the first amino acid sequence. Most preferably the C-tag is at the C-terminus.

[0037] When a polypeptide according to the invention comprises a first recognition tag and a second recognition tag, it is preferred that at least one of the recognition tags is a C-tag or a Strep- tag. When a C-tag is present it is preferably C-terminal. When a Strep-tag is present it is preferably N-terminal. When a second recognition tag is present it can be a His-tag. When a His-tag is present it is preferably N-terminal.

[0038] Within the polypeptide according to the invention, the first amino acids sequence and any further sequences such as recognition tags can be directly adjacent to one another, together forming a contiguous sequence. These different elements can also be separated by a linker. For ease of manufacture, synthesis, or handling, it can be convenient to use such a linker, and a linker can conveniently consist of one or more amino acid residues within the polypeptide. Linker sequences can consist of a mere single amino acid such as a single glycine residue, and they can also be longer sequences. The function of a linker sequence is to separate relevant elements from one another, and linkers are therefore often relatively simple in their design. A skilled person can recognize a linker sequence. Provided are polypeptides according to the invention, wherein the polypeptide comprises a first recognition tag, wherein the first recognition tag and the first amino acid sequence are separated by a linker sequence, wherein the linker sequence preferably comprises 1-40 amino acids.

[0039] In preferred embodiments, the linker comprises only glycine, alanine, serine, and / or threonine residues, more preferably only glycine, alanine, and / or serine residues, most preferably only glycine and / or serine residues. Preferably a linker comprises predominantly glycine residues, such as comprising at least 50% glycine residues. For reasons related to solvent interactions, serine or threonine residues are attractive. Preferred linkers comprise at least one serine or threonine residue. More preferably, a linker comprises at least 10%, more preferably at least 25% serine residues. When a linker has a length of at least 4 amino acids, the linker preferably has at least 1 serine or threonine residue. Suitable linkers are represented by GG, GGG, AG, AGG, GS, and GGS.

[0040] In preferred embodiments a linker sequence comprises 1-20 amino acids, preferably 1-15 amino acids, more preferably 1-10 amino acids, even more preferably 1-6 amino acids. Preferably the linker comprises 1 , 2, 3, 4, 5, or 6 amino acids, more preferably 1 , 2, 3, or 4 amino acids, even more preferably 2 or 3 amino acids, most preferably 2 amino acids. Most preferably a linker is GG, AG, GA, SG, or GS, and good results were obtained with AG and GS. When multiple linkers are present, these can be individually selected.

[0041] When a first recognition tag is present, a linker preferably separates the first amino acid sequence and the first affinity tag. When a second recognition tag is present, a linker preferably separates the first amino acid sequence and the second affinity tag. When a linker is N-terminal to the first amino acid sequence, it preferably comprises AG, more preferably consists of AG. When a linker is C-terminal to the first amino acid sequence, it preferably comprises GS, more preferably consists of GS.

[0042] SEQ ID NOs: 48-61 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has a C-terminal first recognition tag (here: a C-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, XX).

[0043] SEQ ID NOs: 63-76 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has a C-terminal first recognition tag (here: a C-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, GS).

[0044] SEQ ID NOs: 78-91 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has an N-terminal first recognition tag (here: a His-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, XX).

[0045] SEQ ID NOs: 93-106 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has an N-terminal first recognition tag (here: a His-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, AG).

[0046] SEQ ID NOs: 108-121 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has a C-terminal first recognition tag (here: a C-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, XX) and has an N-terminal second recognition tag (here: a His-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, XX).

[0047] SEQ ID NOs: 123-136 provide examples of polypeptides according to the invention wherein the first amino acid sequence (for the SEQ ID NOs the first amino acid sequence consists of subdomains 1-14, respectively) has a C-terminal first recognition tag (here: a C-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, GS) and has an N-terminal second recognition tag (here: a His-tag) that is separated from the first amino acid sequence by a linker (here: two amino acids, AG).

[0048] Preferred embodiments of the invention are polypeptides represented by SEQ ID NOs: 3- 16, particularly SEQ ID NO: 14 which encodes D12. More preferred embodiments of the invention are polypeptides represented by SEQ ID NOs: 48-61 , particularly SEQ ID NO: 59 which comprises D12. Even more preferred embodiments of the invention are polypeptides represented by SEQ ID NOs: 63-76, particularly SEQ ID NO: 74 which comprises D12. Still more preferred embodiments of the invention are polypeptides represented by SEQ ID NOs: 108-121 , particularly SEQ ID NO: 119 which comprises D12. Most preferred embodiments of the invention are polypeptides represented by SEQ ID NOs: 123-136, particularly SEQ ID NO: 134 which comprises D12.

[0049] Tachibana et al. describe how their polypeptides related to domain 12 do not lead to transmission blocking, and this is linked to these polypeptides not being in a native conformation. Polypeptides according to the present invention do lead to transmission blocking. Analysis of the polypeptides according to the invention reveals that reducing conditions lead to a shift in mass. This can indicate that the polypeptides according to the invention comprise one or more dithiol bridges. Accordingly, preferred polypeptides according to the invention comprise one or more dithiol bridges. Preferred polypeptides according to the invention are in their native conformation. Preferred polypeptides according to the invention are folded. Preferred polypeptides according to the invention are folded as in the native Pfs230 protein. Preferred polypeptides according to the invention are obtained by a production method according to the invention. Preferred polypeptides according to the invention are obtainable by a production method according to the invention.

[0050] It was found that polypeptides as described in the prior art did not achieve transmissionreducing activity (TRA) to any useful extent. The present invention demonstrates how for example polypeptides related to domain 12 do achieve transmission-reducing activity. In preferred embodiments the polypeptide according to the invention exhibits transmission-reducing activity of at least 20%, preferably at least 30%, more preferably at least 40%, still more preferably at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, most preferably at least about 90%. TRA can be assessed using a standard membrane feeding assay (SMFA). A suitable SMFA is an assay wherein mice sera are diluted in FCS and mixed with mature P. falciparum NF54 gametocytes and NHS. Anopheles stephensi mosquitoes can then be fed blood meals, after which unfed and partially fed mosquitoes can be removed. A suitable number such as 20 mosquitoes per condition can be dissected 6-8 days after the blood meal to collect their midguts. The midguts can be stained, for instance with mercurochrome, and oocysts can be counted. TRA can be defined as the reduction in oocyst intensity (oocysts per mosquito midgut) in a test condition compared to a negative control in which no mice sera (FBS control) was added. In preferred embodiments the polypeptide according to the invention induces transmission-reducing activity in a vaccinated subject. Transmission-reducing activity is preferably as compared to transmission of parasites in mosquitoes who fed on non-vaccinated subjects. TRA can further be assessed as detailed in the Examples. SMFA can further be performed as detailed in the Examples. In preferred embodiments the polypeptide according to the invention induces a reduction in oocyst numbers. Preferably the polypeptide according to the invention is for use as a medicament, wherein the medicament is for reduction of oocyst numbers. Oocysts are preferably reduced in mosquitoes. Reduction is preferably by at least 20%, preferably at least 30%, more preferably at least 40%, still more preferably at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, most preferably at least about 90%. Oocysts are preferably Plasmodium, more preferably Plasmodium falciparum oocysts.

[0051] In preferred embodiments the polypeptide according to the invention does not comprise a His-tag that is C-terminal to the first amino acid sequence. In more preferred embodiments the polypeptide according to the invention does not comprise a His-tag that is C-terminal to the first amino acid sequence wherein the His-tag consists of six consecutive histidine residues (SEQ ID NO: 528). In even more preferred embodiments, when the polypeptide according to the invention comprises a His-tag that is C-terminal to the first amino acid sequence, the His-tag comprises SEQ ID NO: 529 (H7) or SEQ ID NO: 530 (H8).

[0052] In preferred embodiments the polypeptide according to the invention does not comprise a sequence represented by SEQ ID NO: 535.

[0053] Further Products

[0054] The invention provides a composition comprising a polypeptide according to the invention and a pharmaceutically acceptable excipient. Preferably, such a composition is formulated as a pharmaceutical composition. Preferably such a composition is formulated for oral administration or for injection, preferably parenteral injection such as subcutaneous injection. A preferred excipient is water, preferably purified water, more preferably ultrapure water. Further preferred excipients are adjuvants, binders, desiccants, or diluents. Further preferred compositions additionally comprise additional medicaments for treating malaria or for treating fever or inflammation. Preferred additional medicaments in this regards are further distinct malaria vaccines. The invention provides a composition comprising a polypeptide according to the invention and a pharmaceutically acceptable excipient, comprising one or more further polypeptides comprising a first amino acid sequence that has at least 80% sequence identity with Plasmodium falciparum surface protein Pfs230 subdomain, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the first amino acid sequence of the polypeptide and of the further polypeptide do not have at least 80% sequence identity with the same subdomain.

[0055] Thus the invention provides a composition comprising a polypeptide according to the invention and a pharmaceutically acceptable excipient, optionally comprising one or more further polypeptides comprising a first amino acid sequence that has at least 80% sequence identity with Plasmodium falciparum surface protein Pfs230 subdomain, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is a domain that is not domain 12. In some embodiments the one or more further polypeptides are not optional.

[0056] Also provided is a nucleic acid construct comprising a first nucleotide sequence encoding a polypeptide according to the invention. A skilled person knows how to obtain a nucleic acid sequence that encodes any particular polypeptide. Preferably the first nucleotide sequence is codon-optimised.

[0057] The first nucleotide sequence preferably has at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NOs: 258-527, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, still more preferably 98%, most preferably 100% sequence identity. Particularly preferred are SEQ ID NOs: 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 435, 450, 465, 480, 495, 510, and 525, which encode domain 12. Of these, SEQ ID NOs: 405, 420, 435, 450, 465, 480, 495, 510, and 525 are preferred for also encoding a BiP-tag as described later herein. Of these, SEQ ID NOs: 450, 465, 480, 495, 510, and 525 are more preferred for also encoding a BiP-tag and further also encoding a first recognition tag (here: a C-tag). In preferred embodiments the sequence identity is over the entire length of the sequence. In preferred embodiments the first nucleotide sequence has the same length as the SEQ ID NO it has sequence identity with. The nucleic acid construct preferably does not encode SEQ ID NO: 535. When the nucleic acid construct encodes a polypeptide wherein the first amino acid sequence is followed by a His-tag, the His-tag is preferably represented by SEQ ID NOs: 529 or 530.

[0058] Also provided is an expression vector comprising the nucleic acid construct as described above. In such an expression vector the first nucleotide sequence is preferably operably linked to a promoter. Also provided is a cell such as a host cell comprising the expression vector as described above or comprising the nucleic acid construct as described above. Host cells are preferably for multiplication of other products according to the invention, in which case the host cell can be any suitable microbial cell, such as E. coll. Excellent results were obtained when the host cell was a Drosophila melanogaster cell, particularly an S2 cell (Schneider 2 cell). Thus a preferred embodiment is a Drosophila melanogaster cell comprising an expression vector as described above or comprising the nucleic acid construct as described above.

[0059] A "nucleic acid construct" or "nucleic acid vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology or synthetic techniques. The term "nucleic acid construct" therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention.

[0060] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

[0061] As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. An inducible promoter may also be present but not induced.

[0062] Preferred nucleic acid constructs are optimised for polypeptide production in suitable cells. For example, preferred nucleic acid constructs according to the invention are those comprising a second nucleotide sequence that is 5’ or 3’, preferably 5’, of the first nucleotide sequence, wherein the second nucleotide sequence encodes a signal peptide such as a binding protein (BiP) signal peptide. A signal peptide (which can also be referred to as a signal sequence, a targeting signal, a localization signal, or other known terms) is a short peptide that is usually 10-30, preferably 15-25 amino acids long and that is generally present at a terminus of a newly synthesized polypeptide when the polypeptide is expressed. Most signal peptides are at the N-terminus. Some signal peptides are at the C-terminus. A signal peptide can direct the polypeptide toward a secretory pathway. For instance, many type I membrane-bound proteins have signal peptides.

[0063] A signal peptide is generally cleaved off of the polypeptide by the action of enzymes present in the host cell, of which signal peptidases are suitable examples. Thus a polypeptide comprising a signal peptide is generally a pre-peptide, and can be cut into the signal peptide and the mature polypeptide. A skilled person can select a suitable signal peptide, and a skilled person can also select a suitable nucleic acid sequence that encodes for a signal peptide. Preferably the signal peptide is directly adjacent to the remainder of the (mature) polypeptide. Accordingly, preferably the second nucleotide sequence is directly adjacent to the first nucleotide sequence.

[0064] A preferred signal peptide is binding protein (BiP). BiP is an endoplasmic reticulum chaperone that plays a role in protein folding and quality control in the endoplasmic reticulum lumen. It is involved in the correct folding of proteins and degradation of misfolded proteins. An example of BiP is SEQ ID NO: 533 (amino acids) and an example of a suitable second nucleotide sequence can therefore be any sequence that encodes SEQ ID NO: 533, such as SEQ ID NO: 534. For nucleic acid constructs BiP is preferably 5’ of the remainder of the nucleic acid construct. For polypeptides BiP is preferably N-terminal to the remainder of the polypeptide.

[0065] Examples of polypeptides comprising BiP are SEQ ID NOs: 137-256, of which SEQ ID NOs: 149, 164, 179, 194, 209, 224, 239, and 254 are preferred for comprising D12. Nucleic acid constructs encoding a polypeptide and a BiP-tag have been described earlier herein.

[0066] Medical use

[0067] As described above, prevention of parasite transmission helps reduce the transmission of malaria. Thus the invention provides the polypeptide according to the invention or the composition according to the invention, for use as a medicament. The medicament is preferably for the prevention of malaria. The medicament is preferably for reducing transmission of parasite to mosquitos, preferably for reducing the number of mosquitoes that become infectious. A preferred mosquito is an Anopheles mosquito. A preferred parasite is a Plasmodium parasite, more preferably Plasmodium falciparum. The medicament is preferably for reducing transmission of parasite gametes and / or zygotes. The medicament is preferably for promoting parasite lysis. In reducing the transmission of parasites, the medicament is for preventing malaria, or for preventing some instances of malaria.

[0068] Also provided is a method for preventing malaria, or for preventing transmission of malaria, the method comprising the step of administering a polypeptide or composition as described above to a subject. The subject is preferably human. The subject is preferably in need of receiving a polypeptide or composition as described above. The polypeptide or composition as described above is preferably administered in an effective amount. The method can also be for preventing mosquitoes from becoming infectious. The medical use and methods as described here can be used to treat various subjects. A preferred subject is a subject in need of treatment, which can be a subject suffering from malaria, or a subject expected to develop, or at risk of developing, malaria, or a subject residing in, or intending to reside in, an area where malaria is endemic.

[0069] Formulation of medicaments, ways of administration, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia.

[0070] Provision of polypeptides

[0071] The polypeptides of the invention can be advantageously produced using a suitable cell line. Accordingly the invention provides a method for providing a polypeptide as defined above, the method comprising the steps of: i) providing a cell as defined above wherein the cell comprises a nucleic acid construct as defined above; ii) culturing the cell under conditions conducive to polypeptide expression to produce the polypeptide; and iii) optionally purifying the produced polypeptide. In preferred embodiments this purification of step iii) is not optional. Such a method is referred to herein as a production method according to the invention.

[0072] The cell as provided in step i) is preferably a Drosophila cell, more preferably a Drosophila melanogaster S2 cell. Such cells are commercially available, for instance from ExpreS2ion Biotechnologies. Culturing of step ii) can be done in any suitable way, as known to the skilled person. For instance, cells can be cultured in shake flasks, preferably with vented cap, in a suitable medium such as for example in EX-CELL420 media (Sigma-Aldrich). Medium is preferably supplemented, such as with antibiotics, preferably 1 % penicillin and streptomycin. Culturing is preferably at about 20-30°C such as 25°C, shaking is preferably performed, for example at 100- 140 rpm, such as at 115 rpm. Cells as provided in step i) preferably comprise a nucleic acid construct as defined earlier herein, more preferably wherein the nucleic acid construct also encodes a signal peptide, more preferably a BiP-tag as described herein. The nucleic acid construct or the can be in a vector as described earlier herein. The nucleic acid construct can be transfected to the cell, for instance using ExpreS2 Insect-TR 5x transfection reagent (ExpreS2ion Biotechnologies), preferably in a T12.5 T-flask. Conditions conducive to polypeptide expression can be incubation at 20-30°C such as at 25°C, and serum such as FBS can be present. A selection agent can be used, such as 4000 pg / mL geneticin. Polypeptide expression can be performed for multiple days, for instance for 5-50 days such as for 20-40 days, preferably for 25-32 days. Expression can be at a suitable density such as 4x105cells / mL to 8x107cells / mL, preferably about 8x106cells / mL.

[0073] Purification of step iii) can be performed using any known means. When the polypeptide comprises a recognition tag, the polypeptide can be purified by using the recognition tag. For instance, when the polypeptide comprises a C-tag, it can be affinity purified, for example using C- tagXL pre-packed columns (Thermo Scientific). More examples and details are provided in the Examples section. General definitions

[0074] In preferred embodiments, compounds and compositions according to the invention are for use in methods according to the invention, or are for use according to the invention. Compounds or compositions are preferably obtainable by or obtained by methods according to the invention. Each embodiment as identified herein may be combined together unless otherwise indicated.

[0075] Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components.

[0076] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0077] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.

[0078] "Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences ortwo or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0079] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0080] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0081] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

[0082] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Vai to ile or leu.

[0083] A “nucleic acid molecule” or “polynucleotide” is represented by a nucleotide sequence. A “polypeptide” is represented by an amino acid sequence. A “polypeptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" can encompass naturally occurring and synthetic residues or molecules.

[0084] In the context of this invention, a cell or a sample can be a cell or a sample from a sample obtained from a subject. Such an obtained sample can be a sample that has been previously obtained from a subject. Such a sample can be obtained from a human subject. Such a sample can be obtained from a non-human subject.

[0085] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0086] Description of drawings

[0087] Fig. 1A - Schematic representation of full-length Pfs230. Domain boundaries are indicated by amino acid numbers and are based on predictions made by Gerloff et al. (2005). Single domain fragments with these boundaries were expressed in D. melanogaster S2 cells.

[0088] Fig. 1 B - Analysis of recombinant Pfs230 domain protein constructs produced in S2 cells, by western blot analysis of S2 cell supernatants. Supernatants of D1 through D14 were harvested from stable transfected cell lines. Supernatants were separated by SDS-PAGE, transferred to western blot, and stained with 1 / 1000 C-tag antibody and 1 :2500 IRDye Streptavidin 680LT. + Ctrl: Pro-CS3- 6C; - Ctrl: supernatant from un-transfected cells.

[0089] Fig. 1 C - Coomassie-stained SDS PAGE gel of purified single domain fragments (specifically D1 , D5, D6 and D8) produced in Drosophila melanogaster S2 cells under reducing conditions (R) and non-reducing conditions (NR).

[0090] Fig. 1 D - Coomassie-stained SDS PAGE gel of purified single domain fragments (specifically D9, D10, D12 and D13) produced in Drosophila melanogaster S2 cells under reducing conditions (R) and non-reducing conditions (NR).

[0091] Fig. 1 E - Western blot of recombinant fragments with a-C-tag antibody under non-reducing conditions. + and - controls are C-tagged protein and untransfected S2 cell line, respectively.

[0092] Fig. 1 F - Glycosylation-stained SDS PAGE gel of recombinant fragments under non-reducing conditions. + and - controls are control proteins provided with the PierceTM glycoprotein staining kit.

[0093] Fig. 2A - Antigen and parasite recognition by mouse antibodies raised against single Pfs230 domains (as in Fig. 2B and 2C). Midpoint titers from antigen specific ELISA. Each dot represents an individual mouse and bars represent median values.

[0094] Fig. 2B - Gametocyte extract ELISA with pooled mouse sera, tested at 1 :100 dilution. Values are means from two independent experiments with three technical replicates each and error bars represent s.e.m. Pre-I, pre-immune serum. Statistical analysis is done by comparing test groups to pre-immune group using ordinary one-way ANOVA with a Dunnett’s multiple comparison test using Pre-I as a reference (ns=not significant, ***=p<0.001 , ****=p<0.0001).

[0095] Fig. 2C - Female gamete binding assay with pooled mouse sera, tested at 1 :40 dilution. Values are means (MFI= Mean Fluorescence Intensity) from two independent experiments with two technical replicates each, and error bars indicate s.e.m. The data was normalised against 230CMB to allow averaging across experiments. Statistical analysis is performed as in Fig. 2B.

[0096] Fig. 3A - Antigen specific ELISA with sera from immunized mice with 230CMB. The sera of each individual mouse were titrated along with the pooled pre-immune sera. Sigmoidal curve fits were used to calculate EC50 values that are shown in Fig. 2A.

[0097] Fig. 3B - As in Fig. 3A, but following immunization with D1 .

[0098] Fig. 3C - As in Fig. 3A, but following immunization with D5.

[0099] Fig. 3D - As in Fig. 3A, but following immunization with D6.

[0100] Fig. 3E - As in Fig. 3A, but following immunization with D8.

[0101] Fig. 3F - As in Fig. 3A, but following immunization with D9.

[0102] Fig. 3G - As in Fig. 3A, but following immunization with D10.

[0103] Fig. 3H - As in Fig. 3A, but following immunization with D12.

[0104] Fig. 3I - As in Fig. 3A, but following immunization with D13.

[0105] Fig. 4A - Functional activity of mouse antibodies raised against single Pfs230 domains (as in Fig. 4B-4E). Transmission reducing activity (TRA) of pooled mouse sera in standard membrane feeding assay (SMFA) with cultured Plasmodium falciparum NF54 gametocytes and Anopheles stephensi mosguitoes. Values are estimates from two independent SMFA experiments with oocyst counts for 20 fully-fed mosguitoes per condition each. Dotted line indicates 80% TRA, which has previously been established as threshold for clinical development (Sauerwein and Bousema, 2015). Error bars indicate 95% confidence intervals. Pooled sera were tested at a final dilution of 1 :9 in the presence of active human complement.

[0106] Fig. 4B - TRA of pooled mouse sera in SMFA, in the presence of active (+) or heat-inactivated (-) human complement. Pooled mouse sera were tested at a final dilution of 1 :9. Values are estimates from two independent experiments with oocysts counts for 20 fully-fed mosguitoes per condition per experiment.

[0107] Fig. 4C - C1 deposition on the surface of female gametes in the presence of 2.5% pooled mouse serum, as assessed by flow cytometry. Values are means from two independent experiments with two technical replicates each and error bars indicate s.e.m. Data was normalised against 230CMB to allow averaging across experiments. Pre-I, pre-immune serum. Statistical analysis was done by comparing test groups to pre-immune group using ordinary one-way ANOVA and accounted for multiple comparisons by Dunnett’s multiple comparison test (ns=not significant, *=p<0.05, **=p<0.01 , ****=p<0.0001).

[0108] Fig. 4D - Female gamete lysis assay with 10% pooled mouse serum and active human complement. Values are means from two independent experiments with two technical replicates each and error bars indicate s.e.m. 0% lysis is defined as the number of live gametes present after incubation with human complement only. Statistical analysis was performed as in Fig. 4C. Fig. 4E - Pooled mouse sera that showed strong lysis in Fig. 4D were titrated in the gamete lysis assay. Values are means from two independent experiments with two technical replicates each and error bars indicate s.e.m.

[0109] Fig. 5A - Transmission-reducing activity of Pfs230D12 sera in direct membrane feeding assay (DMFA) with naturally circulating gametocyte strains from a volunteer in Burkina Faso. Gametocytes from a volunteer were fed to Anopheles coluzzii mosguitoes, in the presence of mouse sera (day 57) and human complement. Pre-I, pre-immune serum. Data are shown as violin plots with the median indicated as a line and the interguartile range (IQR) (Q1 and Q3 guartiles) as dotted lines. Values above bars indicate percentage TRA, and the number of infected mosguitoes and the total number of mosguitoes between brackets (# infected mosguitoes / # total mosguitoes). Fig. 5B - As in Fig. 5A, for a second volunteer from Burkina Faso.

[0110] Fig. 5C - Estimate TRA values from two independent experiments (Fig. 5A and 5B) combined. Bars are estimated means and error bars indicate 95% confidence intervals.

[0111] Fig. 6A - Recognition of Pfs230D12 by plasma from volunteers naturally exposed to Plasmodium falciparum (as in Fig. 6B-6D). Antibody levels against D12 in Ugandan plasma samples, as determined by ELISA. AU are arbitrary units calculated using a highly reactive plasma pool from Tanzania as reference. Naive samples are pooled plasma samples from malaria-naive Dutch donors (n=8). Groups were compared by one-sided Mann-Whitney test (****=p<0.0001). The threshold for positivity is marked and defined as the mean of naive controls plus three standard deviations (AV + 3*SD). Percentage of positive samples is indicated above the graph. The number of positive samples and the total number of samples are depicted in between brackets (# positve samples / # total samples). Data are shown as violin plots with the median indicated as a line and the interguartile range (IQR) (Q1 and Q3 guartiles) as dotted lines (as in Fig. 6B and 6D).

[0112] Fig. 6B - Antibody levels stratified by age (in years). Groups were compared by Kruskal-Wallis test with Dunn’s correction for multiple testing (ns=not significant, ****=p<0.0001). Number of samples per age group are shown below the graph.

[0113] Fig. 6C - Correlation between antibody levels against D12 and 230CMB (Spearman’s p = 0.4073, p < 0.0001). Arbitrary units against 230CMB are calculated using serum from an individual that had high levels of antibodies against D12 (Donor A). Five individuals with 230CMB-specific antibody levels below 0.1 AU are not shown on graph.

[0114] Fig. 6D - Antibody levels stratified by TRA. Groups were compared by Kruskal-Wallis test with Dunn’s correction for multiple testing (ns=not significant). Number of individuals per group are shown below the graph.

[0115] Examples

[0116] Example 1 - Abstract

[0117] Malaria transmission-blocking vaccines (TBV) target sexual stage parasites that are transmitted to mosguitoes and critical for spread of the pathogen. The clinically most advanced TBV candidate contains part of the Pro-domain (Pro) and Domain 1 (D1) of Plasmodium falciparum surface protein Pfs230. Subunit vaccines that contain other domains of Pfs230 have so far failed to induce functional antibodies. We produced eight single domain fragments of Pfs230 were produced in Drosophila melanogaster S2 cells and assessed their immunogenicity in mouse immunizations. In addition to D1 -specific antibodies, antibodies raised against Domain 12 (D12) showed strong recognition of Pfs230 on the surface of female parasites. Importantly, D12-specific antibodies showed strong functional transmission-reducing activity in membrane feeding assays with cultured parasites, an activity that was complement-dependent. Murine D12-specific antibodies further reduced mosquito transmission of parasites acquired from naturally infected parasite carriers from Burkina Faso. The D12 antigen was recognized by sera from an all-age cohort of individuals who had been naturally exposed to Plasmodium falciparum with antibody levels increasing with age. In conclusion, we identified the suitability of Pfs230D12 as a transmission-blocking vaccine.

[0118] Example 2 - Results

[0119] We expressed all single Pfs230 domains in Drosophila melanogaster S2 cells that have been successfully used for expression of the 6-Cys domain protein Pfs48 / 45. We obtained eight pure single domain fragments that were used to immunize mice. Of these fragments, Domain 12 induced antibodies with strong TRA in membrane feeding assays with lab cultured parasites and as well as membrane feeding assays with naturally circulating parasites from human donors. We also show that people with natural exposure to malaria parasites possess antibodies that recognize Pfs230D12. These unexpected results position Pfs230D12 as a promising new TBV.

[0120] Production of single domain Pfs230 protein fragments

[0121] We expressed single domain protein fragments with a C-terminal C-tag in D. melanogaster S2 cells (Table S1) and mass spectrometry results confirmed Pfs230-D12 identity. Nine of the fourteen constructs showed clear expression in S2 cell supernatants by western blot with an a-C-tag antibody (Fig. 1 B).

[0122] Table S1. Characteristics of all Pfs230 single domain constructs. NetOGIyc version 4.0 (DTU Health Tech, Denmark) and NetNglyc version 1.0 (DTU Health Tech, Denmark) were used to predict the O-linked and N-linked glycosylation sites.

[0123] The cell lines that showed clear expression, i.e. lines expressing D1 , D3, D5, D6, D8, D9, D10, D12 and D13, were scaled up and proteins were purified using C-tag purification followed by size exclusion chromatography. Unlike the other domains, D3 and D9 showed strong aggregation by size exclusion chromatography. Using the mild detergent Empigen BB, we resolved aggregation for D9. D3 remained largely aggregated in the presence of Empigen BB and was excluded from further analyses.

[0124] We obtained pure D1 , D5, D6, D8, D9, D10, D12 and D13 proteins as determined by SDS-PAGE (Fig. 1 C and Fig. 1 D) and western blot (Fig. 1 E) analyses. The proteins showed a change in apparent mass between reducing and non-reducing conditions, indicating that they form disulphide bonds, as can be expected for 6-Cys domain proteins (Fig. 1 C and 1 D, Table S1). D6 appeared as a dimeric protein on SDS-PAGE, which was resolved by the addition of reducing agent, indicating intermolecular disulphide formation (Fig. 1 C). All the antigens appear to be glycosylated, albeit to different extents (Fig. 1 F). Altogether, we obtained eight Pfs230 single domain antigens in sufficient quantity and purity for mouse immunizations.

[0125] Pfs230D12 mouse antibodies recognize native Pfs230

[0126] For each selected polypeptide, a group of five mice was immunized three times (on day 0, 21 and 43) subcutaneously with 20 pg antigen formulated in Montanide ISA720 and blood was collected 14 days after the third immunization (on day 57). A positive control group was immunized with 230CMB (amino acids 444-730, SEQ ID NO: 532), a plant-produced protein containing the Prodomain and D1 , that was previously shown to induce strong transmission reducing activity (TRA) in rabbits. All mice generated antibody responses against the immunogen they were immunized with (Fig. 2A, Fig. 3A-3I). One mouse in the D12 group showed very low antibody responses (Fig. 3H) and sera from this mouse were therefore excluded from further analyses. Antibodies in pooled mouse sera recognized native Pfs230 in ELISA with gametocyte extract at different intensities (Fig. 2B). Sera raised against D1 , D5, D9, D10, D12 and D13 showed statistically significantly higher recognition compared to pre-immune sera. Recognition by sera against D6 and D8 was weaker and not statistically significant. We also tested recognition of native Pfs230 on the surface of purified live female gametes. Strikingly, only sera generated against 230CMB, D1 and D12 bound to the surface of female gametes (Fig. 2C). Together, the results indicate that while sera raised against most single domain constructs recognize Pfs230 in parasite extract, only sera against D1 and D12 recognize Pfs230 on live female gametes.

[0127] Pfs230D12 antibodies block transmission of Plasmodium falciparum NF54

[0128] To assess functional activity of the domain-specific antibodies, we tested these in the standard membrane feeding assay (SMFA). In this assay, we let laboratory-reared Anopheles stephensi mosquitoes feed on a mixture of cultured P. falciparum NF54 gametocytes and sera from immunized mice, and after 6-8 days we count oocysts in the mosquito midgut to calculate TRA. In line with previous studies, sera raised against 230CMB and D1 showed strong TRA, reducing oocyst formation by 98.3% (95% Cis: 97.2-99.0) and 78.5% (95% Cis: 70.3-84.4) respectively (Fig. 4A). Interestingly, D12 sera also showed strong TRA (95.2% TRA, 95%Cls: 93.1-96.6). Sera raised against other domains showed very low or no TRA. Mass spectrometry confirmed purity of the D12 immunogen, and western blots with gametocyte extract and single domain fragments further confirmed that the functional antibodies were D12-specific.

[0129] Since the vast majority of functional Pfs230 antibodies described to date are complementdependent, we tested sera against 230CMB, D1 , and D12 in SMFA with either active or heat- inactivated human complement (Fig. 4B). Sera showed increased TRA in the presence of active complement, demonstrating that complement plays a role for the antibodies against these domains. To confirm that the complement-related activity is mediated by classical pathway activation, the ability of the mouse antibodies to fix C1q on the parasite surface was assessed in a flow cytometry assay with live female gametes. Antibodies against 230CMB, D1 , and D12 were able to mediate C1q deposition, while antibodies against D6, D8, and D9 failed to do so (Fig. 4C), in line with the SMFA results. We then tested whether the deposition of C1q leads to lysis of the female gamete in a flow cytometry lysis assay (Fig. 4D and 4E). In this assay purified female gametes are incubated with mouse sera and human complement, and after incubation the percentage lysis is determined by live / dead staining. Sera against 230CMB, D1 , and D12 showed over 80% lysis when tested at 1 :10 dilution (Fig. 4D). Other sera showed little or no lysis, consistent with results from the C1q deposition assay and SMFA. Titration of mice sera demonstrates similar potency for 230CMB and D12 sera, while D1 sera has slightly lower potency, similar to the trend observed in SMFA. Taken together, these data indicate that the D12 antigen can induce functional antibodies in mice capable of reducing transmission of lab-cultured malaria parasites to mosquitoes.

[0130] Pfs230D12 is genetically conserved and rodent antibodies reduce transmission of naturally circulating gametocytes

[0131] To assess the functionality of D12 antibodies against naturally circulating gametocyte strains, we performed direct membrane feeding assays (DMFA) using blood of naturally infected gametocyte carriers from Burkina Faso. After removal of autologous plasma, gametocyte-infected red blood cells were mixed with pooled mouse sera and normal human serum containing complement, and fed to mosquitoes. After seven days oocysts were counted and TRA was calculated as the reduction in oocyst intensity compared to a negative control. While D5 sera did not reduce oocyst formation, in line with SMFA results, the 230CMB and D12 sera reduced oocyst formation across two independent experiments (Fig. 5A and 5B). The estimated TRA values were 96.8% (95% Cis: 94.4- 98.2) and 82.1 % (95% Cis: 72.7-88.3) for D12 and 230CMB sera respectively (Fig. 5C). The DMFA results thus show that D12 antibodies have strong TRA against naturally circulating gametocytes.

[0132] Pfs230D12 is recognized by sera from individuals naturally exposed to P. falciparum

[0133] To assess natural antibody responses to D12, we screened plasma samples from an all-age cohort of individuals residing in Tororo, an area in eastern Uganda where at the time of sampling transmission intensity was intense and perennial. Purified IgG samples of these cohort participants were tested for transmission-reducing immune responses in the SMFA, as described above, as part of a larger study on naturally acquired transmission-reducing immunity. We detected significantly higher antibody levels in Ugandan samples compared to naive control samples from Dutch donors (Mann-Whitney test, p < 0.0001) (Fig. 6A). 139 out of 189 (73.2 %) individuals were seropositive for D12. Antibody intensity was significantly higher in >15 years old individuals compared to school aged children (5-15 years old) and younger children (<5 years old) (Kruskal-Wallis test, p <0.0001) (Fig. 6B). We observed a statistically significant correlation between antibody responses against 230CMB and D12 (Spearman’s, p = 0.4073, p < 0.0001) (Fig. 6C). There were no significant differences in D12-specific antibody levels between individuals that had no / weak (<80%) or strong (>80%) TRA (Fig. 6D). Whilst numbers were too small for meaningful statistical comparisons, we observed strongest responses in two individuals whose plasma showed strong TRA in SMFA. Together, we found that individuals exposed to gametocytes can generate antibodies against D12 and that antibody levels increase with age.

[0134] Example 3 - Discussion

[0135] We successfully expressed eight individual domains of Pfs230, a P. falciparum protein that is involved in parasite transmission. Antibodies raised against the D12 domain showed potent binding to live female gametes in vitro and have strong functional TRA. Functional activity was demonstrated against lab-cultured gametocytes and genetically diverse gametocytes from naturally-infected parasite carriers from Burkina Faso. Furthermore, sera from Ugandan donors naturally exposed to P. falciparum showed immune recognition of D12 in an age-dependent manner. This establishes D12 as a basis for a new TBV.

[0136] TBVs could be valuable tools for the elimination and eradication of malaria. Several TBV candidates have been identified, of which the Pro-D1 fragment of Pfs230 has progressed furthest in terms of clinical testing. This study aimed to comprehensively examine constructs outside Pro-D1. Earlier immunization studies included fragments containing D12, produced in Escherichia coll and wheat germ cell-free extract (Williamson et al., 1995; Tachibana et al., 2019). These constructs induced antibodies that recognized native Pfs230 on gametes, but the antibodies did not reduce transmission to mosquitoes. This is in contrast with our results that show high TRA for mouse antibodies raised against D12 produced in D. melanogaster S2 cells. Gel electrophoresis under reducing conditions showed how our D12 polypeptides were properly folded, displaying dithiol bridge formation. Without wishing to be bound to theory, it can be speculated that the expression system plays an important role with D. melanogaster S2 cells producing (more) properly folded D12, which can be relevant for raising functional antibodies (Fig. 1 B). This would be in line with preclinical studies with another 6-Cys family protein, namely Pfs48 / 45 (Sauerwein et al., 2022). There it was shown that the host expression system and the proper conformation of the antigen are relevant for inducing functional responses.

[0137] While antibodies raised against the D12 antigen showed strong gamete recognition and functional TRA, we did not observe functional responses against D5, D6, D8, D9, D10 and D13 (Fig. 4A). Strikingly, antibodies against most of these domains recognized native Pfs230 in gametocyte extract, but did not recognize Pfs230 on live gametes (Fig. 2C-D) suggesting that epitopes on these domains are occluded by other parasite surface proteins that interact with, or are in close proximity of, Pfs230. Alternatively, epitopes on these domains may be close to the parasite membrane and therefore not accessible to antibodies.

[0138] The activity of most if not all functional Pfs230 antibodies described to date are related to complement. The antibodies we raised against D12 are no exception. The influence of complement on the D12 antibodies was shown in membrane feeding assays where antibodies showed reduced TRA in the presence of heat-inactivated complement (Fig. 4B). The D12 antibodies can activate the classical pathway by fixing C1q (Fig. 4C) and induce complement-mediated gamete lysis in vitro (Fig. 4D and 4E), which is the presumed effector mechanism in the mosquito midgut.

[0139] D12 polypeptides have shown themselves to be particularly promising for development into vaccines. In general, vaccine candidates should target conserved functional epitopes to generate cross-strain protection, should be able to induce highly potent antibodies so lower overall antibody responses are needed for protection, and should be immunogenic in humans. Like other sexual stage Plasmodium proteins, Pfs230 is well conserved and D12 in particular contains fewer non- synonymous single nucleotide polymorphisms than the leading TBV candidate ProD1 (de Jong et al., 2021). The D12-specific mouse antibodies do not only block transmission of the reference strain P. falciparum NF54, but also block transmission of naturally circulating gametocytes that may be genetically diverse (Fig. 5).

[0140] We observed for the first time that many individuals in a Ugandan cohort were seropositive for D12, demonstrating that the D12 antigen is immunogenic in humans and that vaccine-induced antibody levels may be boosted by natural exposure and vice versa. Naturally acquired transmission reducing immunity is a rare phenomenon, at least the high levels of TRA that can be reproducibly demonstrated in the SMFA (Stone et al., 2018). Whilst now allowing for a formal assessment of a possible role of D12-specific antibodies in naturally acquired TRA, we made use of a larger cohort study where plasma samples were available alongside TRA estimates. Two individuals with high levels of TRA also had high levels of antibodies against D12. Our findings demonstrate that D12 antibodies are naturally acquired in an age-dependent manner - probably reflecting cumulative exposure to P. falciparum gametocytes - in a manner that is similar to antibodies to other Pfs230 domains, and that individuals with naturally acquired TRA can have D12 antibodies. This further suggests that our D12 polypeptides have the same folding as naturally occurring D12 while it is comprised in the full Pfs230 protein.

[0141] In our immunisation studies we included 230CMB, containing Pro-D1 , as positive control. D12 polypeptides induced at least similar levels of TRA and gamete lysis activity as Pro-D1 , which has already been under development for some time. In conclusion, our work shows how provision of well-folded D12 polypeptides are a promising new tool for combating malaria.

[0142] Example 4 - Material & Methods

[0143] Protein construct design

[0144] Expression plasmids were created for all fourteen subdomains of Pfs230 (Fig. 1 A). Boundaries of the domains were based on previous research and the linkers domains between subdomains were excluded. Sequences were codon optimized for expression in Drosophila melanogaster and synthesized (BaseClear). The Pfs230 single domains were cloned with an N-terminal BiP signal peptide, His6-tag and Alanine-serine linker, and a C-terminal glycine-serine linker followed by a C- tag into the pExpreS2.2 plasmid (ExpreS2ion Biotechnologies), downstream of the Actin+HSP70 promoter. The plasmids were verified by Sanger sequencing (Baseclear). The sequences of the inserts can be found in SEQ ID NOs: 514-527.

[0145] Drosophila melanogaster S2 cell transfection and culture

[0146] The D. melanogaster S2 cell line (ExpreS2ion Biotechnologies) was used for the expression of all Pfs230 protein constructs. S2 cells were cultured in shake flasks with vented cap in EX-CELL420 media (Sigma-Aldrich), supplemented with 1 % penicillin and streptomycin, at 25°C shaking 115 rpm. Cells were counted twice a week and resuspended to 8x106cells / mL alternately by dilution or centrifugation. For transfections, 2.5 mL cell suspension was mixed with 6.25 pg plasmid DNA and 25 pL ExpreS2 Insect-TR 5x transfection reagent (ExpreS2ion Biotechnologies) in a T12.5 T-flask. The transfected cells were then incubated at 25°C, and 1 mL of FBS was added after 3 hours. 4000 pg / mL geneticin was added as a selection agent after 24 h. Approximately 26 days after the transfection, the cultures were scaled up to shake flasks. During this step FBS and geneticin were removed by centrifugation and resuspending cells in EX-CELL420 to 8x106cells / mL. Supernatant was harvested 5 days after the cells were diluted, for protein expression analysis on western blot and protein purification.

[0147] Protein purification

[0148] The S2 cell supernatant was concentrated from 200-300 mL to approximately 50 mL using the Masterflex EasyLoad (Masterflex). The Pfs230 single domain protein constructs were affinity purified with CaptureSelect C-tagXL pre-packed columns (Thermo Scientific) on an AKTA start (Cytiva), using 20 mM Tris wash buffer (pH 7.4) and 20 mM Tris + 2 M MgCh (pH 7.4) elution buffer. Peak fractions from the chromatogram were pooled and dialysed overnight in PBS. The sample was filtered and concentrated to approximately 600 pL. Subsequently, the sample was further purified using a Superdex75 10 / 300 GL column (Cytiva) with filtered and degassed PBS as running buffer. For Pfs230D9 and Pfs230D3 0.2% Empigen BB (Sigma-Aldrich) was added to all purification buffers to decrease aggregation of the proteins. Superdex fractions containing pure monomer protein were pooled and the protein concentration measured using a Nanodrop spectrophotometer (Thermo Scientific). Samples were frozen in liquid nitrogen and stored at -70°C.

[0149] SDS-PAGE analysis

[0150] For analysis of proteins from S2 expression, samples were mixed with 4x NuPAGE LDS sample buffer (Invitrogen), heated at 70°C for 10 min before loading on a 4-20% bis-tris polyacrylamide gel (GenScript). In the case of purified protein 1 pg protein was loaded per condition and the Precision Plus Dual Color protein marker (Bio-Rad) was used as size standard. The gels were stained for 30 minutes using Instant Blue Coomassie Protein Stain (Abeam). To reduce disulphide bonds, a final concentration of 10 mM dithiothreitol (DDT) was added in the preparation of the sample.

[0151] For protein analysis with parasite extract, P. falciparum NF54 gametocyte extract (Theisen et al., 2014) was diluted to the equivalent of 500,000 gametocytes per well. A final concentration of 10 mM dithiothreitol (DTT) was added for reducing conditions. Gametocyte extract and 230CMB 18 samples were mixed with 4x NuPAGE™ LDS sample buffer and heated for 10 minutes at 70°C before loading on a 4-20% Bis-Tris gel (GenScript). 20 ng 230CMB was loaded per well and the Precision Plus Dual Color protein marker (Bio-Rad) was used as size standard.

[0152] For analysis of proteins expressed in wheat germ cell-free extract, purified proteins (Inklaar et al., 2023 and Tachibana et al., 2019) were mixed with SDS-sample buffer and TCEP-HCI (Pierce™), and denatured at 37°C for 30 minutes before loading on a 12.5% PAGE Tris gel (ATTO, Tokyo, Japan). 0.5 pg of each protein was loaded per well and Precision Plus Protein All blue standard (Bio-rad) was used as the size standard.

[0153] Western blot analysis

[0154] For western blot analysis of proteins expressed in S2 cells, a positive control with C-tag (Pro-CS3- 6C, kindly gifted by Susheel Singh, 36.3kDa) and negative control (Pf3D7_1306500C no C-tag, transfected in S2 cells) was included. The bis-tris gels were blotted on a 0.45 pm nitrocellulose membrane using the TurboBlot system (Bio-Rad). The membranes were washed in between steps with PBS supplemented with 0.05% Tween20 (PBST), blocked overnight in 5% skimmed milk PBS (mPBS) at 4°C, and incubated for 1 hour at room temperature (RT) with the CaptureSelect Biotin Anti-C-tag conjugate (1 / 1000, Cat. No. 7103252100, Thermo Scientific) in 1 % mPBST. Thereafter the membranes were incubated with 1 / 2500 IRDye Streptavidin 680LT (Cat. No. 926-68031 , Ll- COR) in 1 % mPBST for 1 hour at RT. The blot was developed with Clarity Max Western ECL substrate (Bio-Rad) and imaged with the Odyssey CLX (LI-COR).

[0155] For western blots with gametocyte extract, gels were transferred to a 0.45 pm nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The blots were blocked with 5% skimmed milk in PBS before incubation with 1 / 5000 polyclonal serum from mice immunized with Pfs230D12. After washing, the strips were incubated with 1 / 3000 diluted polyclonal rabbit antiMouse IgG HRP (Cat. No. P0260, DAKO). Blots were developed with Clarity Max Western ECL substrate (Bio-Rad) and imaged on the ImageQuant™ LAS 4000 (GE Healthcare).

[0156] For western blot analysis of proteins expressed in wheat germ cell-free extract, the SDS- PAGE gels were transferred to an Amersham Hybond P Low fluorescence 0.2 pm PVDF membrane (Cytiva) using the Trans-blot SD semidry transfer cell (Bio-rad) (25 V, 126 mA / gel, 75 minutes). The blots were blocked with 5% skimmed milk in PBST before incubation with 1 / 1000 polyclonal serum from mice immunized with Pfs230D12 (diluted in PBST) for 1 hour at RT and overnight at 4°C. After washing, the blots were incubated with 1 / 10,000 polyclonal Sheep anti-Mouse IgG HRP (Cat. No. NA931 VS, Cytiva). Blots were developed with Immobilon Western chemiluminescent HRP substrate (Cat. no. WBKLS0500, Millipore) and imaged on the LAS-4000 (FUJIFILM, Tokyo, Japan) for 13 minutes.

[0157] Glycosylation staining

[0158] Glycosylation of Pfs230 domains was assessed using the Pierce Glycoprotein Staining Kit (ThermoFisher Scientific). 5 pg of each Pfs230 protein construct was loaded on gel and the gel was stained following manufacturer’s instructions.

[0159] Mass spectrometry

[0160] The identity of recombinant Pfs230D12 was confirmed by mass spectrometry. 5 pg of purified protein was denatured in 4M urea, 100 mM Tris-HCI (pH 8.0) and disulfide bonds were reduced using 10 mM DTT for 30 minutes at RT. Cysteines were alkylated using 50 mM lodoacetamide for 30 minutes, and samples were diluted to 2M Urea, using 100 mM Tris-HCI (pH 8.0). Samples were digested overnight with 0.5 pg Trypsin at 25°C. Next day, samples were desalted using StageTips.

[0161] Peptides were analyzed using an Easy nLC 1000 equiped with a 30 cm reverse phase column, coupled on-line to a Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). A 60 minute gradient of buffer B (80% acetonitrile, 0.1 % formic acid) was applied and the mass spectrometer was operated in TopS mode with a dynamic exclusion of 60 seconds.

[0162] RAW data was analyzed using Maxquant version 1.6.6.0 with a Drosophila database supplemented with sequences for single domain constructs of Pfs230. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD039716.

[0163] Mice immunization

[0164] 45 female 6-8 weeks old CD-1 mice (Charles Rivers), divided in groups of 5 mice, were immunized with 230CMB, D1 , D5, D6, D8, D9, D10, D12 and D13. 230CMB, a construct comprising aa 444- 730 of Pfs230 produced in a plant-based expression system (Farrance et al., 201 1) and known to induce transmission reducing capacity was included as positive control. Mice were injected subcutaneously with 100 pL of 0.2 mg / mL antigen in 70% Montanide ISA720 (SEPPIC) at day 0, day 21 and day 43. Pre-bleed samples were collected at day -1 and final bleeding was performed at day 57 before sacrificing. Blood was allowed to clot at RT for 30 minutes, and serum was collected after centrifugation and was stored at -20°C. For each group of mice sera, samples were pooled for further analysis.

[0165] Enzyme-Linked Immunosorbent Assays (ELISA)

[0166] For antigen ELISA to assess antigen-specific antibody responses in mice, Nunc MaxoSorp 96-wells plates (ThermoFisher) were coated with 100 pL of 1 pg / mL antigen and incubated overnight at 4°C. Plates were washed three times with PBS in between incubation steps. Plates were blocked with 5% mPBS for 1 hour. Plates were incubated with serum samples diluted in 1 % mPBST, for 3 hours at RT. Subsequently, the plates were incubated with polyclonal Rabbit Anti-Mouse HRP (1 / 3000 dilution, Cat. No. P0260, DAKO) for 2 hours at RT. The ELISA was developed by adding 100 pL tetramethylbenzidine (TMB). The color reaction was stopped by adding 50 pL 0.2 M H2SO4 and the optical density was read at 450 nm on an iMark™ microplate absorbance reader (Bio-Rad).

[0167] For the gametocyte ELISA, P. falciparum NF54 gametocyte extracts were prepared as described previously (Theisen et al., 2014). 100 pL lysate per well, equivalent to 75,000 gametocytes, was pipetted into Nunc MaxiSorp™ 96-wells plates (ThermoFisher) and incubated overnight at 4°C. The next steps of the ELISA were performed as described above.

[0168] D12-specific antibody levels in sera from individuals exposed to malaria parasites were assessed using an antigen ELISA as described above. Goat anti-Human IgG (H+L) HRP (1 / 40,000 dilution, Cat. No. 31412, Invitrogen) was used for detection. A titration of pooled hyperimmune serum from gametocyte carriers in Tanzania was used to calculate arbitrary units using ADAMSEL FPL (available on the internet at www.malariaresearch.eu / content / software).

[0169] Gamete purification

[0170] To obtain purified female gametes for flow cytometry assays, we collected N-acetyl glucosamine treated 16-day old P. falciparum NF54 gametocyte cultures. The cultures were centrifuged for 10 minutes at 2,000xg at RT, to be resuspended in FBS using a volume that equals half the original culture volume. Gametocytes were placed on a roller bank for 45 minutes at RT for activation and thereafter centrifuged for 10 minutes at 2,000xg at 4°C. The pelleted gametes were resuspended in 1 mL PBS, loaded onto a 7 mL layer of 11 % w / v Accudenz (Accurate Chemical) and centrifuged for 30 minutes at 7,000xg at 4°C without brake (Sorvall RC-5B Superspeed Centrifuge with HB-4 swing-out rotor). The female gametes present in the top layer were collected, transferred to a 50 mL tube and PBS was added up to 50 mL total volume. A final centrifugation for 5 minutes at 2,000xg at 4°C was done to pellet the gametes, which were resuspended in 1 mL PBS and counted using a Burker-Turk counting chamber. Flow cytometry

[0171] For the assessment of gamete antibody binding, gamete C1q deposition and gamete lysis we used similar flow cytometry assays with specific adjustments that are described in this paragraph. All gamete incubations were carried out in PBS supplemented with 2% FBS and 0.02% sodium azide. For all three assays 50,000 purified gametes were used per well in a V-bottom non-treated 96-well plate (Costar) and were incubated for 1 hour at RT with mice sera. In the case of a lysis assay, there is an addition of 20% normal human serum (NHS) and the incubation is reduced to 30 minutes at RT. For a C1q deposition assay this is reduced to 10% NHS (30 minutes incubation at RT). Plates were centrifuged at 2,000xg for 3 minutes at 4°C and washed three times with PBS. The C1q deposition assay includes additional steps; first PBS supplemented with 10 mM EDTA is added for 5 minutes at 4°C to inactivate complement. Second, after 3 washes with PBS, 1 / 5000 anti-C1q goat anti-human polyclonal serum (Complement Technology) is added for 30 minutes incubation at RT. Gametes were washed and then incubated with either 1 / 200 Alexa Fluor™ 488 Chicken anti-Mouse IgG (H+L) (Invitrogen) (binding assay), 1 / 200 anti-Pfs47 (rat mAb 47.1) labelled with DyLight™ 650 NHS ester (Thermo Scientific) (lysis assay) or 1 / 200 Alexa Fluor™ 488 Donkey anti-Goat IgG (H+L) (Invitrogen). 1 / 1000 eBioscience™ Fixable Viability Dye eFluor™ 780 (Invitrogen) was added in all assays and gametes were incubated for 30 minutes at RT. After washing with PBS, samples were resuspended in 150 pl PBS. Antibody binding to gametes, lysis of gametes and C1q deposition on gametes were assessed by flow cytometry by analysing a minimum of 2,000 gametes with the Gallios™ 10-color system (Beckman Coulter) and analyzed with FlowJo (BD, version 10.7.1).

[0172] SMFA

[0173] Mice sera were diluted in FCS and mixed with mature P. falciparum NF54 gametocytes and NHS. To inactivate NHS for conditions where inactive complement is required, it was heated for 30 minutes at 56°C prior to mixing with the gametocytes. Anopheles stephensi mosquitoes from a colony maintained at Radboudumc (Nijmegen, the Netherlands) were fed blood meals. Unfed and partially fed mosquitoes were removed. 20 mosquitoes per condition were dissected 6-8 days after the blood meal to collect their midguts. The midguts were stained with mercurochrome and oocysts were counted. TRA was defined as the reduction in oocyst intensity (oocysts per mosquito midgut) in a test condition compared to a negative control in which no mice sera (FBS control) was added. All samples were tested in two independent SMFA experiments.

[0174] DMFA

[0175] Gametocyte-infected blood from patients residing in the villages surrounding Bobo-Dioulasso was collected in heparin tubes, 5 mL per tube. Immediately after blood collection, the blood was centrifuged at 3000xg for 5 minutes, and plasma was removed. 120 pl of the remaining RBC pellet was transferred to tubes containing 90 pl of naive AB serum and 30 pl of mice sera (or FCS as negative control). The total of 240 pl was carefully mixed by pipetting and the content of each tube was transferred to an individual feeder maintained at 37°C to allow Anopheles coluzzii mosquito feeding for 30 minutes. Unfed mosquitoes were removed; fullyfed mosquitoes were kept for 7 days post feeding. All surviving mosquitoes were dissected for each condition, midguts were stained with mercurochrome for oocyst detection and oocysts were counted.

[0176] Statistical analysis

[0177] Transmission reducing activity (TRA) was calculated as the reduction in oocysts compared to a negative control, using an online tool (Ramjith et al., 2022). All other statistical analyses were performed using GraphPad Prism (version 10.1.0).

[0178] References

[0179] Amen, A. et al. (2024). BioRxiv, doi: 10.1101 / 2023.1 1.03.565335.

[0180] De Jong, R. M. et al. (2021). NPJ Vaccines doi:10.1038 / s41541-021-00366-9.

[0181] Inklaar, M. R. et al. (2023). NPJ Vaccines doi:10.1038 / s41541-023-00784-x.

[0182] Tachibana et al. (2019). Vaccine, doi:10.1016 / j.vaccine.2019.02.021 .

[0183] Gerloff, D. L. et al. (2005). PNAS, doi:10.1073 / pnas.0502378102.

[0184] Sauerwein, R. W. and Bousema, T. (2015). Vaccine, doi:10.1016 / j.vaccine.2O15.08.073.

[0185] Theisen, M. et al. (2014). Vaccine doi:10.1016 / j. vaccine.2014.03.020.

[0186] Farrance, C. E. et al. (2011). Clin Vaccine Immunol, doi:10.1128 / CVI.05105-11 .

[0187] Ramjith, J. et al. (2022). Front / mmuno / doi:10.3389 / fimmu.2022.899615.

[0188] Williamson, K. C. et al. (1995). Mol Biochem Parasitol, doi:10.1016 / 0166-6851 (95)02507-3.

[0189] Sauerwein, R. et al. (2022). Am J Trop Med Hyg, doi:10.4269 / ajtmh.21-1320

[0190] Tang, W. K. et al. (2023). Immunity, doi:10.1016 / j.immuni.2023.01 .012.

[0191] Ivanochko, D. et al. (2023). Immunity, doi:10.1016 / j.immuni.2023.01 .013.

[0192] Stone, W. J. R. et al. (2018). Nat commun, doi: 10.1038 / S41467-017-02646-2.

Claims

Claims1 . A polypeptide comprising a first amino acid sequence that has at least 80% sequence identity with a subdomain of Plasmodium falciparum surface protein Pfs230, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is domain 12.

2. The polypeptide according to claim 1 , wherein the first amino acid sequence has a length of 120-200 amino acids, preferably of 145-165 amino acids, or wherein the polypeptide has a length of 160-180 amino acids, preferably 165-175 amino acids.

3. The polypeptide according to claim 1 or 2, wherein the first amino acid sequence comprises at least 100 consecutive amino acids from Pfs230.

4. The polypeptide according to any one of claims 1-3, wherein the first amino acid sequence has at least 90% sequence identity with SEQ ID NO: 14, preferably at least 95%, more preferably 100%.

5. The polypeptide according to any one of claims 1 -4, wherein the polypeptide further comprises a first recognition tag, wherein the first recognition tag is preferably C-terminal to the first amino acid sequence, wherein the first recognition tag preferably consists of the sequence EPEA.

6. The polypeptide according to any one of claims 1-5, wherein the polypeptide does not comprise a sequence represented by SEQ ID NO: 535.

7. The polypeptide according to claim 5 or 6, wherein the first recognition tag and the first amino acid sequence are separated by a linker sequence, wherein the linker sequence preferably comprises 1-40 amino acids.

8. The polypeptide according to any one of claims 1-7, wherein it exhibits transmission-reducing activity of at least 40%, preferably at least 50%, more preferably at least 80%, wherein transmission-reducing activity is assessed using a standard membrane feeding assay (SMFA).

9. Composition comprising a polypeptide according to any one of claims 1-8 and a pharmaceutically acceptable excipient, optionally comprising one or more further polypeptides comprising a first amino acid sequence that has at least 80% sequence identity with Plasmodium falciparum surface protein Pfs230 subdomain, wherein the polypeptide has a length of 100-300 amino acids, wherein the first amino acid sequence has a length of at least 100 amino acids, and wherein the subdomain is a domain that is not domain 12.

10. The polypeptide according to any one of claims 1-8 or the composition according to claim 9, for use as a medicament, wherein the medicament is preferably for the prevention of malaria.

11. Nucleic acid construct comprising a first nucleotide sequence encoding a polypeptide as defined in any one of claims 1-8.

12. The nucleic acid construct according to claim 11 , comprising a second nucleotide sequence that is 5’ of the first nucleotide sequence, wherein the second nucleotide sequence encodes a signal peptide such as a binding protein (BiP) signal peptide.

13. Expression vector comprising the nucleic acid construct according to claim 11 or 12.

14. Cell comprising the nucleic acid construct according to claim 11 or 12 or comprising the expression vector according to claim 13.

15. Method for providing a polypeptide as defined in any one of claims 1-8, the method comprising the steps of: i) providing a cell as defined in claim 14 wherein the cell comprises a nucleic acid construct as defined in claim 12; ii) culturing the cell under conditions conducive to polypeptide expression to produce the polypeptide; and iii) optionally purifying the produced polypeptide.

16. A polypeptide comprising a first amino acid sequence that has 100% sequence identity with SEQ ID NO: 14, wherein the first amino acid sequence has a length of 145-165 amino acids, and wherein the polypeptide has a length of 150-190 amino acids.

17. The polypeptide according to claim 16, wherein the polypeptide comprises a first recognition tag, wherein the first recognition tag is C-terminal to the first amino acid sequence, and wherein the first recognition tag consists of the sequence EPEA.

18. The polypeptide according to claim 16 or 17, wherein the polypeptide is represented by SEQ ID NO: 134.

19. The polypeptide according to claim 16 or 17, wherein the polypeptide is represented by SEQ ID NO: 254.

Images