Compositions for delivery of plasmodium antigens and related methods

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for delivery of malarial protein antigens and related technologies (e.g., components thereof and / or methods relating thereto). Among other things, the present disclosure provides polyribonucleotides encoding malarial protein, Pfs230.

Description

COMPOSITIONS FOR DELIVERY OF PLASMODIUM ANTIGENSAND RELATED METHODS BACKGROUND

[0001] Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the Plasmodium genus. According to the World Health Organization, an estimated 3.4 billion people in 92 countries are at risk of being infected with the malaria parasite and developing disease.SUMMARY

[0002] The present disclosure provides technologies (e.g., compositions, methods, etc.) for delivery of P / asmodium antigens (also referred to herein as "malaria antigens" or "malarial antigens"). Challenges to mounting an effective immune response against Plasmodium include the parasite's morphologically different life stages and changing mosaic of antigens on the parasite surface. These features of the Plasmodium life cycle mean adaptive immune responses formed against antigens present in one stage of the parasite's life cycle are often ineffective in parasites in a different stage of the life cycle, which can allow the parasite to evade the immune response, e.g., after the initial infection. This challenge increases the need for therapeutics that target different stages, and particularly the lesser targeted sexual stage, of the parasite's life cycle.

[0003] Targeting Plasmodium parasite's transition from host to mosquito vector may block fertilization and / or the invasion of the mosquito midgut by parasites, thereby preventing onward transmission. Despite the possible benefits that can be achieved by targeting Plasmodium in its sexual stage, there are no authorized therapeutics that result in an adaptive immune response that targets Plasmodium parasites at the sexual stage. Thus, there remains a need for a therapeutic approach that prevents transmission from human to mosquito, as this could reduce the incidence of infection (e.g., in vaccinated populations) and assist in the eradication of malaria.

[0004] Pfs230 is an approximately 300 kDa protein expressed from stage II of gametocytogenesis onward and is localized to on the surface of gametocytes and gametes. Seropositivity of human sera against Pfs230 is a predictor for transmission-blocking immunity. The presence of Pfs230 on the gametocyte surface during the sexual stage of the Plasmodium life cycle and its essential role in fertilization help make Pfs230 an ideal target for immune responses that can achieve transmission blocking.

[0005] Provided herein is a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more Plasmodium Pfs230 polypeptide regions or portions thereof.

[0006] The present disclosure provides a recognition of several challenges involved in development and administration of polypeptide therapies. First, development of protein-based therapies can be time consuming and expensive. For example, polypeptide development is challenged by demanding and costly production, including purification and formulation methods. Second, protein-based therapies present an increased number of regulatory challenges. In addition to demonstrating that a protein therapy will be safe and efficacious, there is tight regulation over manufacture of protein replacement therapies (e.g., assessing post-translational modifications, etc.) and quality control during storage and administration complicate the use of protein therapies. Third, administration of protein therapies to a subject can be painful and time consuming. Finally, protein therapies can have a short serum half-life. The present disclosure provides insights that, among other things, address these challenges, making it possible to deliver one or more therapies, as described herein, to a subject safely, reliably, and with strong potency.

[0007] According to a first aspect, the present invention concerns a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more antigenic portions of Plasmodium Pfs230, and a membrane-anchoring region, wherein antigenic portions of Plasmodium Pfs230 have an amino acid sequence selected from any one of SEQ ID NOs: 2 to 14 and 504 to 509 or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2 to 14 and 504 to 509.

[0008] In certain embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 3.

[0009] In certain embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 2.

[0010] In certain embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid substitution at one or more N-linked glycosylation sites, wherein the amino acid substitution prevents glycosylation at the one or more, preferably all N-linked glycosylation sites. In some embodiments, the Plasmodium Pfs230 antigenic portion comprises an amino acid substitution at one or more of positions 585, 821, 829, 889, 961, 1079, and 1089, or any combination thereof, as numbered according to SEQ ID NO: 1. In some embodiments, the amino acid substitution comprises an NX[T / S] to QX[T / S] substitution.

[0011] In certain embodiments, the polypeptide comprises a secretory signal. In some embodiment, the secretary signal comprises an amino acid sequence selected from any one of SEQ ID NOs: 15 to 57 or an amino acid sequence encoded by a nucleotide sequence selected from any one of SEQ ID NOs: 58 to 95. In some embodiments, the secretory signal consists of an amino acid sequence according to SEQ ID NO: 15.

[0012] In certain embodiments, the membrane-anchoring region comprises an amino acid sequence selected from any one of SEQ ID NOs: 96 to 106. In some embodiments, the transmembrane region consists of an amino acid sequence according to SEQ ID NO: 96.

[0013] In certain embodiments, the polypeptide comprises one or more linker. In some embodiments, the one or more linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 107 to 117. In some embodiments, the one or more linker consists of an amino acid sequence according to SEQ ID NO: 107 or 108.

[0014] In certain embodiments, the polypeptide comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 119 to 131 or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 119 to 131.

[0015] In certain embodiments, the polypeptide consists of an amino acid sequence according to SEQ ID NO: 119.

[0016] In certain embodiments, the polypeptide consists of an amino acid sequence according to SEQ ID NO: 120.

[0017] In certain embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 123.

[0018] In certain embodiments, the polypeptide consists of an amino acid sequence according to SEQ ID NO: 121.

[0019] In certain embodiments, the polypeptide consists of an amino acid sequence according to SEQ ID NO: 122.

[0020] In certain embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 123.

[0021] In certain embodiments, a nucleic acid sequence selected from any one of SEQ ID NOs: 132 to 185 or a nucleic acid sequence at least 90% identical to any one of SEQ ID NOs: 132 to 158.

[0022] According to another aspect, the present invention concerns RNA construct comprising in 5' to 3' order:(i) a 5' cap;(ii) a 5' UTR;(iii) a polyribonucleotide disclosed herein;(iv) a 3' UTR; and(v) a polyA tail sequence.

[0023] In some embodiments, the 5' UTR consists of a nucleic acid sequence according to SEQ ID NO: 187. In some embodiments, the 3' UTR consists of a nucleic acid sequence according to SEQ ID NO: 194. In some embodiments, the polyA tail sequence consists of a nucleic acid sequence according to SEQ ID NO: 190. In some embodiments, the polyribonucleotide includes modified uridines in place of uridines. In some embodiments, the polyribonucleotide includes modified uridines in place of all uridines. In some embodiments, the the modified uridines are each Nl-methyl-pseudouridine.

[0024] According to another aspect, the present invention concerns a pharmaceutical composition comprising a polyribonucleotide or RNA construct disclosed herein.

[0025] In some embodiments, a pharmaceutical composition comprises:(i) a first polyribonucleotide or RNA construct encoding a Plasmodium Pfs230 polypeptide construct disclosed herein; and(ii) a second polyribonucleotide encoding a second Plasmodium polypeptide construct, wherein the second Plasmodium polypeptide construct comprises a CSP antigen, a T cell antigen or an RH5 antigen.

[0026] In some embodiments, the pharmaceutical composition comprises:(i) a first polyribonucleotide or RNA construct encoding a Plasmodium Pfs230 polypeptide construct disclosed herein;(ii) a second polyribonucleotide encoding a second Plasmodium polypeptide construct, wherein the second Plasmodium polypeptide construct comprises a CSP antigen, a T cell antigen or an RH5 antigen; and(iii) a third polyribonucleotide encoding a third Plasmodium polypeptide construct, wherein the third Plasmodium polypeptide construct comprises an a CSP antigen, a T cell antigen or an RH5 antigen.

[0027] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a CSP antigen.

[0028] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a T cell antigen.

[0029] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises an RH5 antigen.

[0030] In some embodiments of the pharmaceutical composition, the third Plasmodium polypeptide construct comprises a CSP antigen.

[0031] In some embodiments of the pharmaceutical composition, the third Plasmodium polypeptide construct comprises a T cell antigen.

[0032] In some embodiments of the pharmaceutical composition, the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0033] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a first CSP antigen and the third Plasmodium polypeptide construct comprises a second CSP antigen. In some embodiments, the first and the second CSP antigen share no overlap. In some embodiments, the first and second CSP antigens partially overlap.

[0034] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a CSP antigen and the third Plasmodium polypeptide construct comprises a T cell antigen.

[0035] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a CSP antigen and the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0036] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a first set of T-cell antigens and the third Plasmodium polypeptide construct comprises a second set of T cell antigens. In some embodiments, the first and the second set of T cell antigens comprisedifferent T cell antigens. In some embodiments, the T cell antigens in the first and second set of T cell antigens share some T cell antigens with one another whereas they each comprise T cell antigens not shared by the respective other set of T cell antigens.

[0037] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a T cell antigen and the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0038] In some embodiments of the pharmaceutical composition, the second Plasmodium polypeptide construct comprises a first RH5 antigen and the third Plasmodium polypeptide construct comprises a second RH5 antigen. In some embodiments, the first and the second RH5 antigens partially overlap.

[0039] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the (first, second and / or third) polyribonucleotides or RNA constructs are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0040] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a cryoprotectant. In some embodiments, the pharmaceutically acceptable excipient comprises an aqueous buffered solution. In some embodiments, the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.

[0041] According to an additional aspect, the present invention concerns a combination comprising:(i) a first pharmaceutical composition comprising a first polyribonucleotide or an RNA construct encoding a Plasmodium Pfs230 polypeptide construct disclosed herein; and(ii) a second pharmaceutical composition comprising a second polyribonucleotide encoding a second Plasmodium polypeptide construct, wherein the second Plasmodium polypeptide construct comprises a CSP antigen, a T cell antigen or an RH5 antigen.

[0042] In some embodiments, the combination comprises(i) a first pharmaceutical composition comprising a first polyribonucleotide or an RNA construct encoding a Plasmodium Pfs230 polypeptide construct disclosed herein;(ii) a second pharmaceutical composition comprising a second polyribonucleotide encoding a second Plasmodium polypeptide construct, wherein the second Plasmodium polypeptide construct comprises a CSP antigen, a T cell antigen or an RH5 antigen; and(iii) a third pharmaceutical composition comprising a third polyribonucleotide encoding a third Plasmodium polypeptide construct, and the third Plasmodium polypeptide construct comprises a CSP antigen, a T cell antigen or an RH5 antigen.

[0043] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a CSP antigen.

[0044] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a T cell antigen.

[0045] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises an RH5 antigen.

[0046] In some embodiments of the combination, the third Plasmodium polypeptide construct comprises a CSP antigen.

[0047] In some embodiments of the combination, the third Plasmodium polypeptide construct comprises a T cell antigen.

[0048] In some embodiments of the combination, the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0049] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a first CSP antigen and the third Plasmodium polypeptide construct comprises a second CSP antigen. In some embodiments, the first and the second CSP antigen share no overlap. In some embodiments, the first and second CSP antigens partially overlap.

[0050] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a CSP antigen and the third Plasmodium polypeptide construct comprises a T cell antigen.

[0051] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a CSP antigen and the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0052] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a first set of T-cell antigens and the third Plasmodium polypeptide construct comprises a second set of T cell antigens. In some embodiments, the first and the second set of T cell antigens comprise different T cell antigens. In some embodiments, the T cell antigens in the first and second set of T cell antigens share some T cell antigens with one another whereas they each comprise T cell antigens not shared by the respective other set of T cell antigens.

[0053] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a T cell antigen and the third Plasmodium polypeptide construct comprises an RH5 antigen.

[0054] In some embodiments of the combination, the second Plasmodium polypeptide construct comprises a first RH5 complex antigen and the third Plasmodium polypeptide construct comprises a second RH5 complex antigen. In some embodiments, the first and the second RH5 antigens partially overlap.

[0055] In some embodiments, the pharmaceutical composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the (first, second and / or third) polyribonucleotides or RNA constructs are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0056] In some embodiments, the first, second and third pharmaceutical compositions comprise at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a cryoprotectant. In some embodiments, the pharmaceutically acceptable excipient comprises an aqueous buffered solution. In some embodiments, the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.

[0057] In some embodiments, the combination is a pharmaceutical kit.

[0058] In some embodiments, the kit comprises instructions for use of the kit in treating or preventing a malaria infection.

[0059] In some embodiments of the pharmaceutical composition or combination disclosed herein, the second or third polypeptide construct comprising a CSP antigen comprises an amino acid sequence selected from 203, 206, 208, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 264, 269, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 305-321, 334-345.

[0060] In some embodiments of the pharmaceutical composition or combination disclosed herein, the second or third polypeptide construct comprising a T cell antigen comprises an amino acid sequence selected from 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, 409, 412, 415, 418, 421, 424, 427.

[0061] In some embodiments of the pharmaceutical composition or combination disclosed herein, the second or third polypeptide construct comprising a Rh5 antigen comprises an amino acid sequence selected from 429, 431, 433, 435, 437, 439, 441, 443, 445, 447-503 and 510-525.

[0062] According to another aspect, the present invention concerns a method for treating or preventing a malaria infection, the method comprising administering a polyribonucleotide, RNA construct, pharmaceutical composition or combination disclosed herein to a subject.

[0063] According to another aspect, the present invention concerns the use of the polyribonucleotide, RNA construct, pharmaceutical composition or combination disclosed herein in the treatment or prevention of a malaria infection.

[0064] According to another aspect, the present invention concerns the use of the polyribonucleotide, RNA construct, pharmaceutical composition or combination disclosed herein for the manufacture of a medicament for the treatment or prevention of a malaria infection.

[0065] According to another aspect, the present invention concerns a polyribonucleotide, RNA construct, pharmaceutical composition or combination disclosed herein for use in a method for treating or preventing a malaria infection.

[0066] According to another aspect, the present invention concerns a polypeptide encoded by a polyribonucleotide disclosed herein.

[0067] According to another aspect, the present invention concerns a host cell comprising a polyribonucleotide or RNA construct disclosed herein.

[0068] According to another aspect, the present invention concerns a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more Plasmodium sexual stage polypeptides or antigenic portions thereof.

[0069] In some embodiments of the polyribonucleotide, the one or more Plasmodium sexual stage polypeptides or antigenic portions thereof comprise one or more Plasmodium Pfs230 polypeptides or antigenic portions thereof. In some embodiments the one or more Plasmodium sexual stage polypeptides or antigenic portions thereof comprise an antigenic portion of Plasmodium Pfs230. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 11.

[0070] In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises one or more N-linked glycosylation sites. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises one N-linked glycosylation site.

[0071] In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, theantigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid sequence according to SEQ ID NO: 11.

[0072] In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid substitution at one or more N-linked glycosylation sites, wherein the amino acid substitution prevents glycosylation at the one or more N-linked glycosylation sites.

[0073] In some embodiments, the antigenic portion of Plasmodium Pfs230 comprises an amino acid substitution at all of the N-linked glycosylation sites, wherein the amino acid substitution prevents glycosylation at all of the N-linked glycosylation sites.

[0074] In some embodiments, the Plasmodium Pfs230 antigenic portion comprises an amino acid substitution at position 585, as numbered according to SEQ ID NO: 1, wherein the amino acid substitutions prevents glycosylation at all of the N-linked glycosylation sites.

[0075] In some embodiments the Plasmodium Pfs230 antigenic portion comprises amino acid substitutions at positions 585, 821, 829, 889, 961, 1079, and 1089, or any combination thereof, as numbered according to SEQ ID NO: 1, wherein the amino acid substitutions prevent glycosylation at all of the N-linked glycosylation sites.

[0076] In some embodiments, the Plasmodium Pfs230 antigenic portion comprises amino acid substitutions at positions 585, 821, 829, 889, 961, 1079, and 1089, as numbered according to SEQ ID NO: 1, wherein the amino acid substitutions prevent glycosylation at all of the N-linked glycosylation sites. In some embodiments, the amino acid substitution comprises an NX[T / S] to QX[T / S] substitution.

[0077] In some embodiments, the polypeptide comprises a secretory signal. In some embodiments, the secretory signal comprises or consists of a heterologous secretory signal. In some embodiments, the heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, the viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, the HSV secretory signal comprises or consists of an HSV-1 or HSV-2 secretory signal. In some embodiments, the HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, wherein the HSV gD secretory signal consists of an amino acid sequence according to SEQ ID NO: 15. In some embodiments, the secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, the Plasmodium secretory signal comprises or consists of an amino acid sequence according to SEQ ID NOs: 15-38. In some embodiments, the secretory signal is located at the N-terminus of the polypeptide.

[0078] In some embodiments, the polypeptide comprises a transmembrane region. In some embodiments, the transmembrane region is a heterologous transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, the viral transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, the HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NOS: 96, 102, 103, 104, 105 or 106.

[0079] In some embodiments, the polypeptide comprises one or more linkers. In some embodiments, the one or more linkers comprise one or more glycine-serine linkers. In some embodiments, the one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NOs: 107-117. In some embodiments, the one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 107. In some embodiments, the one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 108. In someembodiments, the polypeptide comprises a linker after an amino acid sequence of the one or more Plasmodium sexual stage polypeptides or antigenic portions thereof.

[0080] In some embodiments, Plasmodium is Plasmodium falciparum. In some embodiments, the one or more Plasmodium sexual stage polypeptides or antigenic portions thereof are one or more P. falciparum sexual stage polypeptides or antigenic portions thereof. In some embodiments, the one or more Plasmodium sexual stage polypeptides or antigenic portions thereof are a P. falciparum Pfs230 polypeptide or antigenic portion thereof. In some embodiments, Plasmodium falciparum is Plasmodium falciparum isolate 3D7.

[0081] In some embodiments, the polyribonucleotide is an isolated polyribonucleotide.

[0082] In some embodiments, the polyribonucleotide is an engineered polyribonucleotide.

[0083] In some embodiments, the polyribonucleotide is a codon-optimized polyribonucleotide.

[0084] In some embodiments, the polypeptide comprises:

[0085] (i) a secretory signal; and

[0086] (ii) a Plasmodium Pfs230 polypeptide or antigenic portion thereof.

[0087] In some embodiments, the polypeptide comprises:

[0088] (i) a secretory signal;

[0089] (ii) a Plasmodium Pfs230 polypeptide or antigenic portion thereof; and

[0090] (iii) a transmembrane region.

[0091] In some embodiments, the polypeptide comprises:

[0092] (i) a secretory signal;

[0093] (ii) a Plasmodium Pfs230 polypeptide or antigenic portion thereof;

[0094] (iii) a linker; and

[0095] (iv) a transmembrane region.

[0096] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 122. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 122.

[0097] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 123. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 123.

[0098] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 124. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 124.

[0099] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 125. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 125.

[0100] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 126. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 126.

[0101] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 121. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 121.

[0102] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 123. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 123.

[0103] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 124. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 124.

[0104] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 125. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 125.

[0105] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 126. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 126.

[0106] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 119. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 119.

[0107] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 120. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 120.

[0108] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 13. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 13.

[0109] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to a sequence comprising SEQ ID NO: 14. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of SEQ ID NO: 14.

[0110] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 127. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence according to SEQ ID NO: 127.

[0111] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according toSEQ ID NO: 128. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence according to SEQ ID NO: 128.

[0112] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 129. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence according to SEQ ID NO: 129.

[0113] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 130. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence according to SEQ ID NO: 130.

[0114] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 131. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of an amino acid sequence according to SEQ ID NO: 131.

[0115] In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 542-736 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 542-731 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 564-731 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 579-731 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 587-731 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 443-1132 of SEQ ID NO: 1. In some embodiments, the Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises or consists of amino acids 542-1132 of SEQ ID NO: 1.

[0116] In some embodiments, the secretory signal is a viral secretory signal. In some embodiments, the viral secretory signal is an HSV glycoprotein D (gD) secretory signal. In some embodiments, the HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 96.

[0117] In some embodiments, the linker comprises a glycine-serine linker. In some embodiments, the linker comprises or consists of an amino acid sequence according to SEQ ID NO: 107-117. In some embodiments, the linker comprises or consists of an amino acid sequence according to SEQ ID NO: 107. In some embodiments, the one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 108.

[0118] In some embodiments, the transmembrane region is a viral transmembrane region. In some embodiments, the viral transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 106. In some embodiments, the transmembrane region is a HSV gD transmembrane region. In some embodiments, the HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 96, 102, 103, 104, 105, or 106.

[0119] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 122 or 121. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 122 or 121.

[0120] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 123. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 123.

[0121] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 124. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0122] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 125. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 125.

[0123] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 126. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 126.

[0124] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 119. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 119.

[0125] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 120. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 120.

[0126] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 127. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 127.

[0127] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 128. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 128.

[0128] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 129. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 129.

[0129] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 130. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 130.

[0130] In some embodiments, the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 131. In some embodiments, the polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 131.

[0131] In another aspect, the invention concerns an RNA construct comprising in 5' to 3' order:

[0132] (i) a 5' UTR;

[0133] (ii) a polyribonucleotide disclosed herein;

[0134] (iii) a 3' UTR; and

[0135] (iv) a polyA tail sequence.

[0136] In some embodiments of the RNA construct:

[0137] (i) the 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR; and

[0138] (ii) the 3' UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0139] In some embodiments, the 5’ UTR consists of a ribonucleic acid sequence according to SEQ ID NO: 188.

[0140] In some embodiments, the 3’ UTR consists of a ribonucleic acid sequence according to SEQ ID NO:

[0141] In some embodiments, the polyA tail sequence is a split polyA tail sequence. In some embodiments, the split polyA tail sequence consists of a ribonucleic acid sequence according to SEQ ID NO: 190.

[0142] In some embodiments, the RNA construct further comprises a 5' cap.

[0143] In some embodiments, the RNA construct further comprises a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide.

[0144] In some embodiments, the 5' cap comprises or consists of m7(3'OMeG)(5')ppp(5')(2'OMeAl)pG2, wherein Al is position +1 of the polyribonucleotide, and G2 is position +2 of the polyribonucleotide.

[0145] In some embodiments, the cap proximal sequence comprises Al and G2 of the Capl structure, and a sequence comprising: A3A4U5 (SEQ ID NO: 186) at positions +3, +4 and +5 respectively of the polyribonucleotide.

[0146] In some embodiments, the polyribonucleotide includes modified uridines in place of all uridines. In some embodiments, modified uridines are each Nl-methyl-pseudouridine.

[0147] In another aspect, the invention concerns a composition comprising one or more polyribonucleotides disclosed herein.

[0148] In another aspect, the invention concerns a composition comprising one or more RNA constructs disclosed herein.

[0149] In some embodiments, the composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the one or more polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0150] In some embodiments, the composition further comprises lipid nanoparticles, wherein the one or more polyribonucleotides are encapsulated within the lipid nanoparticles.

[0151] In another aspect, the invention concerns a pharmaceutical composition comprising a composition disclosed herein and at least one pharmaceutically acceptable excipient.

[0152] In some embodiments, the pharmaceutical comprises a cryoprotectant.

[0153] In some embodiments, the pharmaceutical comprises an aqueous buffered solution.

[0154] In some embodiments, the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.

[0155] In another aspect, the invention concerns a combination comprising:

[0156] (i) a first pharmaceutical composition comprising a first polyribonucleotide disclosed herein; and

[0157] (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium T-cell antigens.

[0158] In some embodiments:

[0159] (a) the first polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 1-14; and

[0160] (b) the second polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NO: 409 and 415.

[0161] In another aspect, the invention concerns a combination comprising:

[0162] (i) a first pharmaceutical composition comprising a first polyribonucleotide disclosed herein; and

[0163] (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, the second polypeptide comprises one or more Plasmodium CSP polypeptides or antigenic portions thereof.

[0164] In some embodiments:

[0165] (a) the first polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 1-14; and

[0166] (b) the second polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NO: 231 and 279.

[0167] In another aspect, the invention concerns a combination comprising:

[0168] (i) a first pharmaceutical composition comprising a first polyribonucleotide disclosed herein; and

[0169] (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, the second polypeptide comprises Plasmodium Rh5 invasion complex polypeptides or antigenic portions thereof.

[0170] In some embodiments:

[0171] (a) the first polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 1-14; and

[0172] (b) the second polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NO: 429, 431, 433, 435, 437, 439, 441, 443, 445, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, and 458.

[0173] In another aspect, the invention concerns a method comprising administering a polyribonucleotide disclosed herein to a subject.

[0174] In another aspect, the invention concerns a method comprising administering an RNA construct disclosed herein to a subject.

[0175] In another aspect, the invention concerns a method comprising administering a composition disclosed herein to a subject.

[0176] In another aspect, the invention concerns a method comprising administering one or more doses of the pharmaceutical composition disclosed herein to a subject.

[0177] In another aspect, the invention concerns a pharmaceutical composition disclosed herein for use in the treatment of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

[0178] In another aspect, the invention concerns a pharmaceutical composition disclosed herein for use in the prevention of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

[0179] In another aspect, the invention concerns a method disclosed herein or a pharmaceutical composition for use dislosed herein, the method or use comprising administering two or more doses of the pharmaceutical composition to a subject.

[0180] In another aspect, the invention concerns a method disclosed herein or a pharmaceutical composition for use dislosed herein, the method or use comprising administering three or more doses of the pharmaceutical composition to a subject.

[0181] In another aspect, the invention concerns a method comprising administering a combination of disclosed herein to a subject. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on different days. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject at different locations on the subject's body.

[0182] In some embodiments, the method is a method of treating a malaria infection. In some embodiments, the method is a method of preventing a malaria infection. In some embodiments, the subject has or is at risk of developing a malaria infection. In some embodiments, the subject is a human. In some embodiments, administration induces an anti-malaria immune response in the subject. In some embodiments, the anti-malaria immune response in the subject comprises an adaptive immune response. In some embodiments, the anti-malaria immune response in the subject comprises a T-cell response. In some embodiments, the T-cell response is or comprises a CD4+ T cell response. In some embodiments, the T-cell response is or comprises a CD8+ T cell response. In some embodiments, the anti-malaria immune system response comprises a B-cell response. In some embodiments, the anti-malaria immune system response comprises the production of antibodies directed against the one or more Plasmodium antigens.

[0183] In another aspect, the invention concerns the use of the pharmaceutical composition disclosed herein in the treatment of a malaria infection.

[0184] In another aspect, the invention concerns the use of the pharmaceutical composition disclosed herein in the prevention of a malaria infection.

[0185] In another aspect, the invention concerns the use of the pharmaceutical composition disclosed herein in inducing an anti-malaria immune response in a subject.

[0186] In another aspect, the invention concerns a polypeptide encoded by a polyribonucleotide disclosed herein.

[0187] In another aspect, the invention concerns a polypeptide encoded by an RNA construct disclosed herein.

[0188] In another aspect, the invention concerns a host cell comprising a polyribonucleotide disclosed herein.

[0189] In another aspect, the invention concerns a host cell comprising an RNA construct disclosed herein.

[0190] In another aspect, the invention concerns a host cell comprising a polypeptide disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0191] FIG. 1 includes schematics of exemplary polypeptides (e.g., RNA Constructs 1 8) encoded by DNA constructs provided herein. All amino acid (aa) references included refer to the P. falciparum Pfs230 sequence set out in SEQ ID NO: 1. As shown, RNA Construct 1 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 443-1132) (e.g., SEQ ID NO: 504), a glycine-serine linker (e.g., SEQ ID NO: 107), a Hibit tag (SEQ ID NO: 118), another glycine-serine linker (e.g., SEQ ID NO: 107), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 2 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 542-1132) (e.g., SEQ ID NO: 505), a glycine-serine linker (e.g., SEQ ID NO: 107), a Hibit tag (SEQ ID NO: 118), another glycine-serine linker (e.g., SEQ ID NO: 107), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 3 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 542-736) (e.g., SEQ ID NO: 506), a glycine-serine linker (e.g., SEQ ID NO: 107), a Hibit tag (SEQ ID NO: 118), another glycine-serine linker (e.g., SEQ ID NO: 107), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 4 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 542-736) (e.g., SEQ ID NO: 506), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 5 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 507), a Pfs230 region (aa 542-731) (e.g. SEQ ID NO: 3), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 6 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 508), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 7 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 509), a Pfs230 region (aa 579-731) (e.g. SEQ ID NO: 5), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV gD transmembrane region (e.g.,SEQ ID NO: 96). RNA Construct 8 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 542-736) (e.g. SEQ ID NO: 6), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV gD transmembrane region (e.g., SEQ ID NO: 96). RNA Construct 9 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 9), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV1 gD 41aa KRR transmembrane region (e.g., SEQ ID NO: 102). RNA Construct 10 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 9), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV1 gD 54aa transmembrane region (e.g., SEQ ID NO: 103). RNA Construct 11 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 9), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV1 gD 34aa transmembrane region (e.g., SEQ ID NO: 104). RNA Construct 12 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 9), a glycine-serine linker (e.g., SEQ ID NO: 108), and an HSV1 gD 34aa KRR transmembrane region (e.g., SEQ ID NO: 105). RNA Construct 13 encodes a polypeptide comprising an HSV glycoprotein D (gD) secretory signal (e.g., SEQ ID NO: 15), a Pfs230 region (aa 564-731) (e.g. SEQ ID NO: 9), a glycine-serine linker (e.g., SEQ ID NO: 108), and an H1N1 transmembrane region (e.g., SEQ ID NO: 106).

[0192] FIGS. 2A and 2B depict in-vitro expression of select RNA constructs encoding Plasmodium polypeptides as described herein by host cells (e.g., HEK293T cells). In-vitro expression was measured by detecting the protein associated with cells, either intracellular or surface bound, or the protein detected in the culture medium (e.g., exported). FIG. 2A depicts total cell-associated protein expression determined with a HiBit luminescence assay for RNA constructs 1, 2, and 3, and FIG. 2B depicts total exported protein detected in the culture medium for RNA constructs 1, 2, and 3.

[0193] FIGS. 3A-3C depict binding specificity of antibodies generated from mice immunized with different RNA constructs to Pfs230 protein in Plasmodium falciparum gametocyte lysates. FIG. 3A shows binding between antibodies generated from mice immunized with RNA Construct 2 and Pfs230 protein in the gametocyte lysates as assessed by absorbance (OD450). FIG. 3B shows binding between antibodies generated from mice immunized with RNA Construct 3 and Pfs230 protein in the gametocyte lysates as assessed by absorbance (OD450). FIG. 3C depicts binding of antibodies generated from mice immunized with a negative control.

[0194] FIGS. 4A and 4B depict the measurement of transmission reducing activity of antibodies generated from mice immunized with RNA Constructs 2 or 3, as measured by luminescence (RLU), in a standard membrane feeding assay. FIG. 4A shows one representative replicate at 20% serum, and FIG. 4B shows another representative replicate at 5%, 10%, and 20% serum.

[0195] FIGS. 5A and 5B depict the measurement of transmission reducing activity of antibodies generated from mice immunized with RNA Constructs 2 or 3, as measured by prevalence percent of mosquitoes infected with P / asmodium parasites, in a standard membrane feeding assay (SMFA). FIG.5A shows one representative replicate, and FIG. 5B shows another representative replicate.

[0196] FIGS. 6A and B depict in-vitro cell viability of host cells (e.g., HEK293T cells) following transfection with polyribonucleotide constructs encoding Plasmodium polypeptides as described herein. FIG. 6A depicts the percentage of cells that are viable after staining for intracellular protein expression for both transfected and nontransfected (NT) cells. FIG. 6B depicts the percentage of cells that are viable after staining for surface expressed protein for both transfected and non-transfected cells.

[0197] FIGS. 7A and B depict the transfection rate of polyribonucleotide constructs encoding Plasmodium polypeptides as described herein to host cells (e.g., HEK293T cells). FIG. 7A depicts the transfection rate of indicated polyribonucleotide constructs as measured by percentage of total host cell population that are positive for cell associated protein, both intracellular and surface. FIG. 7B depicts the transfection rate of indicated polyribonucleotide constructs as measured by percentage of total host cell population that are positive for presence of surface expressed protein.

[0198] FIGS. 8A and B depict expression of Plasmodium polypeptides encoded by polyribonucleotide constructs as described herein by host cells (e.g., HEK293T cells). FIG. 8A depicts total cell associated proteinexpression (both intracellular and surface) as measured by median fluorescence intensity of the total host cell population for both transfected and non-transfected cells. FIG. 8B depicts total surface protein expression as measured by median fluorescence of the total host cell population for both transfected and non-transfected cells. Numbers depicted at x-axis indicate RNA construct number. NT stands for non-transfected.

[0199] FIG. 9A and B depict non-debris cell viability of host cells (e.g., HEK293T cells) following transfection with polyribonucleotide constructs encoding Plasmodium polypeptides as described herein. FIG. 9A depicts the percentage of viable non-debris host cells that are positive for presence of intracellularly expressed protein for both transfected and non-transfected (NT) cells. FIG. 9B depicts the percentage of viable non-debri host cells that are positive for presence of surface expressed protein for both transfected and non-transfected cells.

[0200] FIGS. 10A and 10B depict transfection rate and in-vitro expression of select RNA constructs encoding Plasmodium polypeptides as described herein by host cells (e.g., HEK293T cells). In-vitro expression was measured as the percentage of total host cell population that are positive for presence of protein using monoclonal antibody 4F12 and a serum immune to construct 3. FIG. 10A depicts the transfection rate achieved with constructs 3, 5, 6, 7 and 8 formulated in lipids, and FIG. 10B depicts total cell associated protein expression (both intracellular and surface) as measured by median fluorescence intensity of the total host cell population for both transfected and non-transfected cells for RNA constructs 3, 5, 6, 7 and 8 formulated in lipids.

[0201] FIGS. 11A to 11H depict the binding specificity of antibodies generated from mice immunized with RNA constructs 3, 4, 5, 6, 7 and 8 to recombinantly expressed Pfs230 protein (SEQ ID NO: 197). FIG. 11A shows binding between antibodies generated from mice immunized with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed by absorbance (OD450) on day 14 after first dose. FIG. 11B shows binding between antibodies generated from mice immunized with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed by absorbance (OD450) on day 21 after first dose. FIG. 11C shows binding between antibodies generated from mice immunized with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed by absorbance (OD450) on day 28 after first dose. FIG. 11D shows binding between antibodies generated from mice immunized with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed by absorbance (OD450) on day 35 after first dose. FIG. 11E shows the reciprocal serum titer achieved in response to inoculation with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed on day 21 after first dose. FIG. 11F shows the reciprocal serum titer achieved in response to inoculation with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed on day 35. FIG. 11G shows titration curves illustrating the relative levels of antibody binding to Pfs230 protein, as measured by absorbance (OD450), for serum samples collected on day 21 post first dose from mice immunized with RNA constructs 3, 5, 6, 7, and 8. FIG. 11H shows titration curves demonstrating the relative strength of binding between antibodies generated from mice immunized with RNA constructs 3, 5, 6, 7 and 8 and Pfs230 protein as assessed by absorbance (OD450) on day 35 after first dose.

[0202] FIGS. 12A to 12C depict the measurement infection status and transmission reduction of individual mosquitoes fed with infectious P. falciparum gametocytes in a standard membrane feeding assay (SMFA). FIG.12A shows infection intensity of mosquitoes when fed gametocytes with sera of mice immunized twice with lug of formulated constructs 3, 5, 6, 7 and 8 at different dilutions (5%, 1.6%, 0.5%, 0.16%) as a function of Luminescence (RLU) FIG. 12B shows transmission blocking activity of sera generated with constructs 3, 5, 6, 7 and 8 at different dilutions (5%, 1.6%, 0.5%, 0.16%) as a function of % transmission inhibition, with the negative control condition of mosquitoes fed with 5% serum from mice immunized with construct 142 set as 100% transmission. FIG. 12C shows transmission reduction activity of sera generated with constructs 3, 5, 6, 7 and 8 as a function of % transmission inhibition at increasing serum %.DEFINITIONS

[0203] Embodiments of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. In this application, unless otherwise clear from context, (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms"about" and "approximately" may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. As used herein, the following definitions shall apply unless otherwise indicated.

[0204] About. The term "about", when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0205] Agent. As used herein, the term "agent," may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term "therapeutic agent" refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or any combination or complex thereof.

[0206] Amino acid.- In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. " Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. " Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0207] Antigen: ne term "antigen", as used herein, refers to an agent that (i) elicits an immune response; and / or (ii) an agent that binds to a T cell receptor {e.g., when presented by an MHC molecule) or to an antibody.

[0208] Anti-malaria immune response. The term "anti-malaria immune response", as used herein, refers to an immune response directed to one or more antigens derived from Plasmodium.

[0209] Associated. Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0210] Characteristic portion-. As used herein, the term "characteristic portion", in the broadest sense, refers to a portion of a polypeptide or region thereof whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the polypeptide or region thereof. In some embodiments, a characteristic portion of a polypeptide or region thereof is a portion that is found in the polypeptide or region thereof and in related polypeptide or region thereof that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact polypeptide or region thereof. For example, in some embodiments, a "characteristic portion" of a polypeptide or region thereof is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of the polypeptide or region thereof. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a polypeptide or region thereof is one that, in addition to the sequence and / or structural identity specified above, shares at least one functional characteristic with the relevant intact polypeptide or region thereof. In some embodiments, a characteristic portion may be biologically active. In some embodiments, a fragment as described herein can be a portion. Accordingly, in some embodiments, a characteristic fragment can be a "characteristic portion."

[0211] Combination therapy. As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in any combination composition.

[0212] Comparable.- As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0213] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware ofvarious sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term "corresponding to" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0214] Dosing regimenr. Those skilled in the art will appreciate that the term "dosing regimen" (or "therapeutic regimen") may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

[0215] Encode.- As used herein, the term "encode" or "encoding" refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0216] Expression-. As used herein, the term "expression" of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0217] Heterologous: As used herein, the term "heterologous", with respect to secretory signal or transmembrane region, refers to a secretory signal or transmembrane region from a virus or an organism other than Plasmodium.

[0218] Homology. As used herein, the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.

[0219] Identity. As used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be "substantially identical" to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna. CMP matrix.

[0220] Increased, Induced, or Reduced. As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be "increased" relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term "reduced" or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term "reduced" or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0221] In order: As used herein with reference to a polynucleotide or polyribonucleotide, "in order" refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, "in order" refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. " In order" does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being "in order, Feature A, Feature B, and Feature C," this description does not exclude, e.g., Feature D being located between Features A and B.

[0222] Isolated.- The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0223] Linker. As used herein, the term "linker" refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0224] Lipid. As used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0225] Multimerization region: As used herein, the term "multimerization region" refers to a region that directs assembly of multimers into a complex, where each multimer comprises a polypeptide associated with the multimerization region.

[0226] RNA lipid nanoparticle.- As used herein, the term " RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0227] Neubalizationr. As used herein, the term "neutralization" refers to an event in which binding agents (such as antibodies) bind to a biological active site of a parasite (such as a receptor binding protein), thereby inhibiting the parasitic infection of cells. In some embodiments, binding agents eliminate or significantly reduce ability of infecting cells. In some embodiments, neutralization includes prevention of a fertilization event by a parasite. In some embodiments, neutralization includes delaying a fertilization event by a parasite. In some embodiments, neutralization includes prevention of Plasmodium sexual stage parasites from infecting (e.g., traversing into) a mosquito.

[0228] Nucleic acid / Polynucleotide.- As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphoroth ioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0229] Pharmaceutically effective amount. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., malaria), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., malaria). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., malaria) and / or interrupting or reversing the progress of the disease (e.g., malaria). In some embodiments, a desired reaction in a treatment of a disease (e.g., malaria) may be or comprise delay or prevention of the onset of a disease (e.g., malaria) or a condition (e.g., a malaria associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., malaria) or a condition (e.g., a malaria associated condition) to be treated, the severity of such a disease (e.g., malaria) or a condition (e.g., a malaria associated condition), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0230] Polypeptide.- As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the presentspecification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a polypeptide is a Plasmodium polypeptide construct described herein. A Plasmodium polypeptide construct is a polypeptide that includes one or more malarial proteins, or one or more portions thereof. In some embodiments, a Plasmodium polypeptide construct described herein includes a Plasmodium Pfs230 polypeptide or an antigenic portion thereof. In some embodiments, a Plasmodium polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), and / or a linker, as described herein.

[0231] Prevent As used herein, the term "prevent" or "prevention" when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. In some embodiments, prevention comprises reducing the risk of developing clinical malaria. In some embodiments, prevent or prevention comprises reducing the occurrence of Plasmodium sexual stage parasites infecting (e.g., traversing into) a mosquito. In some embodiments, prevent or prevention comprises reducing the occurrence of Plasmodium sexual stage parasites moving from a human host to a mosquito vector.

[0232] Reference.- As used herein, the term "reference" describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0233] Ribonucleic acid (RNA) or Polyribonucleotide.- As used herein, the term "ribonucleic acid," " RNA," or "polyribonucleotide" refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of " Nucleic acid / Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, a RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro(e.g., by enzymatic synthesis methods and / or by chemicalsynthesis methods). In some embodiments, a polyribonucleotide encodes a polypeptide, which is preferably is a Plasmodium polypeptide construct.

[0234] Ribonucleotide.- As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0235] Secretory signal: As used herein, the term "secretory signal" refers to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway.

[0236] Subject As used herein, the term "subject" refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0237] Suffering front. An individual who is "suffering from" a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0238] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) due to their presence in an area in which malaria is endemic. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., malaria and / or a malaria-associated condition) may not have been diagnosed with the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will not develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition).

[0239] Therapy. The term "therapy" refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a therapeutic agent or therapy is a medical intervention that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0240] Transmembrane region-. As used herein, the term "transmembrane region" refers to a region of a polypeptide that spans a biological membrane, such as the plasma membrane of a cell.

[0241] Treat As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition).

[0242] Variant: As used herein, the term "variant" refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties {e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid e.g., that are attached to the polypeptide or nucleic acid backbone).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTSI. Malaria

[0243] Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., etai. Malaria. Nat Rev Dis Primers 3, 17050, 2017, which is incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is ingested that contains Plasmodium parasites. If the blood meal contains viable gametocytes, the Anopheles mosquito can become infected, and, upon maturation and migration of sporozoites to the salivary glands, the infectious mosquito can subsequently bite and transmit the parasites to a new host.

[0244] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many malaria parasites have developed resistance to available therapeutics. According to Malaria Eradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination.

[0245] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as " RTS, S", a milestone in malaria vaccine development. In 2019, the World Health Organization launched pilot programs that provide RTS, S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS, S / AS01 is an adjuvanted protein subunit vaccine that consists of a portion of the major repeat region and the C-terminus of CSP from Plasmodium falciparum fused to the Hepatitis B surface antigen (HBsAg). The vaccine is a mix of this / CSP-HBsAg compound with HBsAg that forms virus-like particles (RTS, S / AS01; Mosquirix™). RTS, S is administered according to a regimen that requires four doses: an initial 3-dose schedule given at least 1 monthapart, and a 4th dose 15-18 months after dose 3 (see, for example, Vandoolaeghe 8i Schuerman Expert Rev Vaccines. 15:1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, each of which is incorporated herein by reference in its entirety). Reports indicate that RTS, S protects approximately 30% to 50% of children from clinical disease over 18 months. RTS, S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T cell responses (see, for example, Moris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). Phase III studies of RTS, S delivered as a three-dose series with a booster after 1 yr (year) showed moderate vaccine efficacy in children aged 5 to 17 months preventing 36% of clinical malaria cases over the full study period with a median follow-up of 4 yrs, with a range of 20% in high to 66% in low transmission settings. Furthermore, published literature suggests that protection wanes over time including reports of potential negative efficacy after 5 yrs in children with high malaria exposure (Olotu et al. 2016, N. Engl. J. Med. 374:2519-29, which is incorporated herein by reference in its entirety). Another malaria vaccine is " R21 / Matrix-M." R21 / Matrix-M (R21) is a pre-erythrocytic protein subunit, candidate malaria vaccine based on RTS, S. R21 lacks the excess HBsAg found in RTS, S and comprises only fusion protein moieties (Datoo, Mehreen S et al. Lancet (London, England) vol. 397,10287 (2021): 1809-1818, which is incorporated herein by reference in its entirety). In a Phase 3 clinical trial, R21 was administered as three doses, four weeks apart, with a booster 12 months after the third dose. Trials were performed in two sites, a site with seasonal Malaria transmission and a site with perennial Malaria transmission. Twelve-month vaccine efficacy was approximately 75% at the seasonal sites and 68% at the standard sites for time to first clinical malaria episode (see, for example Datoo, Mehreen S et aL, which is incorporated herein by reference in its entirety). RTS, S also initially showed a more favorable vaccine efficacy (e.g., 56%), but this efficacy dropped to approximately 36% after 3 years. Both RTS, S and R21 target the same parasite stage (e.g., pre-erythrocytic), and no vaccine currently exists that targets the sexual stage. Thus, an effective malaria vaccine that targets the sexual stage remains an unmet medical need of critical importance for global health.A. Lifecycle

[0246] During a blood meal, infected mosquitoes inject, along with their anticoagulating saliva, sporozoites into the skin. Sporozoites journey through the skin and, into blood vessels, where they can travel in the blood to their target cells, the hepatocytes of the liver. This journey happens very quickly; it can be completed within only a few minutes (Sinnis et al., Parasitol Int. 2007 Sep;56(3):171-8, which is incorporated herein by reference in its entirety). This is a time known to be a stage during which it is challenging for the host immune system to mount an effective response against the parasite, as only a small number (thought to be a few hundred at maximum) of sporozoites are injected by the mosquito, with a small fraction of those parasites establishing infection in the liver (Flores-Garcia et al., mBio. 2018 Nov 20;9(6):e02194-18, which is incorporated herein by reference in its entirety).

[0247] When moving from an inoculation site in the skin to the liver, sporozoites traverse host cells (Mota et aL, Science 2001 Jan 5;291(5501): 141-4). Sporozoites traverse different types of host cells at the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe. 2008 Feb 14;3(2):88-96, which is incorporated herein by reference in its entirety), and the liver sinusoidal barrier, containing liver endothelial cells and Kupffer cells (Frevert et aL, PLoS Biol 3(6): el92. 2005, which is incorporated herein by reference in its entirety) and sinusoidal endothelial cells (Tavares et aL, J Exp Med 2013 May 6;210(5):905-15, which is incorporated herein by reference in its entirety), in order to gain access to hepatocytes. Sporozoites preferentially traverse cells with low-sulfated heparin sulfate proteoglycans (HSPGs) but preferentially invade cells with high-sulfated HSPGs (Coppi et al., Cell Host 8i Microbe 2, 316-327, November 2007, which is incorporated herein by reference in its entirety).

[0248] Cell traversal was first observed as non-phagocytic entry of P. berghei sporozoites into macrophages followed by "escape" from these cells (Vanderberg et aL, J. Euk. Microbiol. 37:528-536, 1990, which is incorporated herein by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being explored. However, it has been suggested by electron microscopy that host cell rupture occurs upon entry and exit from the host cell (Mota et aL, 2001; Tavares et aL, 2013, each of which is incorporated herein by reference in its entirety). It has also been shown that P. yoe / ii sporozoites can enter hepatocytes via a transient vacuole and thathost membrane rupture occurs upon cell exit rather than cell entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov ll;18(5):593-603, which is incorporated herein by reference in its entirety).

[0249] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is incorporated herein by reference in its entirety). Several possibilities emerged as to why this occurs. The first hypothesis suggested that migration through hepatocytes primes parasites for invasion by activating apical exocytosis (Mota et aL, Nat Med 2002 Nov;8(ll): 1318-22, which is incorporated herein by reference in its entirety). The second theory suggested that traversal releases hepatocyte growth factor (HGF), making neighboring hepatocytes more susceptible to infection (Carrolo et al., Nat Med. 2003 Nov;9(ll): 1363-9, which is incorporated herein by reference in its entirety). Lastly, other studies suggest that it takes some time for sporozoites to switch off the machinery for traversal and activate invasion machinery (Amino et al., 2008, Coppi et al., 2007, each of which is incorporated herein by reference in its entirety), and that traversal primarily functions to penetrate cell barriers and avoid phagocytosis en route to the liver (Amino et al., 2008, Coppi et al., 2007, Tavares et aL, 2013, each of which is incorporated herein by reference in its entirety).

[0250] Although it has been shown that sporozoites traverse human cells (Behet et aL, Malar J 2014 Apr 5;13:136; Cha et aL, J Exp Med 2015 Aug 24;212(9): 1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6):e0129623; van Schaijk et aL, PLoS ONE, 3 (10). e3549 2008; Risco-Castillo et aL, 2015, each of which is incorporated herein by reference in its entirety), the molecular basis for the traversal process is largely unstudied.

[0251] Once sporozoites have invaded liver cells, they undergo a phase of massive replication, ultimately differentiating into merozoites, the red blood cell invasive form of the parasite. In approximately one week, a single sporozoite infected hepatocyte can result in the formation of 40,000-60,000 merozoites (Vaughan et al. The Journal of clinical investigation vol. 122(10) 2012 3618-28, which is incorporated herein by reference in its entirety). Merozoites bud from the host hepatocyte in structures called merosomes, which contain up to a thousand merozoites and are hidden from host immune responses due to their host membrane composition. These merosomes ultimately rupture, releasing merozoites into the blood stream, where they invade red blood cells and begin the blood stage of infection, characterized by cyclical ~48 hours asexual replication, rupture and reinvasion, with clinical presentation ~11 days after infectious mosquitos bite. Each mature blood stage parasite can release up to 32 daughter merozoites, which causes parasite density in the blood to increase rapidly.

[0252] Plasmodium spp. parasites gain entry into red blood cells through specific ligand-receptor interactions mediated by proteins on the surface of the parasite that interact with receptors on the host erythrocyte (mature red blood cell) or reticulocyte (immature red blood cell). While P. falciparum can invade and replicate in erythrocytes and reticulocytes, P. vivaxand other species predominantly invade reticulocytes, which are less abundant than erythrocytes. Most of the erythrocyte-binding proteins or reticulocyte-binding proteins that have been associated with invasion exhibit redundancy or are expressed as a family of variant forms; however, for P. falciparum, the PCRCR complex comprised of key proteins Ripr, Rh5, CyRPA, TRAMP, and CSS have been identified as essential for parasite invasion.

[0253] P. vivax and P. ovale can also enter a dormant state in the liver, the hypnozoite.

[0254] The invasion of a red blood cell by the merozoite involves interactions of multiple parasite derived proteins (e.g., ligands) with red blood cell (RBC) proteins (e.g., receptors) (Weiss et al., Pios Path., 2015 Feb 27;10.1371, which is incorporated herein by reference in its entirety). The invasion process begins by the merozoite first attaching to the RBC. Merozoite-RBC interaction is further strengthened through merozoite surface protein-1 (MSP1) and unknown RBC proteins, causing some deformation of the RBC surface. After the deformation, interaction of merozoite EBA and Rh protein families (excluding Rh5) with host receptors like CR1 lead to actindependent deformation of the RBC membrane and reorientation of the merozoites apical end onto the RBC surface (Geoghehan et aL, Nat Commun., 2021 June 15;10.1038 which is incorporated herein by reference in its entirety). This deformation event increases the surface area of interaction between merozoite and RBC membranes, an interaction which can be stabilized by an Rh5 invasion complex comprising Plasmodium thrombospondin-related apical merozoite protein (PTRAMP), cysteine-rich small secreted (CSS), cysteine-rich protective antigen (CyRPA), Rh5-interacting protein (Ripr) and Rh5. This Rh5 invasion complex including the five proteins is referred to as the PCRCR complex.

[0255] The creation of the PCRCR complex is thought to begin in the endoplasmic reticulum, where its constituents cysteine-rich small secreted (CSS) protein and Plasmodium thrombospondin-related apical merozoite protein (PTRAMP) interact to form a heterodimer referred to as PTRAMP-CSS. It is hypothesized that a tetrameric PCRCR complex is formed by PTRAMP-CSS trafficking to a secretory organelle called the microneme, where CSS interacts with Rh5-interacting protein (Ripr) and cysteine-rich protective antigen (CyRPA) to form PCRC. At some point after deformation occurs, the merozoite initiates a polarized secretion process that allows the micronemal PCRC complex opportunity to interact with rhoptry protein Rh5, forming the pentameric complex PCRCR. The PCRCR complex, through Rh5, can then bind a host cell receptor, called basigin, broadly across the merozoite-RBC interphase. PCRCR binding to basigin creates a stable and irreversible platform between the apical end of the merozoite and the deformed RBC surface (Scally et al., Nat Microb., 2022 May 4, which is incorporated herein by reference in its entirety). Once the merozoite-RBC interaction is secured, an open connection between the merozoite's apical tip and the RBC surface is formed which acts as a conduit for Ca2+to flow into the RBC, and for merozoite derived invasion proteins, such as AMA1 and RON2, to help establish the moving junction (Srinivasan et al., Proc Natl Acad Sci., 2011 Jul 25;13275-80, which is incorporated herein by reference in its entirety). It is through this moving junction that the parasite, utilizing its actin-myosin dependent gliding motility, propels itself inside the RBC and establishes a parasitophorous vacuole within which the parasite grows and replicates.

[0256] It has been shown that Rh5 binds basigin with higher affinity when complexed with CyRPA and Ripr to form the RCR complex, and has the highest affinity for basigin when part of the PCRCR complex (Wong et al., Nature., 2019 and Scally et aL, Nat Microb., 2022 May 4, each of which are incorporated herein by reference in its entirety).

[0257] Merozoites that egress from red blood cells can invade other red blood cells to continue to the asexual blood stage of the parasite lifecycle. A small percentage of schizonts are already committed to a different fate and merozoites from these schizonts, following invasion of a new red blood cell, will differentiate into the sexual forms on the parasite's life cycle; either male or female, gametocytes. Gametocytes are taken up by the mosquito vector during blood feeding. In the gut of the mosquito, male gametocytes undergo 3 rapid rounds of mitosis to form 8 flagellate microgametes. Female gametocytes mature into macrogametes, egressing from their red blood cell. Male microgametes are motile forms with flagellae and seek the female macrogamete. The male and female gametocytes fuse, forming a diploid zygote, which matures and differentiates into an ookinete; this motile form secretes chitinases and other lytic proteins such as CelTOS in order to disrupt and traverse through the peritrophic matrix and midgut epithelium to reach the basal lamina where it further differentiates and matures as an oocyst. Oocysts mature over approximately 10 days (depending on the temperature), replicating to form sporozoites that egress the mature oocyst into the hemocoel of the mosquito. Thousands of sporozoites form in a single oocyst and become randomly distributed throughout the hemocoel. These midgut sporozoites have 4 rhoptries, a polarized secretory organelle responsible for releasing factors essential to invasion. It is thought that sporozoites are passively circulated through the mosquito haemolymph until they encounter the salivary glands, where they actively invade the glands. Following invasion of the salivary gland, sporozoites are re-programmed to prepare for liver invasion. It is hypothesized that the salivary gland invasion process utilizes two of the parasite's four rhoptries, reserving the remaining two rhoptries for vertebrate host cell invasion. Evidence of this reprogramming has been demonstrated by the inability of midgut sporozoites (directly from oocysts) to invade hepatocytes, and also by the fact that sporozoites which have successfully invaded a salivary gland are unable to re-invade another salivary gland if presented one. Salivary gland sporozoites alter mosquito behavior and salivary gland function, as less saliva is produced resulting in an increase in mosquito probing behavior, increasing the chances of transmission to a human host via a mosquito bite and continuing the human host's phase of this parasite's life cycle.

[0258] Gametocytes (e.g., sexual stage) are resistant to many drugs used to treat blood stage infections, therefore transmission of malaria through mosquito ingestion of gametocytes remains possible. The risk of malaria transmission remains even after treatment eliminates the symptoms, as asymptomatic individuals can still carry viable gametocytes that mosquitoes ingest which can ultimately lead to onward transmission to another human host. While it is recognized as a promising target for malaria intervention, there is currently a lack of therapeutics which specifically target transmission from humans to mosquitoes and most current therapeutics therefore onlytarget one half of the parasite's life cycle. Parasite transmission from human to mosquito creates a relatively short window for an immune response to prevent transmission of all sexual stage parasites to a mosquito. A lack of immune response during this window represents a missed opportunity to block parasite transmission from human to mosquito and eventually back to human. The clinical importance of this missed opportunity is substantial, as just one successful fertilization event in the mosquito can lead to the formation of thousands of sporozoites, which render mosquitoes even with low infections still infectious to humans. Therefore, targeting the sexual stage of Plasmodium offers a promising approach for interrupting transmission and utimately reducing deaths caused by malaria.

[0259] Malaria symptoms typically develop 4-8 days after initial red blood cell invasion. Replication cycle of asexual parasites within the red blood cells is approximately 48 hours, ending with hemolysis, releasing the merozoites for another round of red blood cell infection. Thus, in synchronous infections (infections that originate from a single infectious bite), fever occurs approximately every 48 hours, due to a release of parasite waste and proteins upon egress and destruction of the RBC host cells.

[0260] Some drugs that prevent Plasmodium spp. invasion or proliferation in the liver have prophylactic activity, drugs that block the red blood cell stage are required for the treatment of the symptomatic phase of the disease, and compounds that inhibit the formation of gametocytes or their development in the mosquito (including drugs that kill mosquitoes feeding on blood) are transmission-blocking agents (Phillips, etai. Malaria. Nat Rev Dis Primers^, 17050 (2017), which is incorporated herein by reference in its entirety).B. Genome

[0261] Since completion of the first sequence of P. falciparum 3D7 genome in 2002, genomic research on malaria parasites has rapidly advanced. Except for a short phase after fertilization in the mosquito midgut, Plasmodium spp. parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases, contain 14 chromosomes, a circular plastid genome of approximately 35 kilobases, and multiple copies of a 6 kilobase mitochondrial DNA. Comparison of genomes from different species showed that homologous genes are often found in synthetic blocks arranged in different orders among different chromosomes.

[0262] The adenine-thymine (AT) content of Plasmodium spp. can also be very different, e.g., -80% AT in P. falciparum, P. reichenowi, and P. gallinaceurrr, -75% AT in rodent malaria parasites; and -60% AT in P. vivax, P. knowiesi, and P. cynomoigi AT content is often higher in introns and intergenic noncoding regions than in protein-coding exons, with an average of 80.6% AT for the whole P. falciparum genome versus 86.5% for noncoding sequences. The high AT content of P. falciparum reflects large numbers of low-complexity regions, simple sequence repeats, and microsatellites, as well as a highly skewed codon usage bias. Polymorphisms of AT-rich repeats provide abundant markers for linkage mapping of drug resistance genes and for tracing the evolution and structure of parasite populations.

[0263] An exemplary polymorphic gene family comprises a group of 14 genes encoding proteins with six cysteines (6-Cys). These proteins often localize on the parasite surface interacting with host proteins and are expressed at different parasite developmental stages. 6-Cys proteins also demonstrate diverse functions and have been shown to play roles in, for example, parasite fertilization, mating interactions (e.g., gamete recognition), evasion of immune responses, and invasion of hepatocytes. The proteins expressed in asexual stages are generally polymorphic and / or under selection, suggesting that they could be targets of the host immune response; however, their functions in parasite development remain largely unknown. P. falciparum sexual stage protein Pfs230 is the first member of the 6-Cys family to be characterized. Pfs230 is also the largest member of the 6-Cys family with a size greater than 300 kDa. Members of the 6-cystein family (e.g., Pfs230) are present (e.g., expressed) during the sexual stages of the parasite life cycle and are responsible for many critical roles in parasite development. For example, knockout studies demonstrated that Pfs230 is critical for male fertility, as Pfs230-deficient male sexual stage parasites (e.g., male gametes) were unable to bind to red blood cells and establish exflagellation centers. (Lyons et. Al., Plasmodium 6-Cysteine Proteins: Functional Diversity, Transmission-Blocking Antibodies and Structural Scaffolds. Front. Cell. Infect. Microbiol. 2022 July 07;(12), which is incorporated herein by reference in its entirety).

[0264] Plasmodium genomes can be highly polymorphic. Early studies demonstrated polymorphisms involving tens to hundreds of kilobases and that the chromosome structure in P. falciparums largely conserved in central regions but extensively polymorphic is both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repetitive sequences and families of genes.C. Plasmodium Proteins1. Plasmodium Gamete ProteinsPfs230

[0265] Pfs230 is an approximately 300 kDa protein expressed from stage II gametocytes and localized on the surface of gametocytes and gametes during the sexual stages of the Plasmodium life cycle. The full-length 230 kDa protein comprises 14 cysteine-rich domains, forming 7 double domains. During gametogenesis, Pfs230 undergoes two separate N-terminal cleavage events, producing two versions of the protein, 300 kDa and 307 kDa in size. It has been reported that Pfs230 is critical for male fertility, with Pfs230 knockout P. falciparum males unable to bind to red blood cells and establish exflagellation centers. Furthermore, seropositivity of human sera against Pfs230 is a predictor for transmission-blocking immunity (Lyons et. al., 2022, which is incorporated herein by reference in its entirety). The presence of Pfs230 on the gametocyte surface during the sexual stage of the Plasmodium life cycle and its essential role in fertilization, help make Pfs230 an ideal target for immune responses that can achieve transmission blocking.

[0266] Pfs230 sequences are known (see, e.g., UniProt accession numbers P68874, P68875, 096175, A0A059WHM4, A0A059WHM8, A0A059WHF8, A0A059WIL0, A0A059WNB1, A0A059WNG3, A0A059WCN2, A0A059WNB7, A0A059WCM8, A0A059WIK5, each of which is incorporated herein by reference in its entirety), and an exemplary Pfs230 amino acid sequence is provided in Table 1.Table 1: Exemplary Sequences for Pfs230 PolypeptidesSEQ ID Protein Sequence (Amino Acid)NO:1 Pfs230 MKKIITLKNLFLIILVYIFSEKKDLRCNVIKGNNIKDDEDKRFHLFYYSHNLFKTPETKEKKN (3D7) KKECFYKNGGIYNLSKEIRMRKDTSVKIKQRTCPFHKEGSSFEMGSKNITCFYPIVGKKER KTLDTIIIKKNVTNDHWSSDMHSNVQEKNMILIRNIDKENKNDIQNVEEKIQRDTYENK DYESDDTLIEWFDDNTNEENFLLTFLKRCLMKIFSSPKRKKTWQKKHKSNFFINSSLKYI YMYLTPSDSFNLVRRNRNLDEEDMSPRDNFVIDDEEEEEEEEEEEEEEEEEEEEEEEEEY DDYVYEESGDETEEQLQEEHQEEVGAESSEESFNDEDEDSVEARDGDMIRVDEYYEDQ DGDTYDSTIKNEDVDEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEEGEEVG EGVGEEVGEEEGEEVGEEEGEYVDEKERQGEIYPFGDEEEKDEGGESFTYEKSEVDKTDL FKFIEGGEGDDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEYGEAVEDGENVI KIIRS VLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPT ESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSK LIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGNRGIVEVYV EPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKI FF 1 QNIYKKNNIYPCYMKLYS GDIGGILFPKNIKSTTCFEEMIPYNKEIKWNKENKSLGNLVNNSWYNKEMNAKYFNVQY VHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEELNFSLFDFYESFVPIKKTIQVAQK NVNNKEHDYTCDFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKCNVNKTQYPNIEIF PKTLKDKKEVLKLDLDIQYQM FSKFFKFNTQNAKYLN LYPYYLIFPFN HIGKKELKN N PTY KNHKDVKYFEQSSVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLNLSINELGSDNNTFS VTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISKQEEKIKGCNFHESKLDYFNENIS SDTH ECTLHAYEN DIIGFNCLETTH PN EVEVEVEDAEIYLQPENCFN NVYKGLN SVDITTI LKNAQTYNIN N KKTPTFLKIPPYN LLEDVEISCQCTIKQWKKIKVIITKN DTVLLKREVQS ESTLDDKIYKCEHENFINPRVNKTFDENVEYTCNIKIENFFNYIQIFCPAKDLGIYKNIQMY YDIVKPTRVPQFKKFN N EELH KUPNSEM LHKTKEM LILYN EEKVDLLH FYVFLPIYIKDIY EFNIVCDNSKTMWKNQLGGKVIYHITVSKREQKVKGCSFDNEHAHMFSYNKTNVKNCII DAKPKDLIGFVCPSGTLKLTNCFKDAIVHTN LTNINGILYLKN N LAN FTYKHQFNYM EIPA LMDNDISFKCICVDLKKKKYNVKSPLGPKVLRALYKKLNIKFDNYVTGTDQNKYLMTYMD LHLSHKRNYLKELFHDLGKKKPADTDANPESIIESLSINESNESGPFPTGDVDAEHLJLEGY DTWESLYDEQLEEVIYNDIESLELKDIEQYVLQVNLKAPKLMMSAQIHNNRHVCDFSKNN LIVPESLKKKEELGGNPVNIHCYALLKPLDTLYVKCPTSKDNYEAAKVNISENDNEYELQVI SLIEKRFHNFETLESKKPGNGDVWHNGWDTGPVLDNSTFEKYFKNIKIKPDKFFEKVIN EYDDTEEEKDLESILPGAIVSPMKVLKKKDPFTSYAAFWPPIVPKDLHFKVECNNTEYKDENQYISGYNGIIHIDISNSNRKINGCDFSTNNSSILTSSVKLVNGETKNCEININNNEVFGISEQ ID Protein Sequence (Amino Acid)NO:ICDN ETN LDPEKCFHEIYSKDN KTVKKFREVIPNIDIFSLHNSN KKKVAYAKVPLDYIN KLL FSCSCKTSHTNTIGTMKVTLNKDEKEEEDFKTAQGIKHNNVHLCNFFDNPELTFDNNKIV LCKIDAELFSEVIIQLPIFGTKNVEEGVQNEEYKKFSLKPSLVFDDNNNDIKVIGKEKNEVS ISLALKGVYGNRIFTFDKNGKKGEGISFFIPPIKQDTDLKFIINETIDNSNIKQRGLIYIFVR KNVSENSFKLCDFTTGSTSLMELNSQVKEKKCTVKIKKGDIFGLKCPKGFAIFPQACFSNV LLEYYKSDYEDSEHINYYIHKDKKYNLKPKDVIELMDENFRELQNIQQYTGISNITDVLHF KNFNLGNLPLNFKNHYSTAYAKVPDTFNSIINFSCNCYNPEKHVYGTMQVESDNRNFDNI KKNENVIKNFLLPNIEKYALLLDDEERQKKIKQQQEEEQQEQILKDQDDRLSRHDDYNKN HTYILYDSN EHICDYEKN ESUSTLPN DTKKIQKSICKINAKALDWTIKCPHTKN FTPKDY FPNSSLITNDKKIVITFDKKNFVTYIDPTKKTFSLKDIYIQSFYGVSLDHLNQIKKIHEEWD DVH LFYPPH N VLH N WLN N HIVN LSSALEGVLFM KSKVTG DETATKKNTTLPTDG VSSIU PPYVKEDITFH LFCGKSTTKKPN KKNTSLALIHIHISSN RNIIHGCDFLYLENQTN DAISN N N N NSYSIFTHN KNTEN N UCDISLIPKTVIGIKCPN KKLN PQTCFDEVYYVKQEDVPSKTTT ADKYNTFSKDKIGNILKNAISINNPDEKDNTYTYLILPEKFEEELIDTKKVLACTCDNKYII HMKIEKSTMDKIKIDEKKnGKDICKYDVTTKVATCEIIDTIDSSVLKEHHTVHYSITLSR WDKUIKYPTN EKTH FEN FFVN PFN LKDKVLYNYN KPINIEHILPGAITTDIYDTRTKIKQY ILRIPPYVHKDIHFSLEFNNSLSLTKQNQNIIYGNVAKIFIHINQGYKEIHGCDFTGKYSHL FTYSKKPLPNDDDICNVTIGNNTFSGFACLSHFELKPNNCFSSVYDYNEANKVKKLFDLST KVELDHIKQNTSGYTLSYIIFNKESTKLKFSCrCSSNYSNYnRITFDPNYIIPEPQSRAIIKYVDLQDKNFAKYLRKLII. Plasmodium polypeptide constructs

[0267] The present disclosure, among other things, utilizes RNA technologies as a modality to express one or more Plasmodium polypeptide constructs that includes one or more malarial proteins, or one or more portions thereof, described herein. For example, in some embodiments, a Plasmodium polypeptide construct comprises one or more Plasmodium gamete polypeptides or portions thereof (e.g., antigenic portions of one or more Plasmodium gamete polypeptides). In some embodiments, one or more polypeptides or portions thereof of a Plasmodium gamete can include one or more polypeptides or portions thereof of a Plasmodium Pfs230. In some embodiments, a portion of a Pfs230 polypeptide can be a characteristic portion of a Pfs230 polypeptide. In some embodiments, a Plasmodium polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), and / or a linker, as described herein.A. Certain Plasmodium Gamete Polypeptides or Portions Thereof1. Pfs230

[0268] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more Plasmodium gamete polypeptides or portions thereof (e.g., antigenic portions thereof) that comprise one or more Plasmodium Pfs230 polypeptides or antigenic portions thereof.

[0269] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more regions or portions (e.g., antigenic portions) of a Pfs230 polypeptide, e.g., Plasmodium Pfs230 polypeptide, e.g., P. falciparum Pfs230 (SEQ ID NO: 1), or a variant thereof (e.g., a glycosylation variant).

[0270] In some embodiments, a Plasmodium polypeptide construct comprises an antigenic portion of Pfs230. In some embodiments, a Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least. 210. at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 510, at least 520, at least 530, at least 540, or at least 550, at least 560, at least 570, at least 580. at least 590, at least 600, at least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least 680, at least 690, at least 700, at least 710, at least 720, at least 730, at least 740, or at least 750 contiguous amino acids of Pfs230, or a variant thereof (e.g., a sequence variant or a glycosylation variant).

[0271] In some embodiments, a Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises at most 700, at most 710, at most 720, at most 730, at most 740, at most 750, at most 760, at most 770, at most 780, at most 790, at most 800, at most 810, at most 820, at most 830, at most 840, or at most 850 contiguous amino acids of Pfs230, or a variant thereof (e.g., a sequence variant or a glycosylation variant).

[0272] In some embodiments, a Plasmodium Pfs230 polypeptide or antigenic portion thereof comprises ranges from 50-850, 50-800, 50-750, 100-700, 100-650, 100-600, 100-500, 100-400, 100-300, 100-200, 150-250, 150-200, 500-800, 550-700, 600-700, or 650-700 contiguous amino acids of Pfs230, or a variant thereof (e.g., a sequence variant or a glycosylation variant).

[0273] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 587-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 6 or a variant thereof (e.g., a glycosylation variant).

[0274] 587-731 portion of Pfs230 (SEQ ID NO. 6) TNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISP TVN EKEN N FKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGN RGIVEVYVEPYG

[0275] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 579-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 5 or a variant thereof (e.g., a glycosylation variant).

[0276] 579-731 portion of Pfs230 (SEQ ID NO. 5) YDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSK LIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGNRGIVEVYVEPYG

[0277] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 579-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 509 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 509 or a variant thereof (e.g., a glycosylation variant).

[0278] 579-731 portion of Pfs230 (SEQ ID NO. 509) YDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSK LIYGLLISPTVN EKEN N FKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKG N RGIVEVYVEPYG

[0279] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 564-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 4 or a variant thereof (e.g., a glycosylation variant).

[0280] 564-731 portion of Pfs230 (SEQ ID NO. 4) YETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYV VLTKEETKLKEKLLSKLIYGLLJSPTVN EKEN N FKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKG N RGIVEVYVEPYG

[0281] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 564-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 508 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 508 or a variant thereof (e.g., a glycosylation variant).

[0282] 564-731 portion of Pfs230 (SEQ ID NO. 508) YETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYV VLTKEETKLKEKLLSKLIYG LLISPTVN EKEN N FKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGN RGIVEVYVEPYG

[0283] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 3 or a variant thereof (e.g., a glycosylation variant).

[0284] 542-731 portion of Pfs230 (SEQ ID NO. 3) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLJKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPVVH KATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYG

[0285] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-731 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 507 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 507 or a variant thereof (e.g., a glycosylation variant).

[0286] 542-731 portion of Pfs230 (SEQ ID NO. 507) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLJSPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYG

[0287] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-736 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 2 or a variant thereof (e.g., a glycosylation variant).

[0288] 542-736 portion of Pfs230 (SEQ ID NO. 2) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKING

[0289] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-736 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 506 or a variant thereof (e.g.,a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 506 or a variant thereof (e.g., a glycosylation variant).

[0290] 542-736 portion of Pfs230 (SEQ ID NO. 506) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKING

[0291] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-1132 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 12 or a variant thereof (e.g., a glycosylation variant).

[0292] 542-1132 portion of Pfs230 (SEQ ID NO. 12) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKLYSGDIGGILFPKNIKS TTCFEEMIPYNKEIKWNKENKSLGNLVNNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEEL N FSLFDFYESFVPIKKTIQVAQKNVN N KEH DYTCDFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKCNVN KTQYPNIEIFPKT LKDKKEVLKLDLDIQYQMFSKFFKFNTQNAKYLNLYPYYLIFPFNHIGKKELKNNPTYKNHKDVKYFEQSSVLSPLSSADSLGKLL NFLDTQETVCLTEKIRYLNLSINELGSDNNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISK

[0293] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 542-1132 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 505 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 505 or a variant thereof (e.g., a glycosylation variant).

[0294] 542-1132 portion of Pfs230 (SEQ ID NO. 505) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKLYSGDIGGILFPKNIKS TTCFEEMIPYNKEIKWNKEQKSLGNLVQNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEE LQFSLFDFYESFVPIKKTIQVAQKNVNNKEHDYTCDFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKCNVQKTQYPNIEIFPK TLKDKKEVLKLDLDIQYQM FSKFFKFNTQNAKYLN LYPYYLIFPFN HIGKKELKN N PTYKN HKDVKYFEQSSVLSPLSSADSLGKL LNFLDTQETVCLTEKIRYLQLSINELGSDQNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELEISK

[0295] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 443-1132 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 11 or a variant thereof (e.g., a glycosylation variant).

[0296] 443-1132 portion of Pfs230 (SEQ ID NO. 11) EYVDEKERQGEIYPFGDEEEKDEGGESFTYEKSEVDKTDLFKFIEGGEGDDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEY GEAVEDGENVIKIIRSVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKC EVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKAT VFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKL YSGDIGGILFPKNIKSTTCFEEMIPYNKEIKWNKENKSLGNLVNNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEELNFSLFDFYESFVPIKKTIQVAQKNVNNKEHDYTCDFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKC NVN KTQYPNIEIFPKTLKDKKEVLKLDLDIQYQM FSKFFKFNTQNAKYLN LYPYYLIFPFN HIG KKELKN N PTYKN HKDVKYFEQS SVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLNLSINELGSDNNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISK

[0297] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of amino acids 443-1132 of a wild-type Pfs230 (SEQ ID NO: 1) or a variant thereof (e.g., a sequence variant or a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 504 or a variant thereof (e.g., a glycosylation variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 504 or a variant thereof (e.g., a glycosylation variant).

[0298] 443-1132 portion of Pfs230 (SEQ ID NO. 504) EYVDEKERQGEIYPFGDEEEKDEGGESFTYEKSEVDKTDLFKFIEGGEGDDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEY GEAVEDGENVIKIIRSVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKC EVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKAT VFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKL YSGDIGGILFPKNIKSTTCFEEMIPYNKEIKWNKEQKSLGNLVQNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEES NLISTSYLVYVSINEELQFSLFDFYESFVPIKKTIQVAQKNVNNKEHDYTCDFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKC NVQKTQYPNIEIFPKTLKDKKEVLKLDLDIQYQMFSKFFKFNTQNAKYLNLYPYYLIFPFNHIGKKELKNNPTYKNHKDVKYFEQS SVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLQLSINELGSDQNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISK

[0299] In some embodiments, one or more Plasmodium Pfs230 antigenic portions comprise one or more N-linked glycosylation sites. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises one, two, three, four, five, six, or seven N-linked glycosylation sites. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises an amino acid substitution at one or more N-linked glycosylation sites, where the amino acid substitution prevents glycosylation. In some embodiments, an amino acid substitution that prevents glycosylation comprises a NX[T / S] to QX[T / S] substitution. In some embodiments, amino acid substitution prevents glycosylation comprises a NX[T / S] to NXA substitution. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises one amino acid substitution at an N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises two amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises three amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises four amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises five amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises six amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises seven amino acid substitutions at N-linked glycosylation sites that prevent glycosylation. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises an amino acid substitution at all of the N-linked glycosylation sites, wherein the amino acid substitution prevents glycosylation.

[0300] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises an asparagine at position 585, as numbered according to SEQ ID NO: 1. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises an asparagine at position 585, position 821, position 829, position 889, position 961, position 1079, position 1089, or any combination thereof, as numbered according to SEQ ID NO: 1. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises a glutamine at position 585, as numbered according to SEQ ID NO: 1. In some embodiments, a Plasmodium Pfs230 antigenic portion comprises a glutamine at position 585, position 821, position 829, position 889, position 961, position 1079, position 1089, or any combination thereof, as numbered according to SEQ ID NO: 1.

[0301] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 10 or a variant thereof (e.g., a sequence variant).

[0302] 579-731 portion of Pfs230 Aglvcan (SEQ ID NO. 10) YDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSK LIYGLLISPTVNEKENNFKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYG

[0303] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 9 or a variant thereof (e.g., a sequence variant).

[0304] 564-731 portion of Pfs230 Aglvcan (SEQ ID NO. 9) YETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLJKVKIICPLKGSVEKLYDNIEYVPKKSPYV VLTKEETKLKEKLLSKLIYGLLISPTVN EKEN N FKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGN RGIVEVYVEPYG

[0305] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 8 or a variant thereof (e.g., a sequence variant).

[0306] 542-731 portion of Pfs230 Aglvcan (SEQ ID NO. 8) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLUSPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYG

[0307] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 7 or a variant thereof (e.g., a sequence variant).

[0308] 542-736 portion of Pfs230 Aglvcan (SEQ ID NO. 7) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKING

[0309] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 14 or a variant thereof (e.g., a sequence variant).

[0310] 542-1132 portion of Pfs230 Aglvcan (SEQ ID NO. 14) SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICP LKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKLYSGDIGGILFPKNIKS TTCFEEMIPYNKEIKWNKEQKSLGNLVQNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEE LQFSLFDFYESFVPIKKTIQVAQKNVNNKEHDYTCDFTDKLDKTVPSTANGKKLITCRKHLKEFDTFTLKCNVQKTQYPNIEIFPK TLKDKKEVLKLDLDIQYQM FSKFFKFNTQNAKYLN LYPYYLIFPFN HIGKKELKN N PTYKN HKDVKYFEQSSVLSPLSSADSLGKL LNFLDTQETVCLTEKIRYLQLSINELGSDQNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISK

[0311] In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 or a variant thereof (e.g., a sequence variant). In some embodiments, a Plasmodium Pfs230 antigenic portion comprises or consists of a sequence according to SEQ ID NO: 13 or a variant thereof (e.g., a sequence variant).

[0312] 443-1132 portion of Pfs230 Aglvcan (SEQ ID NO. 13) EYVDEKERQGEIYPFGDEEEKDEGGESFTYEKSEVDKTDLFKFIEGGEGDDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEY GEAVEDGENVIKIIRSVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKC EVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKAT VFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKL YSGDIGGILFPKNIKSTTCFEEMIPYNKEIKWNKEQKSLGNLVQNSWYNKEMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEES N LISTSYLVYVSIN EELQFSLFDFYESFVPIKKTIQVAQKNVN N KEH DYTCDFTDKLDKTVPSTANGKKLFICRKH LKEFDTFTLKC NVQKTQYPNIEIFPKTLKDKKEVLKLDLDIQYQM FSKFFKFNTQNAKYLN LYPYYLIFPFN HIGKKELKN N PTYKN HKDVKYFEQS SVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLQLSINELGSDQNTFSVTFQVPPYIDIKEPFYFMFGCNNNKGEGNIGIVELLISK

[0313] In some embodiments, an antigenic portion of a Plasmodium Pfs230 polypeptide comprises a transmembrane region (also referred to as a "transmembrane domain"). In some embodiments, an antigenic portion of a Plasmodium Pfs230 polypeptide comprises a transmembrane region at the C-terminus. In some embodiments, an antigenic portion of a Plasmodium Pfs230 polypeptide comprises a HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, an antigenic portion of a Plasmodium Pfs230 polypeptide comprises a transmembrane region that comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of SEQ ID NO: 96. In some embodiments, a PiasmodiumTRNW polypeptide does not comprise a transmembrane region.B. Secretory Signals

[0314] In some embodiments, a Plasmodium polypeptide construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal.

[0315] In some embodiments, an HSV secretory signal comprises or consists of an HSV-1 glycoprotein D (gD) secretory signal.

[0316] In some embodiments, a secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.

[0317] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.

[0318] In some embodiments, a secretory signal is positioned at the N-terminus of a Plasmodium polypeptide construct described herein. In some embodiments, a secretory signal preferably allows transport of a Plasmodium polypeptide construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosoma I- lysosomal compartment.

[0319] In some embodiments, a secretory signal is selected from an S1S2 secretory signal (aa 1-19), an immunoglobulin secretory signal (aa 1-22), a human SPARC secretory signal, a human insulin isoform 1 secretory signal, a human albumin secretory signal, etc. Those skilled in the art will be aware of other secretory signal such as, for example, as disclosed in W02017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1-1115 and 1728, or fragments variants thereof). In some embodiments, a Plasmodium polypeptide construct described herein does not comprise a secretory signal.

[0320] In some embodiments, a secretory signal is one listed in Table 2, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a secretory signal is one listed in Table3, or a secretory signal having 1, 2, 3, 4, 5, 6, 7, 8 or more, 10 or more or up to 15 nucleic acid differences relativethereto. In some embodiments, a signal sequence is selected from those included in the Table 2 below and / or those encoded by the sequences in Table 3 below.Table 2: Exemplary secretory signalsSEQ ID Signal Sequence (Amino Acid)NO:15 HSV-1 gD SP 3 MGGAAARLGAVILFWIVGLHGVRG16 HSV-1 gD SP MGGAAARLGAVILFWIVGLHGVRSKY17 HSV-2 gD SP MGRLTSGVGTAALLWAVGLRWCA18 HSV-2 MGRLTSGVGTAALLWAVGLRWCAKYA19 Csp (isolate 3D7) MMRKLAILSVSSFLFVEA20 Ebola spike glycoprotein GP MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS21 SARS-CoV-2-S M FVFLVLLPLVSSQCVN LThuman Ig heavy chain signal peptide22 MDWIWRILFLVGAATGAHSQM(huSec)23 HuIgGk signal peptide M ETPAQLLFLLLLWLPDTTG24 IgE heavy chain epsilon-lsignal peptide M DWTWILFLVAAATRVHSJapanese encephalitis PRM signal25 MLGSNSGQRWFTILLLLVAPAYSsequence26 VSVg protein signal sequence MKCLLYLAFLFIGVNCAT1 TRIO MCRGLSAVULLVSLSAQLHVWG28 human Ig heavy chain signal peptide 1 MELGLSWIFLLAILKGVQC29 human Ig heavy chain signal peptide 2 MELGLRWVFLVAILEGVQC30 human Ig heavy chain signal peptide 3 M KH LWFFLLLVAAPRWVLS31 human Ig heavy chain signal peptide 4 M DWTWRILFLVAAATGAHS32 human Ig heavy chain signal peptide 5 M DWTWRFLFWAAATGVQS33 human Ig heavy chain signal peptide 6 MEFGLSWLFLVAILKGVQC34 human Ig heavy chain signal peptide 7 MEFGLSWVFLVALFRGVQC35 human Ig heavy chain signal peptide 8 M DLLH KN M KHLWFFLLLVAAPRWVLS36 human Ig kappa chain signal peptide 1 MDMRVPAQLLGLLLLWLSGARC37 human Ig kappa chain signal peptide 2 M KYLLPTAAAG LLLLAAQPAMA38 IL-2 MRMQLLLLIALSLALVTNS39 HSV-1 gD SKY MGGAAARLGAVILFWIVGLHGVRSKY40 HSV-1 gD RG MGGAAARLGAVILFWIVGLHGVRG41 HSV-2 gD MGRLTSGVGTAALLWAVGLRWCA42 HSV-2 gD + KYA MGRLTSGVGTAALLWAVGLRWCAKYA43 Csp (isolate 3D7) MMRKLAILSVSSFLFVEA44 HSV-2 gl + WR MPGRSLQGLAILGLWVCATGLWR45 HSV-2 gl + L MPGRSLQGLAILGLWVCATGL46 HSV-1 gD + KY MGGAAARLGAVILFWIVGLHGVRGKY47 HSV-2 gD + KYAL MGRLTSGVGTAALLWAVGLRWCAKYAL48 HSV-2 gD + KYALA MGRLTSGVGTAALLWAVGLRWCAKYALA49 HSV-2 gC signal peptide MALGRVGLAVGLWGLLWVGWWLANA50 HSV-1 gB + AP MHQGAPSWGRRWFWWALLGLTLGVLVASAAP51 HSV-2 gE + RTS MARGAGLVFFVGVWWSCLAAAPRTS52 Ebola spike glycoprotein GP MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIP53 MHC Class II MAISGVPVLGFFIIAVLMSAQESWA54 MDSKGSSQKGSRLLLLLWSNLLLPQGWG55 Japanese encephalitis JEV MWLVSLAIVTACAGA56 SARS-CoV-2 (short) MFVFLVLLPLVSSQC57 HSV-1 gD (strain KOS) MGGAAARLGAVILFWIVGLHGVRGKYATable 3: Exemplary polynucleotide sequences encoding secretory signalsSEQ ID Signal Sequence (Nucleotide)NO:58 HSV-1 gD SP 3 ATGGGCGGAGCTGCTGCTAGACTGGGAGCCGTGATCCTGTTCGTGGTT ATCGTGGGACTGCACGGCGTGCGCGGA59 HSV-1 gD SP wild-type ATGGGGGGGGCTGCCGCCAGGTTGGGGGCCGTGAI H IG H IG ICGTCATAGTGGGCCTCCATGGGGTCCGCAGCAAATATSEQ ID Signal Sequence (Nucleotide)NO:60 HSV-1 gD SP OptlO nt ATGGGAGGAGCCGCCGCCAGACTGGGAGCCGTGATCCTGTTCGTGGTG sequence ATCGTGGGACTGCATGGAGTGAGAAGCAAGTAC61 SARS-CoV-2-S ATG 1 1 1 G 1 G 1 1 1 C 1 1 G 1 GCTGCTGCCTCTTGTGTCTTCTCAGTGTGTGA ATTTGACA62 human Ig heavy chain signal ATGGATTGGATTTGGAGAATCCTGTTCCTCGTGGGAGCCGCTACAGGAG peptide (huSec) CCCACTCCCAGATG63 human Ig heavy chain signal ATGGAGTTGGGACTGAGCTGGA 1 1 1 ICCI 1 1 I GGCTAI 1 1 IAAAAGGTG peptide 1 TCCAGTGT64 human Ig heavy chain signal ATGGAACTGGGGCTCCGCTGGG 1 1 1 1 CCTTGTTGCTA 1 1 1 IAGAAGGTG peptide 2 TCCAGTGT65 human Ig heavy chain signal ATGAAACACCTGTGG 1 1 Cl 1 CC 1 CCTGCTGGTGGCAGCTCCCAGATGGG peptide 3 TCCTGTCC66 human Ig heavy chain signal ATGGACTGGACCTGGAGGATCC 1 C 1 1 C 1 1 GG 1 GGCAGCAGCAACAGGTG peptide 4 CCCACTCG67 human Ig heavy chain signal ATGGACTGGACCTGGAGGTTCC 1 C 1 1 1 G 1 GG 1 GGCAGCAGCTACAGGTG peptide 5 TCCAGTCC68 human Ig heavy chain signal ATGGAG I 1 1 GGGCTGAGCTGGC 1 1 1 1 IC 1 1 G 1 GGCGATTCTAAAAGGTG peptide 6 TCCAGTGT69 human Ig heavy chain signal ATGGAG I 1 1 GGGCTGAGCTGGG 1 1 1 1 CCTCGTTGC 1 C 1 1 1 1 IAGAGGTG peptide 7 TCCAGTGT70 human Ig heavy chain signal ATGGACCTCCTGCACAAGAACATGAAACACCTGTGG 1 1 Cl I CCI CCTCCT peptide 8 GGTGGCAGCTCCCAGATGGGTGCTGTCC71 human Ig kappa chain signal ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGC peptide 1 TCTCAGGTGCCAGATGT72 human Ig kappa chain signal ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGG peptide 2 CCCAGCCGGCCATGGCC73 IL2 Wild type ATGCGCATGCAGCTGCTGCTGCTGATCGCCCTGTCCCTGGCCCTGGTGA CCAACTCC74 IL2 Version 1 ATGAGAATGCAGCTGCTGCTCCTGATCGCCCTGTCTCTGGCCCTGGTCA CCAAT75 IL2 Version 2 ATGCGCATGCAACTGCTCCTGCTGATTGCGTTGAGCCTTGCCCTGGTGA CCAACAGC76 IL2 Version 2.1 ATGCGAATGCAGC 1 1 C 1 GCTGCTCATTGCC 1 1 G 1 CCCTTGCCTTGGTGA CCAACTCA77 IL-2 Version 3 ATGAGAATGCAGCTGCTGCTGCTGATCGCCCTGTCCCTGGCCCTGGTGA CCAACTCC78 HSV-1 gD_RG Variation 1 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCACGGCGUGCGGGGA79 HSV-1 gD_RG Variation 1.1 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCAUGGCGUGCGGGGA80 HSV-1 gD_RG Variation 1.2 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCAUGGCGUGCGGGGC81 HSV-1 gD_RG Variation 1.4 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCACGGCGUCAGAGGC82 HSV-1 gD_RG Variation 1.4 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCAUGGCGUCAGAGGC83 HSV-1 gD_RG Variation 1.5 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCAUGGCGUGCGGGGU84 HSV-1 gD_RG Variation 1.5 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGU UAUCGUGGGACUGCAUGGCGUUAGAGGC85 HSV-1 gD optimized AUGGGAGGAGCCGCCGCCAGACUGGGAGCCGUGAUCCUGUUCGUGGU nucleotide sequence Variation GAUCGUGGGACUGCAUGGAGUGAGAAGCAAGUAC386 HSV-2 gl Variation 2 AUGCCAGGACGGAGCCUUCAGGGCUUGGCCAUACUGGGGCUUUGGGU GUGUGCAACCGGGUUGGUAGUUCGA87 HSV-2 gl Variation 1 AUGCCUGGCAGAUCUCUGCAAGGACUGGCCAUCCUCGGACUGUGGGU UUGCGCAACAGGCCUGGUUGUUAGA88 HSV-2 gl Variation 3 AUGCCUGGAAGAUCUCUGCAGGGACUGGCAAUUCUGGGACUGUGGGUGUGUGCAACAGGACUGGUGGUGAGASEQ ID Signal Sequence (Nucleotide)NO:89 HSV-1 gD AUGGGAGGCGCAGCUGCCAGACUUGGUGCUGUGAUCCUGUUCGUGGU GAUUGUAGGGCUGCAU90 HSV-1 gD _add AUGGGAGGCGCAGCUGCCAGACUUGGUGCUGUGAUCCUGUUCGUGGU GAUUGUAGGGCUGCAUGGUGUCAGGGGCAAGUAU91 HSV-2 gD AUGGGCCGCCUGACCUCCGGCGUGGGCACCGCCGCCCUGCUGGUGGU GGCCGUGGGCCUGCGCGUGGUGUGCGCC92 HSV-2 gD2 Variant 1 AUGGGCAGACUGACAUCUGGCGUGGGAACAGCUGCUCUGCUGGUGGU UGCUGUGGGCCUGAGAGUCGUGUGUGCC93 HSV-2 gD Variant 2 AUGGGGAGACUCACAUCAGGCGUAGGAACCGCUGCCCUGUUGGUCGU GGCCGUUGGUCUGAGAGUUGUGUGUGCC94 HSV-2 gD Variant 3 AUGGGCAGACUGACCUCCGGCGUGGGCACCGCCGCCCUGCUGGUGGU GGCCGUGGGCCUGAGAGUGGUGUGCGCC95 HSV-2 gD KYA wt AUGGGCCGCCUGACCUCCGGCGUGGGCACCGCCGCCCUGCUGGUGGUGGCCGUGGGCCUGCGCGUGGUGUGCGCCAAGUACGCCC. Membrane-anchoring Regions

[0321] In some embodiments, a Plasmodium polypeptide construct described herein includes membraneanchoring region, i.e. a transmembrane region (also referred to as a "transmembrane domain") or a GPI anchor region. In some embodiments, a utilized transmembrane region or GPI anchor region is a heterologous region.

[0322] In some embodiments, a heterologous transmembrane region does not comprise a hemagglutin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of the first 34, 41, or 54 amino acids of an HSV-1 or HSV-2 transmembrane region (e.g., SEQ ID NOS: 102, 103, 104, 105, or 106). In some embodiments, a heterologous transmembrane region comprises (e.g., the first 34 or 41 amino acids of HSV-1 or HSV-2) or consists of HSV-1 or HSV-2 with mutations at positions H27K, T28R, and Q29R of the sequence, relative to the amino acid numbering of SEQ ID NO: 96. In some embodiments, an HSV transmembrane region comprises or consists of an HSV-1 gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 96).

[0323] In some embodiments, an HSV transmembrane region comprises or consists of an HSV-1 gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRKRRKAPKRIRLPHIR (SEQ ID NO: 102).

[0324] In some embodiments, an HSV transmembrane region comprises or consists of an HSV-1 gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIREDDQPSSHQPLFY (SEQ ID NO: 103).

[0325] In some embodiments, an HSV transmembrane region comprises or consists of an HSV-1 gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKR (SEQ ID NO: 104).

[0326] In some embodiments, an HSV transmembrane region comprises or consists of an HSV-1 gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRKRRKAPKR (SEQ ID NO: 105).

[0327] In some embodiments, a heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, an hDAF-GPI anchor region comprises or consists of an amino acid sequence of PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 97).

[0328] In some embodiments, a transmembrane region comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemiavirus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, Influenza A virus (e.g., H1N1), or a seven transmembrane domain receptor.

[0329] In some embodiments, Hemagglutinin (HA) of Influenza virus transmembrane region comprises or consists of an Influenza A (H INI) transmembrane region, e.g., comprising or consisting of an amino acid sequence of LAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 106).

[0330] In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a utilized transmembrane region is one that is normally associated with CSP in nature. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region is from Plasmodium falciparum. In some embodiments, a Plasmodium CSP GPI anchor region is from Plasmodium falciparum isolate 3D7 (SEQ ID NO: 98).

[0331] In some embodiments, a transmembrane region is located at the N-terminus of a Plasmodium polypeptide construct. In some embodiments, a transmembrane region is located at the C-terminus of a Plasmodium polypeptide construct. In some embodiments, a transmembrane region is not located at the N-terminus or C-terminus of a Plasmodium polypeptide construct.

[0332] Exemplary transmembrane are provided in the following Table 4. In some embodiments, a Plasmodium polypeptide construct described herein includes a transmembrane region or GPI anchor region encoded by a sequence provided in Table 4 or a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto.Table 4: Exemplary transmembrane regionsSEQ ID T ransmembrane Sequence (Amino Acid)NO: Region96 HSV-1 gD GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR97 hDAF-GPI anchor region PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLTPlasmodium CSP GPI FNWNSSIGUMVLSFLFLN98anchor region99 HSV-1 gB MSNPFGALAVGLLVLAGLAAAFFAFRYVMRL100 HSV-2 gB MSNPFGALAVGLLVLAGLVAAFFAFRYVLQL101 VSV-G IASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMN102 HSV1 gD 41aa KRR GLIAGAVGGSLLAALVICGIVYWMRRKRRKAPKRIRLPHIR103 HSV1 gD 54aa GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIREDDQPSSHQPLFY 104 HSV1 gD 34aa GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKR105 HSV1 gD 34 KRR GLIAGAVGGSLLAALVICGIVYWMRRKRRKAPKR106 H1N1 LAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

[0333] In some embodiments, a Plasmodium polypeptide construct described herein does not comprise a transmembrane region.D. Linkers

[0334] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more linkers. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids. In some embodiments, a linker comprises one or more serine (S) amino acids. In some embodiments, a linker comprises a glycine-serine linker. A "glycine-serine linker" as used herein refers to a linker that comprises predominantly (e.g., 80% or more) glycine and serine amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.

[0335] In some embodiments, a linker is or comprises S-G4-S-G4-S. In some embodiments, a linker is or comprises GGSGG (SEQ ID NO: 107). In some embodiments, a linker is or comprises GGSGGGGSGG (SEQ ID NO: 108). In some embodiments, a linker is one presented in Table 5. In some embodiments, a linker is or comprisesa sequence as set forth in W02017 / 081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof).

[0336] In some embodiments, a Plasmodium polypeptide construct described herein comprises a linker between a C-terminal region or portion thereof and a transmembrane region.

[0337] Exemplary linkers are provided in the following Table 5. In some embodiments, a Plasmodium polypeptide construct described herein includes one or more linkers encoded by a sequence provided in Table 5.Table 5: Exemplary linkersSEQ ID NO: Sequence (Amino Acid)107 GGSGG108 GGSGGGGSGG109 SGGGGSGGGGS110 GSPGSGSGS111 GGS112 GGGS113 GGGGSGGGGSGGGGS114 AGNRVRRSVG115 GSGSGS116 GGSLGGGGSG117 SGGE. Tags

[0338] In some embodiments, one or more tags may be used when designing and testing constructs (e.g., Plasmodium polypeptide constructs described herein) in certain contexts (e.g., in vitro, ex vivo, etc.). In some embodiments, one or more tags may be directly connected to a Plasmodium polypeptide construct through a peptide bond (e.g., at the 5'-end or 3'-end of a construct). In some embodiments, one or more tags may be connected to a Plasmodium polypeptide construct through one or more linkers (e.g., one or more linkers described herein). In some embodiments, one or more tags may be internally embedded within a Plasmodium polypeptide construct, wherein the one or more tags are directly connected to the construct through peptide bonds (e.g., at the 5'-end and 3'-end of the one or more tags). In some embodiments, one or more tags may be internally embedded within a Plasmodium polypeptide construct, wherein the one or more tags are connected to the construct through one or more linkers (e.g., one or more linkers described herein).

[0339] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more tags. In some embodiments, a Plasmodium polypeptide construct described herein includes one or more detection tags (e.g., HiBit tag, HA tag, etc.). In some embodiments, a Plasmodium polypeptide construct described herein includes a tag that comprises or consists of a HiBit tag. In some embodiments, a HiBit tag has an amino acid sequence according to VSGWRLFKKIS (SEQ ID NO: 118).

[0340] The present disclosure further recognizes that one or more tags can be removed from constructs provided herein. In some embodiments, one or more tags can be removed from a construct when using a construct provided herein in a composition (e.g., a pharmaceutical composition, an immunogenic composition, e.g., a vaccine). As such, while certain constructs provided herein are shown or described as including one or more tags, the present disclosure contemplates the comparable constructs without the one or more tags.F. Embodiments of Plasmodium polypeptide constructs

[0341] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more Plasmodium gamete polypeptide regions or portions thereof as described above. Exemplary combinations of regions are described below.

[0342] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more regions or portions of a Plasmodium gamete polypeptide from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, a Plasmodium gamete polypeptide is Pfs230.1. Pfs230 Constructs

[0343] In some embodiments, a Pfs230 Construct provided herein can have the following structure:Pfs230 Construct;Sec - Pfs230 Construct;Hetsec - Pfs230 Construct;Virakec - Pfs230 Construct; / / SLSec - Pfs230 Construct; / Z5V-7gDsec - Pfs230 Construct;Pfs230 Construct - TMD;Pfs230 Construct - HetTMD;Pfs230 Construct - l / / / a7TMD;Pfs230 Construct - / / S TMD;Pfs230 Construct - / / Sl / -7gDTMD;Sec - Pfs230 Construct - TMD;Hetsec - Pfs230 Construct - TMD;Virakec - Pfs230 Construct - TMD;HSl ec - Pfs230 Construct - TMD; / Z5V-7gDsec - Pfs230 Construct - TMD;Sec - Pfs230 Construct - HetTMD;Hetsec - Pfs230 Construct - HetTMD;k77a ec - Pfs230 Construct - HetTMD;HSl ec - Pfs230 Construct - HetTMD; / Z5V-7gDsec - Pfs230 Construct - HetTMD;Sec - Pfs230 Construct - k5ra / TMD;Hetsec - Pfs230 Construct - k5ra / TMD;k77a ec - Pfs230 Construct- l / / / a7TMD;HSl^ec - Pfs230 Construct - kira ID; / Z5V-7gDsec - Pfs230 Construct - l / / 7a7TMD;Sec - Pfs230 Construct - / / SLTMD;Hetsec - Pfs230 Construct - / / SITMD;Virakec - Pfs230 Construct - / ZSVTMD;HS ec - Pfs230 Construct - / 75VTMD; / Z5V-7gDsec - Pfs230 Construct - / ZSVTMD;Sec - Pfs230 Construct - / / 5V-7gDTMD;Hetsec - Pfs230 Construct- / / 5V-7gDTMD;Virakec - Pfs230 Construct - / / 5V-7gDTMD;HSVsec - Pfs230 Construct - / 75l / - gDTMD; / Z5V-7gDsec - Pfs230 Construct - / 75l / - gDTMD; Pfs230 Construct - Linker - TMD;Sec - Pfs230 Construct - Linker - TMD;Hetsec - Pfs230 Construct - Linker - TMD;Virakec - Pfs230 Construct - Linker - TMD;HSlkec - Pfs230 Construct - Linker - TMD; / Z5V-7gDsec - Pfs230 Construct - Linker - TMD; Pfs230 Construct - Linker - HetTMD;Sec - Pfs230 Construct - Linker - HetTMD;Hetsec - Pfs230 Construct - Linker - HetTMD; k77a ec - Pfs230 Construct - Linker - HetTMD;HSlkec - Pfs230 Construct - Linker - HetTMD; / Z5V-7gDsec - Pfs230 Construct - Linker - HetTMD; Pfs230 Construct - Linker - l / / 7a7TMD;Sec - Pfs230 Construct - Linker - l / / 7a7TMD;Hetsec - Pfs230 Construct - Linker - l / / 7a7TMD;Virakec - Pfs230 Construct - Linker- l / / 7a7TMD; / / SLSec - Pfs230 Construct - Linker- k5ra / TMD; / Z5V-7gDsec - Pfs230 Construct - Linker - l / / / a7TMD;Pfs230 Construct - Linker - / / SLTMD;Sec - Pfs230 Construct - Linker - / / SLTMD;Hetsec - Pfs230 Construct - Linker - / / SLTMD;Virakec - Pfs230 Construct - Linker - / / SLTMD;HSl ec - Pfs230 Construct - Linker - / / SLTMD; / Z5V-7gDsec - Pfs230 Construct - Linker - / / SLTMDPfs230 Construct - Linker - / ZSV-lgDTMD;Sec - Pfs230 Construct - Linker - / ZSV-lgDTMD;Hetsec - Pfs230 Construct - Linker - / ZSV-lgDTMD;k77a ec - Pfs230 Construct - Linker - / Z5V-lgDTMD;HSl ec - Pfs230 Construct - Linker - / Z5V-lgDTMD; or / Z5V-7gDsec - Pfs230 Construct - Linker - / ZSV-lgDTMD.Pfs230 Construct - Linker - HS lgD41aaKRRVMDSec - Pfs230 Construct - Linker - HSVlgD41aaKRRHSWMD Hetsec - Pfs230 Construct - Linker - HS lgD41aaKRRVMD Virakec - Pfs230 Construct - Linker - HSVlgD41aaKRR tAy, HS ec - Pfs230 Construct - Linker - HSVlgD41aaKRR\\J\\y, HSV-lq s&c - Pfs230 Construct - Linker - HSVlgD41aaKRR Pfs230 Construct - Linker - HSVlgD54aa3,Sec - Pfs230 Construct - Linker - HSVlgD54aa W,Hetsec - Pfs230 Construct - Linker - HSVlgD54aa Wy, Virakec - Pfs230 Construct - Linker - HSVlgD54aa W, HSl ec - Pfs230 Construct - Linker - HS\ZlgD54aaTMDHSV- IqDsec - Pfs230 Construct - Linker - HS lgD54aa MD Pfs230 Construct - Linker - / / SLTMD;Sec - Pfs230 Construct - Linker - HSVlgD34aaTMD;Hetsec - Pfs230 Construct - Linker - HSVlgD34aaTMD;Virakec - Pfs230 Construct - Linker - HSl ec - Pfs230 Construct - Linker - / / SV-ygDsec - Pfs230 Construct - Linker - HS lgD34aa / RR D Pfs230 Construct - Linker - HS lgD34aaKRRVMDSec - Pfs230 Construct - Linker - HS lgD34aaKRRVMD Hetsec - Pfs230 Construct - Linker - HS lgD34aaKRRVMD Virakec - Pfs230 Construct - Linker - HSVlgD34aaKRR, HS ec - Pfs230 Construct - Linker - HSVlgD34aaKRRmD / 75l / - gDsec - Pfs230 Construct - Linker - HS lgD34aa / RR D Pfs230 Construct - Linker - / / 7 / V TMD;Sec - Pfs230 Construct - Linker - / / 7 / V TMD;Hetsec - Pfs230 Construct - Linker - / / 7 / V TMD;k77a ec - Pfs230 Construct - Linker - / / 7 / V TMD;HSl ec - Pfs230 Construct - Linker - / / 7 / V TMD; / Z5V-7gDsec - Pfs230 Construct - Linker - / / 7 / V TMD

[0344] In some embodiments, the Plasmodium polypeptide construct described herein includes Pfs230, or an antigenic portion thereof, from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7 (referred to herein as a " Pfs230 Construct").

[0345] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 542-736 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 542-736 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 542-736 of SEQ ID NO: 1, or the amino acid sequence of positions 542-736 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0346] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 542-731 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 542-731 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 542-731 of SEQ ID NO: 1, or the amino acid sequence of positions 542-731 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0347] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 542-731 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 564-731 of SEQ ID NO: 1 having 1, 2, 3, 4, 5 amino acid substitutions.

[0348] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 542-731 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 564-731 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0349] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 579-731 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 579-731 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 579-731 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0350] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 587-731 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 587-731 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 587-731 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0351] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 443-1132 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of positions 443-1132 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 443-1132 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0352] In some embodiments, an antigenic portion of Pfs230 comprises the amino acid sequence of positions 542-1132 of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to the amino acid sequence of positions 542-1132 of SEQ ID NO: 1. In some embodiments, an antigenic portion of Pfs230 comprises the aminoacid sequence of positions 564-731 of SEQ ID NO: 1, or the amino acid sequence of positions 542-1132 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions.

[0353] In some embodiments, an antigenic portion of Pfs230 can comprise:sequence corresponding to amino acids 542-736 of SEQ ID NO: 1 (542-736 Pfs230 Region); sequence corresponding to amino acids 542-731 of SEQ ID NO: 1 (542-731 Pfs230 Region); sequence corresponding to amino acids 564-731 of SEQ ID NO: 1 (564-731 Pfs230 Region); sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 (579-731 Pfs230 Region); sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 (587-731 Pfs230 Region); sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 (443-1132 Pfs230 Region); sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 (542-1132 Pfs230 Region); sequence corresponding to amino acids 542-736 of SEQ ID NO: 1 with a substitution that prevents glycosylation at position 585 (542-736 Pfs230 Region-A585gly);sequence corresponding to amino acids 542-731 of SEQ ID NO: 1 with a substitution that prevents glycosylation at position 585 (542-731 Pfs230 Region-A585gly);sequence corresponding to amino acids 564-731 of SEQ ID NO: 1 with a substitution that prevents glycosylation at position 585 (564-731 Pfs230 Region-A585gly);sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 with a substitution that prevents glycosylation at position 585 (579-731 Pfs230 Region-A585gly);sequence corresponding to amino acids 443-1132 of SEQ ID NO: 1 with a substitution that prevents glycosylation at positions 585, 821, 829, 889, 961, 1079, and 1089 (443-1132 Pfs230 Region-Agly);sequence corresponding to amino acids 542-1132 of SEQ ID NO: 1 with a substitution that prevents glycosylation at position 585 (542-1132 Pfs230 Region-A585gly);sequence corresponding to amino acids 542-1132 of SEQ ID NO: 1 with a substitution that prevents glycosylation at positions 585, 821, 829, 889, 961, 1079, and 1089 (542-1132 Pfs230 Region-Agly);sequence corresponding to amino acids 542-736 of SEQ ID NO: 1 with a glutamine at position 585 (542-736 Pfs230 Region-N585Q);sequence corresponding to amino acids 542-731 of SEQ ID NO: 1 with a glutamine at position 585 (542-731 Pfs230 Region-N585Q);sequence corresponding to amino acids 564-731 of SEQ ID NO: 1 with a glutamine at position 585 (564-731 Pfs230 Region-N585Q);sequence corresponding to amino acids 579-731 of SEQ ID NO: 1 with a glutamine at position 585 (579-731 Pfs230 Region-N585Q);sequence corresponding to amino acids 443-1132 of SEQ ID NO: 1 with a glutamine at position 585 (443-1132 Pfs230 Region-N585Q);sequence corresponding to amino acids 443-1132 of SEQ ID NO: 1 with a glutamine at positions 585, 821, 829, 889, 961, 1079, and 1089 (443-1132 Pfs230 Region-NtoQ);sequence corresponding to amino acids 542-1132 of SEQ ID NO: 1 with a glutamine at position 585 (542-1132 Pfs230 Region-N585Q); orsequence corresponding to amino acids 542-1132 of SEQ ID NO: 1 with a glutamine at positions 585, 821, 829, 889, 961, 1079, and 1089 (542-1132 Pfs230 Region-NtoQ).

[0354] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more antigenic portions of a P230 polypeptide as described above and also includes a secretory signal (sec). In some embodiments, a secretory signal is a viral secretory signal ( V / rafeec). In some embodiments, a secretory signal is a viral secretory signal is a HSV-lgD secretory signal HSV-lgC^ec).

[0355] In some embodiments, a Plasmodium polypeptide construct described herein includes one or more antigenic portions of a P230 polypeptide as described above and also includes a transmembrane domain (TMD). In some embodiments, a transmembrane domain is an HSV-1 transmembrane domain ( / / 5V- TMD).

[0356] In some embodiments, a Plasmodium polypeptide construct described herein further includes a linker. In some embodiments, a linker is included after antigenic portion of a Pfs230 polypeptide in a polypeptideconstruct. In some embodiments, a linker is included between an antigenic portion of a Pfs230 polypeptide and a transmembrane domain (TMD) in a polypeptide construct.

[0357] Table 6 provides exemplary Pfs230 constructs. The different domains in the Pfs230 constructs in Table 6 are further labeled as follows: secretion signal sequence is underlined, / inker sequence is in italics, and transmembrane domains are in bold.Table 6: Amino Acid Sequences Encoded by Exemplary Pfs230 RNA Constructs as Described HereinSEQ ID RNA Sequence (Amino Acid)NO: Construct119 1 MGGAAARLGAVILFWIVGLHGVRGEYVDEKEROGEIYPFGDEEEKDEGGESFTYEKSE VDKTDLFKFIEGGEGDDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEYGEAVEDGE NVIKIIRSVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCD FTDQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEET KLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTEDDNKK GNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDEHNEKIKMKI FF I QNIYKKN NIYPCYMKLYSGDIGGILFPKNIKSTTCFEEMIPYNKEIKWNKEQKSLGNLVQNSWYNK EMNAKYFNVQYVHIPTSYKDTLNLFCSIILKEEESNLISTSYLVYVSINEELQFSLFDFYES FVPIKKTIQVAQKNVN N KEH DYTCDFTDKLDKTVPSTANGKKLFICRKH LKEFDTFTLKC NVQKTQYPNIEIFPKTLKDKKEVLKLDLDIQYQMFSKFFKFNTQNAKYLNLYPYYUFPFN HIGKKELKNNPTYKNHKDVKYFEQSSVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLQ LSIN ELGSDQNTFSVTFQVPPYIDIKEPFYFM FGCN N N KGEG NIGIVELLISKG'G'SG'GVS GWRLFKKIS6Z7S(S6GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPH IR120 2 MGGAAARLGAVILFWIVGLHGVRGSVLOSGALPSVGVDELDKIDLSYETTESGDTAVSE DSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLY DNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHK ATVFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKINGCAFLDEDEEEEKYGNQIEEDE HNEKIKMKI FF 1 QNIYKKNNIYPCYMKLYSGDIGGILFPKNIKSTTCFEEMIPYNKEIKW N KEQKSLG N LVQNSWYN KEM NAKYFNVQYVHIPTSYKDTLN LFCSIILKEEESN LISTS YLVYVSIN EELQFSLFDFYESFVPIKKTIQVAQKNVN N KEHDYTCDFTDKLDKTVPSTAN GKKLFICRKHLKEFDTFTLKCNVQKTQYPNIEIFPKTLKDKKEVLKLDLDIQYQMFSKFFK FNTQNAKYLN LYPYYLIFPFN HIGKKELKN N PTYKN H KDVKYFEQSSVLSPLSSADSLGK LLN FLDTQETVCLTEKIRYLQLSIN ELGSDQNTFSVTFQVPPYIDIKEPFYFM FGCN N N K GEGNIGIVELLISKG'G'SG'GVSGWRLFKKISG'G'SG'GGLIAGAVGGSLLAALVICGIVY WMRRHTQKAPKRIRLPHIR121 3 MGGAAARLGAVILFWIVGLHGVRGSVLOSGALPSVGVDELDKIDLSYETTESGDTAVSE DSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLY DNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHK ATVFYFICDNSKTEDDN KKGN RGIVEVYVEPYGN KING G'G'SG'GVSGWRLFKKIS GGSG 6GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR122 4 MGGAAARLGAVILFWIVGLHGVRGSVLOSGALPSVGVDELDKIDLSYETTESGDTAVSE DSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLY DNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHK ATVFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKINGG'G'SG'G'G'G'SG'GGLIAGAVGG SLLAALVICGIVYWMRRHTQKAPKRIRLPHIR123 5 MGGAAARLGAVILFWIVGLHGVRGSVLOSGALPSVGVDELDKIDLSYETTESGDTAVSE DSYDKYASQNTNKEYVCDFTDQLKPTESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLY DNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHK AIVFYFICDNSKTEDDNKKGNRGIVEVYVEPYGG'G'SG'G'G'G'SG'GGLIAGAVGGSLLAA LVICGIVYWMRRHTQKAPKRIRLPHIR124 6 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPYGGG5GGGG5G6GLIAGAVGGSLLAALVICGIVYWMRRHTQKAP KRIRLPHIR125 7 MGGAAARLGAVILFWIVGLHGVRGYDKYASONTNKEYVCDFTDOLKPTESGPKVKKCE VKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPT VN EKEN N FKEGVIEFTLPPWH KATVFYFICDNSKTEDDN KKGN RGIVEVYVEPYG GGSGGGG5G6GLIAGAVGGSLUMLVICGIVYWMRRHTQKAPKRIRLPHIRSEQ ID RNA Sequence (Amino Acid)NO: Construct126 8 MGGAAARLGAVILFWIVGLHGVRGTNKEYVCDFTDOLKPTESGPKVKKCEVKVNEPLIK VKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKLKEKLLSKLIYGLLISPTVNEKENNFK EGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGNRGIVEVYVEPYGG'G'SG'G'G'G'SG'GGL IAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR127 9 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPY GGGSGGGGSGGG LIAGAVGGSLLAALVICGIVYWMRRKRRKAP KRIRLPHIR128 10 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPY GGGSGGGGSGGG LIAGAVGGSLLAALVICGIVYWMRRHTQKAP KRIRLPHIREDDQPSSHQPLFY129 11 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPY GGGSGGGGSGGG LIAGAVGGSLLAALVICGIVYWMRRHTQKAP KR130 12 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPY GGGSGGGGSGGG LIAGAVGGSLLAALVICGIVYWMRRKRRKAP KR131 13 MGGAAARLGAVILFWIVGLHGVRGYETTESGDTAVSEDSYDKYASONTNKEYVCDFT DQLKPTESGPKVKKCEVKVNEPUKVKIICPLKGSVEKLYDNIEYVPKKSPYWLTKEETKL KEKLLSKLIYGLLISPTVNEKENNFKEGVIEFTLPPWHKATVFYFICDNSKTEDDNKKGN RGIVEVYVEPY GGGSGGGGSGG LAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

[0358] In some embodiments, a Pfs230 construct described herein has an amino acid sequence provided in Table 6. In some embodiments, a Pfs230 construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of Table 6.

[0359] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to an amino acid sequence of SEQ ID NO: 119. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 119.

[0360] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 120. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 120.

[0361] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 121. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 121.

[0362] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 122. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 122.

[0363] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to an amino acid sequence of SEQ ID NO: 123. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 123.

[0364] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 124. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 124.

[0365] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 125. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 125.

[0366] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 126. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 126.

[0367] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 127. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 127.

[0368] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 128. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 128.

[0369] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 129. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 129.

[0370] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 130. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 130.

[0371] In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence of SEQ ID NO: 131. In some embodiments, a Pfs230 construct comprises or consists of an amino acid sequence of SEQ ID NO: 131.Table 7: DNA Sequences Corresponding to Exemplary Pfs230 RNA Constructs as Described HereinSEQ ID RNA Sequence (DNA)NO: Construct132 1 ATGGGCGGAGCTGCTGCTAGACTGGGAGCCGTGATCCTGTTCGTGGTTATCGTGGG ACTGCACGGCGTGCGCGGAGAGTACGTGGACGAGAAGGAGCGCCAAGGCGAGATCT ACCCATTTGGGGATGAGGAGGAGAAGGACGAGGGCGGAGAGAGCTTTACCTATGAG AAGTCCGAAGTGGACAAGACCGACCTGTTCAAATTTATTGAGGGTGGCGAAGGAGAT GACGTGTATAAGGTCGATGGCAGTAAGGTCCTGTTGGACGATGACACCATCAGCCGC GTGTCCAAAAAGCATACTGCCCGCGATGGGGAGTACGGCGAATACGGCGAAGCTGT GGAGGACGGAGAGAATGTGATCAAAATTATCAGATCAGTGCTGCAGTCAGGTGCTCT GCCTTCCGTTGGCGTGGACGAGCTGGATAAGATCGACCTGTCCTACGAGACTACCGA ATCCGGCGACACTGCAGTGTCCGAGGACTCCTACGATAAGTACGCCTCCCAAAATAC TAACAAAGAATATGTATGCGACTTCACCGACCAGCTTAAGCCTACCGAGTCTGGCCC TAAAGTGAAGAAATGCGAGGTGAAAGTGAACGAGCCGCTGATCAAGGTGAAGATCAT TTGCCCGCTGAAGGGCAGCGTGGAGAAGCTGTATGACAACATCGAATACGTGCCTAA AAAGAGCCCATATGTGGTCCrCACCAAGGAGGAAACGAAATTGAAGGAGAAACTCCr GTCCAAACTTATCTACGGCCTCCTGATCAGCCCCACTGTGAACGAGAAAGAAAATAA CTTCAAGGAGGGGGTAATCGAGTTCACTCTGCCACCCGTGGTCCACAAGGCCACTGT TTTCTACTTTATTTGCGATAACTCCAAGACCGAGGACGATAACAAAAAGGGCAATCGTGGCATTGTCGAGGTGTACGTTGAGCCTTACGGAAACAAAATCAATGGTTGTGCCTTSEQ ID RNA Sequence (DNA)NO: Construct CCTGGATGAGGACGAGGAGGAGGAGAAGTATGGTAACCAGATCGAGGAGGACGAGC ATAACGAGAAAATCAAAATGAAAACCTTTTTCACCCAAAACATCTACAAAAAGAATAA CATCTACCCATGCTACATGAAACTCTACTCCGGTGACATCGGAGGCATCCTGTTCCCT AAGAACATCAAGTCCACAACCTGCTTCGAGGAAATGATCCCTTATAACAAGGAGATCA AATGGAACAAAGAACAAAAGTCCCTGGGTAACCTGGTGCAGAACTCCGTAGTCTACA ACAAGGAGATGAATGCCAAGTATTTCAACGTGCAGTACGTGCACATTCCTACCAGTT ATAAGGATACCCTCAACCTGTTCTGTTCCATCATTCTCAAAGAGGAGGAGTCCAACCT GATTTCCACC 1 C 1 1 ATCTTGTCTATGTGTCTATTAACGAGGAGCTGCAATTCTCCCTG TTCGACI 1 1 1 ATGAAAGCTTCGTGCCGATTAAAAAGACAATCCAAGTGGCACAGAAG AACGTGAATAACAAGGAGCACGACTACACATGCGACTTCACCGACAAACTGGATAAG ACCGTGCCCAGCACAGCCAATGGGAAAAAGC I l l i CATTTGCCGCAAACACCTGAAG GAGTTTGATACTTTCACATTGAAGTGTAACGTTCAAAAGACCCAGTACCCTAACATCG AGAI 1 1 I CCCCAAGACACTGAAGGACAAGAAAGAGGTACTGAAACTGGATCTGGACA TCCAGTACCAGATGTTTAGCAAGTTTTTCAAATTCAACACCCAGAACGCCAAGTACCT GAACCTGTACCCGTACTATCTGATCTTCCCCTTCAACCATATCGGTAAAAAGGAGCTG AAAAATAACCCAACGTACAAGAACCACAAGGATGTGAAGTAI 1 1 1 GAGCAATCTTCCG TTCTGAGCCCAC 1 H U I ICCGCCGATTCTCTGGGAAAGCTCCTGAACTTCCTGGATAC CCAGGAGACTGTTTGCCTCACCGAAAAGATCCGTTATTTGCAGCTGTCCATCAACGA ACTCGGCTCTGACCAGAACACCTTCrCTGTGACATTCCAGGTGCCGCCCTACATCGA TATCAAGGAGCCTTTTTACTTCATGTTCGGATGCAACAATAACAAGGGTGAGGGGAA CATCGGCATCGTGGAGC 1 1 U 1 GATCTCTAAAGGAGGCTCTGGCGGTGGACrTATTGC CGGCGCTGTTGGAGGATCTCTGCTGGCCGCTCTGGTCATCTGTGGCATCGTGTACTG GATGCGGCGGCACACACAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATG ATGA133 2 ATGGGCGGAGCTGCTGCTAGACTGGGAGCCGTGATCCTGTTCGTGGTTATCGTGGG ACTGCACGGCGTGCGCGGATCAGTGCTGCAGTCAGGTGCTCTGCCrTCCGTTGGCG TGGACGAGCTGGATAAGATCGACCTGTCCTACGAGACTACCGAATCCGGCGACACTG CAGTGTCCGAGGACrCCTACGATAAGTACGCCTCCCAAAATACTAACAAAGAATATGT ATGCGACTTCACCGACCAGCrTAAGCCTACCGAGTCTGGCCCTAAAGTGAAGAAATG CGAGGTGAAAGTGAACGAGCCGCTGATCAAGGTGAAGATCATTTGCCCGCTGAAGG GCAGCGTGGAGAAGCTGTATGACAACATCGAATACGTGCCTAAAAAGAGCCCATATG TGGTCCrCACCAAGGAGGAAACGAAATTGAAGGAGAAACTCCTGTCCAAACrTATCT ACGGCCTCCTGATCAGCCCCACTGTGAACGAGAAAGAAAATAACTTCAAGGAGGGGG TAATCGAGTTCACTCTGCCACCCGTGGTCCACAAGGCCACTGTTTTCTACTTTATTTG CGATAACTCCAAGACCGAGGACGATAACAAAAAGGGCAATCGTGGCATTGTCGAGGT GTACGTTGAGCCTTACGGAAACAAAATCAATGGTTGTGCCTTCCTGGATGAGGACGA GGAGGAGGAGAAGTATGGTAACCAGATCGAGGAGGACGAGCATAACGAGAAAATCA AAATGAAAACCTTTTTCACCCAAAACATCTACAAAAAGAATAACATCTACCCATGCTA CATGAAACTCTACTCCGGTGACATCGGAGGCATCCTGTTCCCTAAGAACATCAAGTC CACAACCTGCTTCGAGGAAATGATCCCTTATAACAAGGAGATCAAATGGAACAAAGA ACAAAAGTCCCTGGGTAACCTGGTGCAGAACTCCGTAGTCTACAACAAGGAGATGAA TGCCAAGTATTTCAACGTGCAGTACGTGCACATTCCTACCAGTTATAAGGATACCCTC AACCTGTTCTGTTCCATCATTCTCAAAGAGGAGGAGTCCAACCTGATTTCCACC 1 C 1 1 ATCTTGTCTATGTGTCTATTAACGAGGAGCTGCAATTCTCCCTGTTCGACI 1 1 IATGA AAGCTTCGTGCCGATTAAAAAGACAATCCAAGTGGCACAGAAGAACGTGAATAACAA GGAGCACGACTACACATGCGACTTCACCGACAAACTGGATAAGACCGTGCCCAGCAC AGCCAATGGGAAAAAGC I l l i CATTTGCCGCAAACACCTGAAGGAGTTTGATACTTT CACATTGAAGTGTAACGTTCAAAAGACCCAGTACCCTAACATCGAGAI 1 1 I CCCCAAG ACACTGAAGGACAAGAAAGAGGTACTGAAACTGGATCTGGACATCCAGTACCAGATG TTTAGCAAG 1 1 1 1 1 CAAATTCAACACCCAGAACGCCAAGTACCTGAACCTGTACCCGT ACTATCrGATCTTCCCCTTCAACCATATCGGTAAAAAGGAGCTGAAAAATAACCCAAC GTACAAGAACCACAAGGATGTGAAGTA 1 1 1 1 GAGCAATCTTCCGTTCTGAGCCCACT TTCTTCCGCCGATTCTCrGGGAAAGCrCCrGAACTTCCrGGATACCCAGGAGACrGT TTGCCTCACCGAAAAGATCCGTTATTTGCAGCTGTCCATCAACGAACTCGGCTCTGA CCAGAACACCTTCTCTGTGACATTCCAGGTGCCGCCCTACATCGATATCAAGGAGCC TTTTTACTTCATGTTCGGATGCAACAATAACAAGGGTGAGGGGAACATCGGCATCGT GGAGC 1 1 C 1 GATCTCTAAAGGAGGCTCTGGCGGTGGACTTATTGCCGGCGCTGTTG GAGGATCTCTGCTGGCCGCTCTGGTCATCTGTGGCATCGTGTACTGGATGCGGCGGCACACACAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATGATGASEQ ID RNA Sequence (DNA)NO: Construct134 3.01 ATGGGCGGAGCTGCTGCTAGACTGGGAGCCGTGATCCTGTTCGTGGTTATCGTGGG ACTGCACGGCGTGCGCGGATCAGTGCTGCAGTCAGGTGCTCTGCCrTCCGTTGGCG TGGACGAGCTGGATAAGATCGACCTGTCCTACGAGACTACCGAATCCGGCGACACTG CAGTGTCCGAGGACrCCTACGATAAGTACGCCTCCCAAAATACTAACAAAGAATATGT ATGCGACTTCACCGACCAGCrTAAGCCTACCGAGTCTGGCCCTAAAGTGAAGAAATG CGAGGTGAAAGTGAACGAGCCGCTGATCAAGGTGAAGATCATTTGCCCGCTGAAGG GCAGCGTGGAGAAGCTGTATGACAACATCGAATACGTGCCTAAAAAGAGCCCATATG TGGTCCrCACCAAGGAGGAAACGAAATTGAAGGAGAAACTCCTGTCCAAACrTATCT ACGGCCTCCTGATCAGCCCCACTGTGAACGAGAAAGAAAATAACTTCAAGGAGGGGG TAATCGAGTTCACTCTGCCACCCGTGGTCCACAAGGCCACTGTTTTCTACTTTATTTG CGATAACTCCAAGACCGAGGACGATAACAAAAAGGGCAATCGTGGCATTGTCGAGGT GTACGTTGAGCCTTACGGAAACAAAATCAATGGTGGAGGCTCTGGCGGTGTGTCAG GATGGCGTCTGTTCAAAAAGATCAGCGGAGGCrCrGGCGGTGGACTTATTGCCGGC GCTGTTGGAGGATCTCTGCTGGCCGCTCTGGTCATCTGTGGCATCGTGTACTGGATC CGGCGGCACACACAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATGATGA135 3.02 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGAAGCGTGCTGCAAAGCGGAGCACTGCCrTCAGTGGGAG TCGACGAGCTGGACAAGATCGACCTGAGCTACGAGACAACCGAGAGCGGCGATACA GCCGTGTCCGAGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAG TGCGAAGTGAAAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAG GGCAGCGTGGAAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTAC GTGGTGCTGACCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATC TACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGC GTTATCGAGTTCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCATT TGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGA AGTGTACGTGGAACCCTACGGCAACAAGATCAACGGCGGAGGATCAGGTGGCGTGT CAGGATGGCGTCTGTTCAAAAAGATCAGCGGAGGAAGCGGAGGCGGACTGATTGCA GGTGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTG GATGAGAAGGCACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATG ATGA136 3.03 ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I CI C I 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTAGCGTACTGCAATCTGGCGCACTCCCTTCTGTAGGGGT AGACGAACTGGACAAAATTGACCTGTCATACGAGACTACGGAGTCAGGGGATACAGC CGTGTCCGAGGATTCCTACGACAAATACGCTTCCCAGAATACCAATAAGGAGTACGT GTGCGACTTTACCGATCAGCTGAAGCCCACAGAATCCGGGCCAAAGGTGAAGAAGTG CGAAGTGAAAGTCAACGAGCCTCTGATCAAGGTCAAGATCATCTGTCCGCTGAAAGG CAGTGTTGAGAAGCTTTACGACAACATCGAGTACGTCCCCAAGAAAAGCCCCTACGT GGTTCTGACCAAGGAAGAAACCAAGCTGAAAGAGAAACTCCTGTCTAAACTCATCTA CGGACTGCTGATTAGCCCCACTGTCAACGAGAAAGAGAACAATTTCAAGGAAGGCGT TATTGAGTTTACTCTGCCACCTGTGGTGCATAAGGCTACCGTTTTCTACTTCATCTGC GATAACAGCAAGACAGAGGACGATAACAAGAAGGGGAATAGGGGTATCGTCGAAGT GTACGTTGAACCATACGGGAACAAGATAAACGGAGGCGGATCAGGTGGTGTGTCAG GATGGCGTCTGTTCAAAAAGATCAGCGGAGGCAGTGGAGGCGGCTTGATTGCCGGA GCTGTGGGTGGCAGCC 1 C 1 I GGCAGCCC I 1 G 1 GATCTGTGGGATTGTGTACTGGAT GAGAAGGCACACACAGAAAGCCCCTAAACGCATACGGTTGCCACACATTCGTTGATGA137 3.04 ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCAGCGTGCTGCAGAGCGGCGCCCTGCCAAGCGTGGGCG TGGACGAGCTGGACAAGATCGACCTGAGCTACGAGACCACCGAGAGCGGCGACACC GCCGTGAGCGAGGACAGCTACGACAAGTACGCCAGCCAGAATACCAATAAGGAGTAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAG TGCGAGGTGAAGGTGAACGAGCCTCTGATCAAGGTGAAGATCATCTGCCCTCTGAAG GGCAGCGTGGAGAAACTGTACGACAATATCGAGTACGTGCCTAAGAAGTCCCCTTAC GTGGTGCTGACCAAGGAGGAGACCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGAT CTACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAGGAGAATAATTTCAAGGAGGG CGTGATCGAGTTCACCCTGCCTCCTGTGGTGCACAAGGCCACCGTGTTCTACTTCAT CTGCGACAATAGCAAGACCGAGGACGACAATAAGAAGGGCAATAGAGGCATCGTGG AGGTGTACGTGGAGCCTTACGGCAATAAGATCAACGGCGGCGGCAGCGGCGGAGTG TCAGGATGGCGTCTGTTCAAAAAGATCAGCGGAGGAAGCGGAGGCGGACTGATCGCCGGAGCCGTGGGAGGCAGCCTGCTGGCCGCCCTGGTGATCTGCGGCATCGTGTACTSEQ ID RNA Sequence (DNA)NO: Construct GGATGAGAAGACACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCTCACATCAGAT GATGA138 4.01 OptG ATGGGCGGAGCTGCTGCTAGACTGGGAGCCGTGATCCTGTTCGTGGTTATCGTGGG ACTGCACGGCGTGCGCGGATCAGTGCTGCAGTCAGGTGCTCTGCCrTCCGTTGGCG TGGACGAGCTGGATAAGATCGACCTGTCCTACGAGACTACCGAATCCGGCGACACTG CAGTGTCCGAGGACTCCTACGATAAGTACGCCTCCCAAAATACTAACAAAGAATATGT ATGCGACTTCACCGACCAGCrTAAGCCTACCGAGTCTGGCCCTAAAGTGAAGAAATG CGAGGTGAAAGTGAACGAGCCGCTGATCAAGGTGAAGATCATTTGCCCGCTGAAGG GCAGCGTGGAGAAGCTGTATGACAACATCGAATACGTGCCTAAAAAGAGCCCATATG TGGTCCTCACCAAGGAGGAAACGAAATTGAAGGAGAAACTCCTGTCCAAACTTATCT ACGGCCTCCTGATCAGCCCCACTGTGAACGAGAAAGAAAATAACTTCAAGGAGGGGG TAATCGAGTTCACTCTGCCACCCGTGGTCCACAAGGCCACTGTTTTCTACTTTATTTG CGATAACTCCAAGACCGAGGACGATAACAAAAAGGGCAATCGTGGCATTGTCGAGGT GTACGTTGAGCCTTACGGAAACAAAATCAATGGTGGAGGCrCTGGCGGTGGAGGCT CTGGCGGTGGACrTATTGCCGGCGCTGTTGGAGGATCTCTGCTGGCCGCTCTGGTC ATCTGTGGCATCGTGTACTGGATGCGGCGGCACACACAGAAGGCCCCTAAGAGAATC AGACTGCCCCACATCAGATGATGA139 4.02 Opt 1 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG prime ACTGCACGGCGTCAGAGGAAGCGTGCTGCAAAGCGGAGCACTGCCTTCAGTGGGAG TCGACGAGCTGGACAAGATCGACCTGAGCTACGAGACAACCGAGAGCGGCGATACA GCCGTGTCCGAGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAG TGCGAAGTGAAAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAG GGCAGCGTGGAAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTAC GTGGTGCTGACCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATC TACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAAGAGAACAACrTCAAAGAGGGC GTTATCGAGTTCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCATT TGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGA AGTGTACGTGGAACCCTACGGCAACAAGATCAACGGCGGAGGATCAGGTGGCGGAG GAAGCGGAGGCGGACTGATTGCAGGTGCAGTTGGAGGATCACTGCTGGCCGCACTG GTCATTTGCGGCATCGTGTATTGGATGAGAAGGCACACCCAGAAGGCCCCTAAGAGA ATCAGACTGCCCCACATCAGATGATGA140 4.03 Opt3m ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I C I CI 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTAGCGTACTGCAATCTGGCGCACTCCCTTCTGTAGGGGT AGACGAACTGGACAAAATTGACCTGTCATACGAGACTACGGAGTCAGGGGATACAGC CGTGTCCGAGGATTCCTACGACAAATACGCTTCCCAGAATACCAATAAGGAGTACGT GTGCGACTTTACCGATCAGCTGAAGCCCACAGAATCCGGGCCAAAGGTGAAGAAGTG CGAAGTGAAAGTCAACGAGCCTCTGATCAAGGTCAAGATCATCTGTCCGCTGAAAGG CAGTGTTGAGAAGCTTTACGACAACATCGAGTACGTCCCCAAGAAAAGCCCCTACGT GGTTCTGACCAAGGAAGAAACCAAGCTGAAAGAGAAACTCCTGTCTAAACTCATCTA CGGACTGCTGATTAGCCCCACTGTCAACGAGAAAGAGAACAATTTCAAGGAAGGCGT TATTGAGTTTACTCTGCCACCTGTGGTGCATAAGGCTACCGTTTTCTACTTCATCTGC GATAACAGCAAGACAGAGGACGATAACAAGAAGGGGAATAGGGGTATCGTCGAAGT GTACGTTGAACCATACGGGAACAAGATAAACGGAGGCGGATCAGGTGGTGGAGGCA GTGGAGGCGGCTTGATTGCCGGAGCTGTGGGTGGCAGCC 1 C 1 I GGCAGCCCI I G I G ATCTGTGGGATTGTGTACTGGATGAGAAGGCACACACAGAAAGCCCCTAAACGCATA CGGTTGCCACACATTCGTTGATGA141 4.04 OptlOLP ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCAGCGTGCTGCAGAGCGGCGCCCTGCCAAGCGTGGGCG TGGACGAGCTGGACAAGATCGACCTGAGCTACGAGACCACCGAGAGCGGCGACACC GCCGTGAGCGAGGACAGCTACGACAAGTACGCCAGCCAGAATACCAATAAGGAGTAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAG TGCGAGGTGAAGGTGAACGAGCCTCTGATCAAGGTGAAGATCATCTGCCCTCTGAAG GGCAGCGTGGAGAAACTGTACGACAATATCGAGTACGTGCCTAAGAAGTCCCCTTAC GTGGTGCTGACCAAGGAGGAGACCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGAT CTACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAGGAGAATAATTTCAAGGAGGG CGTGATCGAGTTCACCCTGCCrCCrGTGGTGCACAAGGCCACCGTGTTCTACTTCAT CTGCGACAATAGCAAGACCGAGGACGACAATAAGAAGGGCAATAGAGGCATCGTGG AGGTGTACGTGGAGCCTTACGGCAATAAGATCAACGGCGGCGGCAGCGGCGGAGGAGGAAGCGGAGGCGGACTGATCGCCGGAGCCGTGGGAGGCAGCCTGCTGGCCGCCCTSEQ ID RNA Sequence (DNA)NO: Construct GGTGATCTGCGGCATCGTGTACTGGATGAGAAGACACACCCAGAAGGCCCCTAAGAG AATCAGACTGCCTCACATCAGATGATGA142 5.02 Optl ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG prime ACTGCACGGCGTCAGAGGAAGCGTGCTGCAAAGCGGAGCACTGCCTTCAGTGGGAG TCGACGAGCTGGACAAGATCGACCTGAGCTACGAGACAACCGAGAGCGGCGATACA GCCGTGTCCGAGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAG TGCGAAGTGAAAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAG GGCAGCGTGGAAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTAC GTGGTGCTGACCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATC TACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGC GTTATCGAGTTCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCATT TGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGA AGTGTACGTGGAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGAC TGATTGCAGGTGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATC GTGTATTGGATGAGAAGGCACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCCCAC ATCAGATGATGA143 5.03 Opt3m ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I CI C I 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTAGCGTACTGCAATCTGGCGCACTCCCTTCTGTAGGGGT AGACGAACTGGACAAAATTGACCTGTCATACGAGACTACGGAGTCAGGGGATACAGC CGTGTCCGAGGATTCCTACGACAAATACGCTTCCCAGAATACCAATAAGGAGTACGT GTGCGACTTTACCGATCAGCTGAAGCCCACAGAATCCGGGCCAAAGGTGAAGAAGTG CGAAGTGAAAGTCAACGAGCCTCTGATCAAGGTCAAGATCATCTGTCCGCTGAAAGG CAGTGTTGAGAAGCTTTACGACAACATCGAGTACGTCCCCAAGAAAAGCCCCTACGT GGTTCTGACCAAGGAAGAAACCAAGCTGAAAGAGAAACTCCTGTCTAAACTCATCTA CGGACTGCTGATTAGCCCCACTGTCAACGAGAAAGAGAACAATTTCAAGGAAGGCGT TATTGAGTTTACTCTGCCACCTGTGGTGCATAAGGCTACCGTTTTCTACTTCATCTGC GATAACAGCAAGACAGAGGACGATAACAAGAAGGGGAATAGGGGTATCGTCGAAGT GTACGTTGAACCATACGGAGGCGGATCAGGTGGTGGAGGCAGTGGAGGCGGCTTGA TTGCCGGAGCTGTGGGTGGCAGCC 1 C 1 IGGCAGCCCI 1 G 1 GATCTGTGGGATTGTG TACTGGATGAGAAGGCACACACAGAAAGCCCCTAAACGCATACGGTTGCCACACATT CGTTGATGA144 5.04 OptlOLP ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCAGCGTGCTGCAGAGCGGCGCCCTGCCAAGCGTGGGCG TGGACGAGCTGGACAAGATCGACCTGAGCTACGAGACCACCGAGAGCGGCGACACC GCCGTGAGCGAGGACAGCTACGACAAGTACGCCAGCCAGAATACCAATAAGGAGTAC GTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAG TGCGAGGTGAAGGTGAACGAGCCTCTGATCAAGGTGAAGATCATCTGCCCrCTGAAG GGCAGCGTGGAGAAACTGTACGACAATATCGAGTACGTGCCTAAGAAGTCCCCTTAC GTGGTGCTGACCAAGGAGGAGACCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGAT CTACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAGGAGAATAATTTCAAGGAGGG CGTGATCGAGTTCACCCTGCCTCCTGTGGTGCACAAGGCCACCGTGTTCTACrTCAT CTGCGACAATAGCAAGACCGAGGACGACAATAAGAAGGGCAATAGAGGCATCGTGG AGGTGTACGTGGAGCCTTACGGCAATAAGATCAACGGCGGCGGCAGCGGCGGAGGA GGAAGCGGAGGCGGACTGATCGCCGGAGCCGTGGGAGGCAGCCTGCTGGCCGCCCT GGTGATCTGCGGCATCGTGTACTGGATGAGAAGACACACCCAGAAGGCCCCTAAGAG AATCAGACTGCCTCACATCAGATGATGA145 6.02 Opt 1 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG prime ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGGTGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGATSEQ ID RNA Sequence (DNA)NO: Construct GAGAAGGCACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATGATGA146 6.03 Opt3m ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I CI C I 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTTACGAGACTACGGAGTCAGGGGATACAGCCGTGTCCG AGGATTCCTACGACAAATACGCTTCCCAGAATACCAATAAGGAGTACGTGTGCGACr TTACCGATCAGCTGAAGCCCACAGAATCCGGGCCAAAGGTGAAGAAGTGCGAAGTGA AAGTCAACGAGCCTCTGATCAAGGTCAAGATCATCTGTCCGCTGAAAGGCAGTGTTG AGAAGCTTTACGACAACATCGAGTACGTCCCCAAGAAAAGCCCCTACGTGGTTCTGA CCAAGGAAGAAACCAAGCTGAAAGAGAAACTCCTGTCTAAACTCATCTACGGACTGC TGATTAGCCCCACTGTCAACGAGAAAGAGAACAATTTCAAGGAAGGCGTTATTGAGT TTACTCTGCCACCTGTGGTGCATAAGGCTACCGTTTTCTACTTCATCTGCGATAACAG CAAGACAGAGGACGATAACAAGAAGGGGAATAGGGGTATCGTCGAAGTGTACGTTG AACCATACGGAGGCGGATCAGGTGGTGGAGGCAGTGGAGGCGGCTTGATTGCCGGA GCTGTGGGTGGCAGCC 1 C 1 I GGCAGCCC I 1 G 1 GATCTGTGGGATTGTGTACTGGAT GAGAAGGCACACACAGAAAGCCCCTAAACGCATACGGTTGCCACACATTCGTTGATGA147 6.04 OptlOLP ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCTACGAGACCACCGAGAGCGGCGACACCGCCGTGAGCG AGGACAGCTACGACAAGTACGCCAGCCAGAATACCAATAAGGAGTACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAGTGCGAGGTG AAGGTGAACGAGCCTCTGATCAAGGTGAAGATCATCTGCCCTCTGAAGGGCAGCGTG GAGAAACTGTACGACAATATCGAGTACGTGCCTAAGAAGTCCCCTTACGTGGTGCrG ACCAAGGAGGAGACCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGATCTACGGCCT GCTGATTAGCCCTACCGTGAACGAGAAGGAGAATAATTTCAAGGAGGGCGTGATCGA GTTCACCCTGCCTCCTGTGGTGCACAAGGCCACCGTGTTCTACTTCATCTGCGACAA TAGCAAGACCGAGGACGACAATAAGAAGGGCAATAGAGGCATCGTGGAGGTGTACG TGGAGCCTTACGGCGGCGGCAGCGGCGGAGGAGGAAGCGGAGGCGGACTGATCGC CGGAGCCGTGGGAGGCAGCCTGCTGGCCGCCCTGGTGATCTGCGGCATCGTGTACT GGATGAGAAGACACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCTCACATCAGAT GATGA148 7.02 Optl ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG prime ACTGCACGGCGTCAGAGGATACGATAAGTACGCCAGCCAGAACACCAACAAAGAATA CGTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAA GTGCGAAGTGAAAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAA GGGCAGCGTGGAAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTA CGTGGTGCTGACCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGAT CTACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAAGAGAACAACrTCAAAGAGGG CGTTATCGAGTTCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCAT TTGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGG AAGTGTACGTGGAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGA CTGATTGCAGGTGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATC GTGTATTGGATGAGAAGGCACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCCCAC ATCAGATGATGA149 7.03 Opt3m ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I CI C I 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTTACGACAAATACGCTTCCCAGAATACCAATAAGGAGTA CGTGTGCGACTTTACCGATCAGCTGAAGCCCACAGAATCCGGGCCAAAGGTGAAGAA GTGCGAAGTGAAAGTCAACGAGCCTCTGATCAAGGTCAAGATCATCrGTCCGCTGAA AGGCAGTGTTGAGAAGCTTTACGACAACATCGAGTACGTCCCCAAGAAAAGCCCCTA CGTGGTTCTGACCAAGGAAGAAACCAAGCTGAAAGAGAAACTCCTGTCTAAACTCAT CTACGGACTGCTGATTAGCCCCACTGTCAACGAGAAAGAGAACAATTTCAAGGAAGG CGTTATTGAGTTTACTCTGCCACCTGTGGTGCATAAGGCTACCGTTTTCTACTTCATC TGCGATAACAGCAAGACAGAGGACGATAACAAGAAGGGGAATAGGGGTATCGTCGA AGTGTACGTTGAACCATACGGAGGCGGATCAGGTGGTGGAGGCAGTGGAGGCGGCT TGATTGCCGGAGCTGTGGGTGGCAGCC 1 C 1 IGGCAGCCCI 1 G 1 GATCTGTGGGATT GTGTACTGGATGAGAAGGCACACACAGAAAGCCCCTAAACGCATACGGTTGCCACAC ATTCGTTGATGA150 7.04 OptlOLP ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCTACGACAAGTACGCCAGCCAGAATACCAATAAGGAGTA CGTGTGCGACTTCACCGACCAGCTGAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAGTGCGAGGTGAAGGTGAACGAGCCTCTGATCAAGGTGAAGATCATCTGCCCTCTGAASEQ ID RNA Sequence (DNA)NO: Construct GGGCAGCGTGGAGAAACTGTACGACAATATCGAGTACGTGCCTAAGAAGTCCCCTTA CGTGGTGCTGACCAAGGAGGAGACCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGA TCTACGGCCTGCTGATTAGCCCTACCGTGAACGAGAAGGAGAATAATTTCAAGGAGG GCGTGATCGAGTTCACCCTGCCTCCTGTGGTGCACAAGGCCACCGTGTTCTACrTCA TCTGCGACAATAGCAAGACCGAGGACGACAATAAGAAGGGCAATAGAGGCATCGTG GAGGTGTACGTGGAGCCTTACGGCGGCGGCAGCGGCGGAGGAGGAAGCGGAGGCG GACTGATCGCCGGAGCCGTGGGAGGCAGCCTGCTGGCCGCCCTGGTGATCTGCGGC ATCGTGTACTGGATGAGAAGACACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCT CACATCAGATGATGA151 8.02 Optl ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG prime ACTGCACGGCGTCAGAGGAACCAACAAAGAATACGTGTGCGACTTCACCGACCAGCT GAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGAAAGTGAACGAGCC CCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGGAAAAGCTGTACGA CAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGACCAAAGAGGAAAC AAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGCTGATTAGCCCTAC CGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGTTCACCCTGCCTCC AGTGGTGCACAAGGCCACCGTGTTCTACTTCATTTGCGACAACAGCAAGACCGAGGA CGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTGGAACCCTACGGCG GAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGGTGCAGTTGGAGG ATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGATGAGAAGGCACAC CCAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATGATGA152 8.03 Opt3m ATGGGCGGGGCAGCCGCTAGACTGGGAGCGGTGAI I CI C I 1 1 G 1 GGTCATAGTGGG CCTTCACGGAGTCCGAGGTACCAATAAGGAGTACGTGTGCGACTTTACCGATCAGCT GAAGCCCACAGAATCCGGGCCAAAGGTGAAGAAGTGCGAAGTGAAAGTCAACGAGC CTCTGATCAAGGTCAAGATCATCTGTCCGCTGAAAGGCAGTGTTGAGAAGCTTTACG ACAACATCGAGTACGTCCCCAAGAAAAGCCCCTACGTGGTTCTGACCAAGGAAGAAA CCAAGCTGAAAGAGAAACTCCTGTCTAAACTCATCTACGGACTGCTGATTAGCCCCAC TGTCAACGAGAAAGAGAACAATTTCAAGGAAGGCGTTATTGAGTTTACTCTGCCACC TGTGGTGCATAAGGCTACCGTTTTCTACTTCATCTGCGATAACAGCAAGACAGAGGA CGATAACAAGAAGGGGAATAGGGGTATCGTCGAAGTGTACGTTGAACCATACGGAG GCGGATCAGGTGGTGGAGGCAGTGGAGGCGGCTTGATTGCCGGAGCTGTGGGTGG CAGCC 1 C 1 1 GGCAGCCC 1 1 G 1 GATCTGTGGGATTGTGTACTGGATGAGAAGGCACAC ACAGAAAGCCCCTAAACGCATACGGTTGCCACACATTCGTTGATGA153 8.04 OptlOLP ATGGGCGGAGCCGCCGCCAGACTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGG CCTGCACGGCGTGAGAGGCACCAATAAGGAGTACGTGTGCGACTTCACCGACCAGCT GAAGCCTACAGAGAGCGGCCCTAAAGTGAAGAAGTGCGAGGTGAAGGTGAACGAGC CTCTGATCAAGGTGAAGATCATCTGCCCTCTGAAGGGCAGCGTGGAGAAACTGTACG ACAATATCGAGTACGTGCCTAAGAAGTCCCCTTACGTGGTGCTGACCAAGGAGGAGA CCAAGCTGAAGGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGCTGATTAGCCCTA CCGTGAACGAGAAGGAGAATAATTTCAAGGAGGGCGTGATCGAGTTCACCCTGCCTC CTGTGGTGCACAAGGCCACCGTGTTCTACTTCATCTGCGACAATAGCAAGACCGAGG ACGACAATAAGAAGGGCAATAGAGGCATCGTGGAGGTGTACGTGGAGCCTTACGGC GGCGGCAGCGGCGGAGGAGGAAGCGGAGGCGGACTGATCGCCGGAGCCGTGGGAG GCAGCCTGCTGGCCGCCCTGGTGATCTGCGGCATCGTGTACTGGATGAGAAGACAC ACCCAGAAGGCCCCTAAGAGAATCAGACTGCCTCACATCAGATGATGA154 9 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCrCCAGTGGTGCACAAGGCCACCGTGTTCTACrTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGG TGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGAT GAGAAGAAAGCGCAGAAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGATGATGASEQ ID RNA Sequence (DNA)NO: Construct155 10 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCrCCAGTGGTGCACAAGGCCACCGTGTTCrACTTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGG TGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGAT GAGAAGGCACACCCAGAAGGCCCCTAAGAGAATCAGACTGCCCCACATCAGAGAGGA CGATCAGCCAAGCTCTCACCAGCCACTGTTCTACTGATGA156 11 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCrCCAGTGGTGCACAAGGCCACCGTGTTCrACTTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGG TGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGAT GAGAAGGCACACCCAGAAGGCCCCTAAGAGATGATGA157 12 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCrCCAGTGGTGCACAAGGCCACCGTGTTCrACTTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGG TGCAGTTGGAGGATCACTGCTGGCCGCACTGGTCATTTGCGGCATCGTGTATTGGAT GAGAAGAAAGCGCAGAAAGGCCCCTAAGAGATGATGACTCGAGCTGGTACTGCATGC ACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCG GGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGA CACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCAC GGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAA CCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAGAGCTCCAACCGGTG TGGTAGCTCCGCCGTTTAACATCGCCCTTCCCAACAGTTGCGCAGCCrGAATGGCGA ATGGAGATCCAAI 1 1 1 IAAG I G IAIAAI G I G I IAAACIAC I GAI I C IAAI I G I 1 I G I G TAI 1 1 1 AGATTCACAGTCCCAAGGCTCATTTCAGGCCCCTCAGTCCTCACAGTCTGTT CATGATCATAATCAGCCATACCACA 1 1 I G IAGAGG I 1 1 1 ACTTGCTTTAAAAAACCTC CCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAAC 1 1 G 1 TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA AGCAI 1 1 1 1 1 I CAC I GCAI I CIAG I IG I GG I 1 1 G 1 CCAAACTCATCAATGTATCTTAA CGCGTAAATTGTAAGCGTTAATA 1 1 1 I G I IAAAAI I CGCG I 1 AAAI 1 1 1 I GTTAAATC AGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAAT AGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGA ACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTAC GTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATC GGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTG GCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGSEQ ID RNA Sequence (DNA)NO: ConstructCGCGTCAGGTGGCAC 1 1 1 1 CGGGGAAATGTGCGCGGAACCCCTA 1 1 IG I 1 IAI 1 1 1 1 CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAA TAATATTGAAAAAGGAAGAATCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTC AGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGC ATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAA GTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCC CATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATT TTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAG TGAGGAGGC I I l l i 1 GGAGGCC 1 AGGC 1 1 1 1 GCAAAGATCGATCAAGAGACAGGATG AGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTG GGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGG 1 1 Cl 1 1 1 1 G 1 CAAGACCGACCT GTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCA CGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGAC TGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCrTGCTCCT GCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCrGCATACGCTTGATCCG GCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGG ATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAACATCAGGGGCTCGC GCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCG TCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGC I l l i CTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCG TTGGCTACCCGTGATATTGCTGAAGAACrTGGCGGCGAATGGGCTGACCGCTTCCTC GTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCC I 1 Cl Al CGCC 1 1 Cl 1 GACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCA ACCTGCCATCACGAGATTTCGATTCCACCGCCGCC 1 1 C 1 ATGAAAGGTTGGGCTTCG GAATCG I l l i CCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG GAGTTCTTCGCCCACCCTAGGGGGAGGCTAACrGAAACACGGAAGGAGACAATACCG GAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGG TCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCA CCGAGACCCCATTGGGGCCAATACGCCCGCG 1 1 1 Cl 1 CC 1 1 1 1 CCCCACCCCACCCCC CAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCC ATAGCCTCAGGTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA AAAGGATCTAGGTGAAGATCCTTTTTGATAATCrCATGACCAAAATCCCTTAACGTGA G 1 1 1 1 CG 1 1 CCAC 1 GAGCG 1 CAGACCCCG 1 AGAAAAGA 1 CAAAGGA 1 C 1 1 C 1 1 GAGA TCCI 1 1 1 1 1 1 C 1 GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG GTGGTTTGTTTGCCGGATCAAGAGCTACCAAC 1 C 1 1 1 1 1 CCGAAGGTAACTGGCTTC AGCAGAGCGCAGATACCAAATACTG 1 I d 1 C 1 AGTGTAGCCGTAGTTAGGCCACCAC TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACrCAAGACGATAGTTAC CGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCrGGTATCTTTATAGTCCTG TCGGGTTTCGCCACCTCrGACTTGAGCGTCGAI 1 1 1 1 G 1 GATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCI 1 1 1 IACGG I I CC I GGCC I 1 1 I GCT GGCCI 1 1 I GCI CACAI G I 1 C 1 1 ICCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT TACCGCCATGCATTAGTTATTAATTAATACGACTCACTATAAGAATAAACTAGTATTC TTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGCGGAGCAGCAGCTAGA CTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGGACTGCACGGCGTCAGAGGATA CGAGACAACCGAGAGCGGCGATACAGCCGTGTCCGAGGACAGCTACGATAAGTACG CCAGCCAGAACACCAACAAAGAATACGTGTGCGACTTCACCGACCAGCTGAAGCCTA CAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGAAAGTGAACGAGCCCCTGATCA AAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGGAAAAGCTGTACGACAATATCG AGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGACCAAAGAGGAAACAAAGCTGA AAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGCTGATTAGCCCTACCGTGAACG AGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGTTCACCCTGCCrCCAGTGGTGC ACAAGGCCACCGTGTTCTACTTCATTTGCGACAACAGCAAGACCGAGGACGACAACA AGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTGGAACCCTACGGCGGAGGATCA GGTGGCGGAGGAAGCGGAGGCGGACTGATTGCAGGTGCAGTTGGAGGATCACTGCT GGCCGCACTGGTCATTTGCGGCATCGTGTATTGGATGAGAAGAAAGCGCAGAAAGGCCCCTAAGAGATGATGASEQ ID RNA Sequence (DNA)NO: Construct158 13 ATGGGCGGAGCAGCAGCTAGACTGGGAGCCGTGATTCTGTTCGTGGTCATCGTTGG ACTGCACGGCGTCAGAGGATACGAGACAACCGAGAGCGGCGATACAGCCGTGTCCG AGGACAGCTACGATAAGTACGCCAGCCAGAACACCAACAAAGAATACGTGTGCGACT TCACCGACCAGCTGAAGCCTACAGAAAGCGGCCCTAAAGTGAAAAAGTGCGAAGTGA AAGTGAACGAGCCCCTGATCAAAGTGAAGATCATTTGCCCTCTGAAGGGCAGCGTGG AAAAGCTGTACGACAATATCGAGTACGTGCCCAAGAAAAGCCCTTACGTGGTGCTGA CCAAAGAGGAAACAAAGCTGAAAGAGAAGCTGCTGAGCAAGCTGATCTACGGCCTGC TGATTAGCCCTACCGTGAACGAGAAAGAGAACAACTTCAAAGAGGGCGTTATCGAGT TCACCCTGCCTCCAGTGGTGCACAAGGCCACCGTGTTCTACTTCATTTGCGACAACA GCAAGACCGAGGACGACAACAAGAAGGGCAATAGAGGCATCGTGGAAGTGTACGTG GAACCCTACGGCGGAGGATCAGGTGGCGGAGGAAGCGGAGGACTGGCCATCTATTC TACAGTGGCCAGCAGCCTGGTGCTCCTGGTGTCTCTGGGAGCCATCAGCI 1 1 I GGATGTGCAGCAACGGCAGCCTGCAGTGCAGAATCTGTATCTGATGA

[0372] In some embodiments, a Pfs230 construct described herein has a nucleic acid sequence provided in Table 7. In some embodiments, a Pfs230 construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to nucleic acid sequence of Table 7.Table 8: RNA Sequences Corresponding to Exemplary Pfs230 RNA Constructs as Described HereinSEQ ID RNA Sequence (RNA)NO: Construct159 1 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGUUAUCGUG GGACUGCACGGCGUGCGCGGAGAGUACGUGGACGAGAAGGAGCGCCAAGGCGAGA UCUACCCAUUUGGGGAUGAGGAGGAGAAGGACGAGGGCGGAGAGAGCUUUACCUA UGAGAAGUCCGAAGUGGACAAGACCGACCUGUUCAAAUUUAUUGAGGGUGGCGAA GGAGAUGACGUGUAUAAGGUCGAUGGCAGUAAGGUCCUGUUGGACGAUGACACCA UCAGCCGCGUGUCCAAAAAGCAUACUGCCCGCGAUGGGGAGUACGGCGAAUACGGC GAAGCUGUGGAGGACGGAGAGAAUGUGAUCAAAAUUAUCAGAUCAGUGCUGCAGU CAGGUGCUCUGCCUUCCGUUGGCGUGGACGAGCUGGAUAAGAUCGACCUGUCCUA CGAGACUACCGAAUCCGGCGACACUGCAGUGUCCGAGGACUCCUACGAUAAGUACG CCUCCCAAAAUACUAACAAAGAAUAUGUAUGCGACUUCACCGACCAGCUUAAGCCU ACCGAGUCUGGCCCUAAAGUGAAGAAAUGCGAGGUGAAAGUGAACGAGCCGCUGA UCAAGGUGAAGAUCAUUUGCCCGCUGAAGGGCAGCGUGGAGAAGCUGUAUGACAA CAUCGAAUACGUGCCUAAAAAGAGCCCAUAUGUGGUCCUCACCAAGGAGGAAACGA AAUUGAAGGAGAAACUCCUGUCCAAACUUAUCUACGGCCUCCUGAUCAGCCCCACU GUGAACGAGAAAGAAAAUAACUUCAAGGAGGGGGUAAUCGAGUUCACUCUGCCACC CGUGGUCCACAAGGCCACUGUUUUCUACUUUAUUUGCGAUAACUCCAAGACCGAG GACGAUAACAAAAAGGGCAAUCGUGGCAUUGUCGAGGUGUACGUUGAGCCUUACG GAAACAAAAUCAAUGGUUGUGCCUUCCUGGAUGAGGACGAGGAGGAGGAGAAGUA UGGUAACCAGAUCGAGGAGGACGAGCAUAACGAGAAAAUCAAAAUGAAAACCUUUU UCACCCAAAACAUCUACAAAAAGAAUAACAUCUACCCAUGCUACAUGAAACUCUACU CCGGUGACAUCGGAGGCAUCCUGUUCCCUAAGAACAUCAAGUCCACAACCUGCUUC GAGGAAAUGAUCCCUUAUAACAAGGAGAUCAAAUGGAACAAAGAACAAAAGUCCCU GGGUAACCUGGUGCAGAACUCCGUAGUCUACAACAAGGAGAUGAAUGCCAAGUAUU UCAACGUGCAGUACGUGCACAUUCCUACCAGUUAUAAGGAUACCCUCAACCUGUUC UGUUCCAUCAUUCUCAAAGAGGAGGAGUCCAACCUGAUUUCCACCUCUUAUCUUGU CUAUGUGUCUAUUAACGAGGAGCUGCAAUUCUCCCUGUUCGACUUUUAUGAAAGC UUCGUGCCGAUUAAAAAGACAAUCCAAGUGGCACAGAAGAACGUGAAUAACAAGGA GCACGACUACACAUGCGACUUCACCGACAAACUGGAUAAGACCGUGCCCAGCACAG CCAAUGGGAAAAAGCUUUUCAUUUGCCGCAAACACCUGAAGGAGUUUGAUACUUUC ACAUUGAAGUGUAACGUUCAAAAGACCCAGUACCCUAACAUCGAGAUUUUCCCCAA GACACUGAAGGACAAGAAAGAGGUACUGAAACUGGAUCUGGACAUCCAGUACCAGA UGUUUAGCAAGUUUUUCAAAUUCAACACCCAGAACGCCAAGUACCUGAACCUGUAC CCGUACUAUCUGAUCUUCCCCUUCAACCAUAUCGGUAAAAAGGAGCUGAAAAAUAA CCCAACGUACAAGAACCACAAGGAUGUGAAGUAUUUUGAGCAAUCUUCCGUUCUGA GCCCACUUUCUUCCGCCGAUUCUCUGGGAAAGCUCCUGAACUUCCUGGAUACCCAG GAGACUGUUUGCCUCACCGAAAAGAUCCGUUAUUUGCAGCUGUCCAUCAACGAACUCGGCUCUGACCAGAACACCUUCUCUGUGACAUUCCAGGUGCCGCCCUACAUCGAUASEQ ID RNA Sequence (RNA)NO: ConstructUCAAGGAGCCUUUUUACUUCAUGUUCGGAUGCAACAAUAACAAGGGUGAGGGGAA CAUCGGCAUCGUGGAGCUUCUGAUCUCUAAAGGAGGCUCUGGCGGUGGACUUAUU GCCGGCGCUGUUGGAGGAUCUCUGCUGGCCGCUCUGGUCAUCUGUGGCAUCGUGU ACUGGAUGCGGCGGCACACACAGAAGGCCCCUAAGAGAAUCAGACUGCCCCACAUC AGAUGAUGA160 2 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGUUAUCGUG GGACUGCACGGCGUGCGCGGAUCAGUGCUGCAGUCAGGUGCUCUGCCUUCCGUUG GCGUGGACGAGCUGGAUAAGAUCGACCUGUCCUACGAGACUACCGAAUCCGGCGAC ACUGCAGUGUCCGAGGACUCCUACGAUAAGUACGCCUCCCAAAAUACUAACAAAGA AUAUGUAUGCGACUUCACCGACCAGCUUAAGCCUACCGAGUCUGGCCCUAAAGUGA AGAAAUGCGAGGUGAAAGUGAACGAGCCGCUGAUCAAGGUGAAGAUCAUUUGCCC GCUGAAGGGCAGCGUGGAGAAGCUGUAUGACAACAUCGAAUACGUGCCUAAAAAGA GCCCAUAUGUGGUCCUCACCAAGGAGGAAACGAAAUUGAAGGAGAAACUCCUGUCC AAACUUAUCUACGGCCUCCUGAUCAGCCCCACUGUGAACGAGAAAGAAAAUAACUU CAAGGAGGGGGUAAUCGAGUUCACUCUGCCACCCGUGGUCCACAAGGCCACUGUU UUCUACUUUAUUUGCGAUAACUCCAAGACCGAGGACGAUAACAAAAAGGGCAAUCG UGGCAUUGUCGAGGUGUACGUUGAGCCUUACGGAAACAAAAUCAAUGGUUGUGCC UUCCUGGAUGAGGACGAGGAGGAGGAGAAGUAUGGUAACCAGAUCGAGGAGGACG AGCAUAACGAGAAAAUCAAAAUGAAAACCUUUUUCACCCAAAACAUCUACAAAAAGA AUAACAUCUACCCAUGCUACAUGAAACUCUACUCCGGUGACAUCGGAGGCAUCCUG UUCCCUAAGAACAUCAAGUCCACAACCUGCUUCGAGGAAAUGAUCCCUUAUAACAA GGAGAUCAAAUGGAACAAAGAACAAAAGUCCCUGGGUAACCUGGUGCAGAACUCCG UAGUCUACAACAAGGAGAUGAAUGCCAAGUAUUUCAACGUGCAGUACGUGCACAUU CCUACCAGUUAUAAGGAUACCCUCAACCUGUUCUGUUCCAUCAUUCUCAAAGAGGA GGAGUCCAACCUGAUUUCCACCUCUUAUCUUGUCUAUGUGUCUAUUAACGAGGAG CUGCAAUUCUCCCUGUUCGACUUUUAUGAAAGCUUCGUGCCGAUUAAAAAGACAAU CCAAGUGGCACAGAAGAACGUGAAUAACAAGGAGCACGACUACACAUGCGACUUCA CCGACAAACUGGAUAAGACCGUGCCCAGCACAGCCAAUGGGAAAAAGCUUUUCAUU UGCCGCAAACACCUGAAGGAGUUUGAUACUUUCACAUUGAAGUGUAACGUUCAAAA GACCCAGUACCCUAACAUCGAGAUUUUCCCCAAGACACUGAAGGACAAGAAAGAGG UACUGAAACUGGAUCUGGACAUCCAGUACCAGAUGUUUAGCAAGUUUUUCAAAUUC AACACCCAGAACGCCAAGUACCUGAACCUGUACCCGUACUAUCUGAUCUUCCCCUU CAACCAUAUCGGUAAAAAGGAGCUGAAAAAUAACCCAACGUACAAGAACCACAAGGA UGUGAAGUAUUUUGAGCAAUCUUCCGUUCUGAGCCCACUUUCUUCCGCCGAUUCU CUGGGAAAGCUCCUGAACUUCCUGGAUACCCAGGAGACUGUUUGCCUCACCGAAAA GAUCCGUUAUUUGCAGCUGUCCAUCAACGAACUCGGCUCUGACCAGAACACCUUCU CUGUGACAUUCCAGGUGCCGCCCUACAUCGAUAUCAAGGAGCCUUUUUACUUCAUG UUCGGAUGCAACAAUAACAAGGGUGAGGGGAACAUCGGCAUCGUGGAGCUUCUGA UCUCUAAAGGAGGCUCUGGCGGUGGACUUAUUGCCGGCGCUGUUGGAGGAUCUCU GCUGGCCGCUCUGGUCAUCUGUGGCAUCGUGUACUGGAUGCGGCGGCACACACAG AAGGCCCCUAAGAGAAUCAGACUGCCCCACAUCAGAUGAUGA161 3.01 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGUUAUCGUG GGACUGCACGGCGUGCGCGGAUCAGUGCUGCAGUCAGGUGCUCUGCCUUCCGUUG GCGUGGACGAGCUGGAUAAGAUCGACCUGUCCUACGAGACUACCGAAUCCGGCGAC ACUGCAGUGUCCGAGGACUCCUACGAUAAGUACGCCUCCCAAAAUACUAACAAAGA AUAUGUAUGCGACUUCACCGACCAGCUUAAGCCUACCGAGUCUGGCCCUAAAGUGA AGAAAUGCGAGGUGAAAGUGAACGAGCCGCUGAUCAAGGUGAAGAUCAUUUGCCC GCUGAAGGGCAGCGUGGAGAAGCUGUAUGACAACAUCGAAUACGUGCCUAAAAAGA GCCCAUAUGUGGUCCUCACCAAGGAGGAAACGAAAUUGAAGGAGAAACUCCUGUCC AAACUUAUCUACGGCCUCCUGAUCAGCCCCACUGUGAACGAGAAAGAAAAUAACUU CAAGGAGGGGGUAAUCGAGUUCACUCUGCCACCCGUGGUCCACAAGGCCACUGUU UUCUACUUUAUUUGCGAUAACUCCAAGACCGAGGACGAUAACAAAAAGGGCAAUCG UGGCAUUGUCGAGGUGUACGUUGAGCCUUACGGAAACAAAAUCAAUGGUGGAGGC UCUGGCGGUGUGUCAGGAUGGCGUCUGUUCAAAAAGAUCAGCGGAGGCUCUGGCG GUGGACUUAUUGCCGGCGCUGUUGGAGGAUCUCUGCUGGCCGCUCUGGUCAUCUG UGGCAUCGUGUACUGGAUGCGGCGGCACACACAGAAGGCCCCUAAGAGAAUCAGAC UGCCCCACAUCAGAUGAUGA162 3.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAAGCGUGCUGCAAAGCGGAGCACUGCCUUCAGUGGG AGUCGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCCGAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAASEQ ID RNA Sequence (RNA)NO: Construct UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAA AAAGUGCGAAGUGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUC UGAAGGGCAGCGUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGC CCUUACGUGGUGCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAA GCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCA AAGAGGGCGUUAUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUU CUACUUCAUUUGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAG GCAUCGUGGAAGUGUACGUGGAACCCUACGGCAACAAGAUCAACGGCGGAGGAUCA GGUGGCGUGUCAGGAUGGCGUCUGUUCAAAAAGAUCAGCGGAGGAAGCGGAGGCG GACUGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGG CAUCGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAAUCAGACUGC CCCACAUCAGAUGAUGA163 3.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUG GGCCUUCACGGAGUCCGAGGUAGCGUACUGCAAUCUGGCGCACUCCCUUCUGUAG GGGUAGACGAACUGGACAAAAUUGACCUGUCAUACGAGACUACGGAGUCAGGGGA UACAGCCGUGUCCGAGGAUUCCUACGACAAAUACGCUUCCCAGAAUACCAAUAAGG AGUACGUGUGCGACUUUACCGAUCAGCUGAAGCCCACAGAAUCCGGGCCAAAGGUG AAGAAGUGCGAAGUGAAAGUCAACGAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCC GCUGAAAGGCAGUGUUGAGAAGCUUUACGACAACAUCGAGUACGUCCCCAAGAAAA GCCCCUACGUGGUUCUGACCAAGGAAGAAACCAAGCUGAAAGAGAAACUCCUGUCU AAACUCAUCUACGGACUGCUGAUUAGCCCCACUGUCAACGAGAAAGAGAACAAUUU CAAGGAAGGCGUUAUUGAGUUUACUCUGCCACCUGUGGUGCAUAAGGCUACCGUU UUCUACUUCAUCUGCGAUAACAGCAAGACAGAGGACGAUAACAAGAAGGGGAAUAG GGGUAUCGUCGAAGUGUACGUUGAACCAUACGGGAACAAGAUAAACGGAGGCGGA UCAGGUGGUGUGUCAGGAUGGCGUCUGUUCAAAAAGAUCAGCGGAGGCAGUGGAG GCGGCUUGAUUGCCGGAGCUGUGGGUGGCAGCCUCUUGGCAGCCCUUGUGAUCUG UGGGAUUGUGUACUGGAUGAGAAGGCACACACAGAAAGCCCCUAAACGCAUACGGU UGCCACACAUUCGUUGAUGA164 3.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCAGCGUGCUGCAGAGCGGCGCCCUGCCAAGCGUGGG CGUGGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACCACCGAGAGCGGCGACA CCGCCGUGAGCGAGGACAGCUACGACAAGUACGCCAGCCAGAAUACCAAUAAGGAG UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAGAGCGGCCCUAAAGUGAA GAAGUGCGAGGUGAAGGUGAACGAGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCU CUGAAGGGCAGCGUGGAGAAACUGUACGACAAUAUCGAGUACGUGCCUAAGAAGU CCCCUUACGUGGUGCUGACCAAGGAGGAGACCAAGCUGAAGGAGAAGCUGCUGAG CAAGCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAGGAGAAUAAUU UCAAGGAGGGCGUGAUCGAGUUCACCCUGCCUCCUGUGGUGCACAAGGCCACCGU GUUCUACUUCAUCUGCGACAAUAGCAAGACCGAGGACGACAAUAAGAAGGGCAAUA GAGGCAUCGUGGAGGUGUACGUGGAGCCUUACGGCAAUAAGAUCAACGGCGGCGG CAGCGGCGGAGUGUCAGGAUGGCGUCUGUUCAAAAAGAUCAGCGGAGGAAGCGGA GGCGGACUGAUCGCCGGAGCCGUGGGAGGCAGCCUGCUGGCCGCCCUGGUGAUCU GCGGCAUCGUGUACUGGAUGAGAAGACACACCCAGAAGGCCCCUAAGAGAAUCAGA CUGCCUCACAUCAGAUGAUGA165 4.01 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUCCUGUUCGUGGUUAUCGUG GGACUGCACGGCGUGCGCGGAUCAGUGCUGCAGUCAGGUGCUCUGCCUUCCGUUG GCGUGGACGAGCUGGAUAAGAUCGACCUGUCCUACGAGACUACCGAAUCCGGCGAC ACUGCAGUGUCCGAGGACUCCUACGAUAAGUACGCCUCCCAAAAUACUAACAAAGA AUAUGUAUGCGACUUCACCGACCAGCUUAAGCCUACCGAGUCUGGCCCUAAAGUGA AGAAAUGCGAGGUGAAAGUGAACGAGCCGCUGAUCAAGGUGAAGAUCAUUUGCCC GCUGAAGGGCAGCGUGGAGAAGCUGUAUGACAACAUCGAAUACGUGCCUAAAAAGA GCCCAUAUGUGGUCCUCACCAAGGAGGAAACGAAAUUGAAGGAGAAACUCCUGUCC AAACUUAUCUACGGCCUCCUGAUCAGCCCCACUGUGAACGAGAAAGAAAAUAACUU CAAGGAGGGGGUAAUCGAGUUCACUCUGCCACCCGUGGUCCACAAGGCCACUGUU UUCUACUUUAUUUGCGAUAACUCCAAGACCGAGGACGAUAACAAAAAGGGCAAUCG UGGCAUUGUCGAGGUGUACGUUGAGCCUUACGGAAACAAAAUCAAUGGUGGAGGC UCUGGCGGUGGAGGCUCUGGCGGUGGACUUAUUGCCGGCGCUGUUGGAGGAUCU CUGCUGGCCGCUCUGGUCAUCUGUGGCAUCGUGUACUGGAUGCGGCGGCACACAC AGAAGGCCCCUAAGAGAAUCAGACUGCCCCACAUCAGAUGAUGA166 4.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAAGCGUGCUGCAAAGCGGAGCACUGCCUUCAGUGGGSEQ ID RNA Sequence (RNA)NO: Construct AGUCGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACAACCGAGAGCGGCGAUA CAGCCGUGUCCGAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAA UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAA AAAGUGCGAAGUGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUC UGAAGGGCAGCGUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGC CCUUACGUGGUGCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAA GCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCA AAGAGGGCGUUAUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUU CUACUUCAUUUGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAG GCAUCGUGGAAGUGUACGUGGAACCCUACGGCAACAAGAUCAACGGCGGAGGAUCA GGUGGCGGAGGAAGCGGAGGCGGACUGAUUGCAGGUGCAGUUGGAGGAUCACUGC UGGCCGCACUGGUCAUUUGCGGCAUCGUGUAUUGGAUGAGAAGGCACACCCAGAA GGCCCCUAAGAGAAUCAGACUGCCCCACAUCAGAUGAUGA167 4.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUG GGCCUUCACGGAGUCCGAGGUAGCGUACUGCAAUCUGGCGCACUCCCUUCUGUAG GGGUAGACGAACUGGACAAAAUUGACCUGUCAUACGAGACUACGGAGUCAGGGGA UACAGCCGUGUCCGAGGAUUCCUACGACAAAUACGCUUCCCAGAAUACCAAUAAGG AGUACGUGUGCGACUUUACCGAUCAGCUGAAGCCCACAGAAUCCGGGCCAAAGGUG AAGAAGUGCGAAGUGAAAGUCAACGAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCC GCUGAAAGGCAGUGUUGAGAAGCUUUACGACAACAUCGAGUACGUCCCCAAGAAAA GCCCCUACGUGGUUCUGACCAAGGAAGAAACCAAGCUGAAAGAGAAACUCCUGUCU AAACUCAUCUACGGACUGCUGAUUAGCCCCACUGUCAACGAGAAAGAGAACAAUUU CAAGGAAGGCGUUAUUGAGUUUACUCUGCCACCUGUGGUGCAUAAGGCUACCGUU UUCUACUUCAUCUGCGAUAACAGCAAGACAGAGGACGAUAACAAGAAGGGGAAUAG GGGUAUCGUCGAAGUGUACGUUGAACCAUACGGGAACAAGAUAAACGGAGGCGGA UCAGGUGGUGGAGGCAGUGGAGGCGGCUUGAUUGCCGGAGCUGUGGGUGGCAGC CUCUUGGCAGCCCUUGUGAUCUGUGGGAUUGUGUACUGGAUGAGAAGGCACACAC AGAAAGCCCCUAAACGCAUACGGUUGCCACACAUUCGUUGAUGA168 4.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCAGCGUGCUGCAGAGCGGCGCCCUGCCAAGCGUGGG CGUGGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACCACCGAGAGCGGCGACA CCGCCGUGAGCGAGGACAGCUACGACAAGUACGCCAGCCAGAAUACCAAUAAGGAG UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAGAGCGGCCCUAAAGUGAA GAAGUGCGAGGUGAAGGUGAACGAGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCU CUGAAGGGCAGCGUGGAGAAACUGUACGACAAUAUCGAGUACGUGCCUAAGAAGU CCCCUUACGUGGUGCUGACCAAGGAGGAGACCAAGCUGAAGGAGAAGCUGCUGAG CAAGCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAGGAGAAUAAUU UCAAGGAGGGCGUGAUCGAGUUCACCCUGCCUCCUGUGGUGCACAAGGCCACCGU GUUCUACUUCAUCUGCGACAAUAGCAAGACCGAGGACGACAAUAAGAAGGGCAAUA GAGGCAUCGUGGAGGUGUACGUGGAGCCUUACGGCAAUAAGAUCAACGGCGGCGG CAGCGGCGGAGGAGGAAGCGGAGGCGGACUGAUCGCCGGAGCCGUGGGAGGCAGC CUGCUGGCCGCCCUGGUGAUCUGCGGCAUCGUGUACUGGAUGAGAAGACACACCC AGAAGGCCCCUAAGAGAAUCAGACUGCCUCACAUCAGAUGAUGA169 5.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAAGCGUGCUGCAAAGCGGAGCACUGCCUUCAGUGGG AGUCGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACAACCGAGAGCGGCGAUA CAGCCGUGUCCGAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAA UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAA AAAGUGCGAAGUGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUC UGAAGGGCAGCGUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGC CCUUACGUGGUGCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAA GCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCA AAGAGGGCGUUAUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUU CUACUUCAUUUGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAG GCAUCGUGGAAGUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAG CGGAGGCGGACUGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUC AUUUGCGGCAUCGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAAU CAGACUGCCCCACAUCAGAUGAUGA170 5.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUGGGCCUUCACGGAGUCCGAGGUAGCGUACUGCAAUCUGGCGCACUCCCUUCUGUAGGGGUAGACGAACUGGACAAAAUUGACCUGUCAUACGAGACUACGGAGUCAGGGGASEQ ID RNA Sequence (RNA)NO: ConstructUACAGCCGUGUCCGAGGAUUCCUACGACAAAUACGCUUCCCAGAAUACCAAUAAGG AGUACGUGUGCGACUUUACCGAUCAGCUGAAGCCCACAGAAUCCGGGCCAAAGGUG AAGAAGUGCGAAGUGAAAGUCAACGAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCC GCUGAAAGGCAGUGUUGAGAAGCUUUACGACAACAUCGAGUACGUCCCCAAGAAAA GCCCCUACGUGGUUCUGACCAAGGAAGAAACCAAGCUGAAAGAGAAACUCCUGUCU AAACUCAUCUACGGACUGCUGAUUAGCCCCACUGUCAACGAGAAAGAGAACAAUUU CAAGGAAGGCGUUAUUGAGUUUACUCUGCCACCUGUGGUGCAUAAGGCUACCGUU UUCUACUUCAUCUGCGAUAACAGCAAGACAGAGGACGAUAACAAGAAGGGGAAUAG GGGUAUCGUCGAAGUGUACGUUGAACCAUACGGAGGCGGAUCAGGUGGUGGAGGC AGUGGAGGCGGCUUGAUUGCCGGAGCUGUGGGUGGCAGCCUCUUGGCAGCCCUUG UGAUCUGUGGGAUUGUGUACUGGAUGAGAAGGCACACACAGAAAGCCCCUAAACGC AUACGGUUGCCACACAUUCGUUGAUGA171 5.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCAGCGUGCUGCAGAGCGGCGCCCUGCCAAGCGUGGG CGUGGACGAGCUGGACAAGAUCGACCUGAGCUACGAGACCACCGAGAGCGGCGACA CCGCCGUGAGCGAGGACAGCUACGACAAGUACGCCAGCCAGAAUACCAAUAAGGAG UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAGAGCGGCCCUAAAGUGAA GAAGUGCGAGGUGAAGGUGAACGAGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCU CUGAAGGGCAGCGUGGAGAAACUGUACGACAAUAUCGAGUACGUGCCUAAGAAGU CCCCUUACGUGGUGCUGACCAAGGAGGAGACCAAGCUGAAGGAGAAGCUGCUGAG CAAGCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAGGAGAAUAAUU UCAAGGAGGGCGUGAUCGAGUUCACCCUGCCUCCUGUGGUGCACAAGGCCACCGU GUUCUACUUCAUCUGCGACAAUAGCAAGACCGAGGACGACAAUAAGAAGGGCAAUA GAGGCAUCGUGGAGGUGUACGUGGAGCCUUACGGCAAUAAGAUCAACGGCGGCGG CAGCGGCGGAGGAGGAAGCGGAGGCGGACUGAUCGCCGGAGCCGUGGGAGGCAGC CUGCUGGCCGCCCUGGUGAUCUGCGGCAUCGUGUACUGGAUGAGAAGACACACCC AGAAGGCCCCUAAGAGAAUCAGACUGCCUCACAUCAGAUGAUGA172 6.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGU GCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACG GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGAC UGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAU CGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAAUCAGACUGCCCC ACAUCAGAUGAUGA173 6.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUG GGCCUUCACGGAGUCCGAGGUUACGAGACUACGGAGUCAGGGGAUACAGCCGUGU CCGAGGAUUCCUACGACAAAUACGCUUCCCAGAAUACCAAUAAGGAGUACGUGUGC GACUUUACCGAUCAGCUGAAGCCCACAGAAUCCGGGCCAAAGGUGAAGAAGUGCGA AGUGAAAGUCAACGAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCCGCUGAAAGGCA GUGUUGAGAAGCUUUACGACAACAUCGAGUACGUCCCCAAGAAAAGCCCCUACGUG GUUCUGACCAAGGAAGAAACCAAGCUGAAAGAGAAACUCCUGUCUAAACUCAUCUA CGGACUGCUGAUUAGCCCCACUGUCAACGAGAAAGAGAACAAUUUCAAGGAAGGCG UUAUUGAGUUUACUCUGCCACCUGUGGUGCAUAAGGCUACCGUUUUCUACUUCAU CUGCGAUAACAGCAAGACAGAGGACGAUAACAAGAAGGGGAAUAGGGGUAUCGUCG AAGUGUACGUUGAACCAUACGGAGGCGGAUCAGGUGGUGGAGGCAGUGGAGGCGG CUUGAUUGCCGGAGCUGUGGGUGGCAGCCUCUUGGCAGCCCUUGUGAUCUGUGG GAUUGUGUACUGGAUGAGAAGGCACACACAGAAAGCCCCUAAACGCAUACGGUUGC CACACAUUCGUUGAUGA174 6.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCUACGAGACCACCGAGAGCGGCGACACCGCCGUGAGC GAGGACAGCUACGACAAGUACGCCAGCCAGAAUACCAAUAAGGAGUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAGAGCGGCCCUAAAGUGAAGAAGUGCGAGG UGAAGGUGAACGAGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCUCUGAAGGGCAGCGUGGAGAAACUGUACGACAAUAUCGAGUACGUGCCUAAGAAGUCCCCUUACGUGGSEQ ID RNA Sequence (RNA)NO: Construct UGCUGACCAAGGAGGAGACCAAGCUGAAGGAGAAGCUGCUGAGCAAGCUGAUCUAC GGCCUGCUGAUUAGCCCUACCGUGAACGAGAAGGAGAAUAAUUUCAAGGAGGGCG UGAUCGAGUUCACCCUGCCUCCUGUGGUGCACAAGGCCACCGUGUUCUACUUCAU CUGCGACAAUAGCAAGACCGAGGACGACAAUAAGAAGGGCAAUAGAGGCAUCGUGG AGGUGUACGUGGAGCCUUACGGCGGCGGCAGCGGCGGAGGAGGAAGCGGAGGCGG ACUGAUCGCCGGAGCCGUGGGAGGCAGCCUGCUGGCCGCCCUGGUGAUCUGCGGC AUCGUGUACUGGAUGAGAAGACACACCCAGAAGGCCCCUAAGAGAAUCAGACUGCC UCACAUCAGAUGAUGA175 7.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAA UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAA AAAGUGCGAAGUGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUC UGAAGGGCAGCGUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGC CCUUACGUGGUGCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAA GCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCA AAGAGGGCGUUAUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUU CUACUUCAUUUGCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAG GCAUCGUGGAAGUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAG CGGAGGCGGACUGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUC AUUUGCGGCAUCGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAAU CAGACUGCCCCACAUCAGAUGAUGA176 7.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUG GGCCUUCACGGAGUCCGAGGUUACGACAAAUACGCUUCCCAGAAUACCAAUAAGGA GUACGUGUGCGACUUUACCGAUCAGCUGAAGCCCACAGAAUCCGGGCCAAAGGUGA AGAAGUGCGAAGUGAAAGUCAACGAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCCG CUGAAAGGCAGUGUUGAGAAGCUUUACGACAACAUCGAGUACGUCCCCAAGAAAAG CCCCUACGUGGUUCUGACCAAGGAAGAAACCAAGCUGAAAGAGAAACUCCUGUCUA AACUCAUCUACGGACUGCUGAUUAGCCCCACUGUCAACGAGAAAGAGAACAAUUUC AAGGAAGGCGUUAUUGAGUUUACUCUGCCACCUGUGGUGCAUAAGGCUACCGUUU UCUACUUCAUCUGCGAUAACAGCAAGACAGAGGACGAUAACAAGAAGGGGAAUAGG GGUAUCGUCGAAGUGUACGUUGAACCAUACGGAGGCGGAUCAGGUGGUGGAGGCA GUGGAGGCGGCUUGAUUGCCGGAGCUGUGGGUGGCAGCCUCUUGGCAGCCCUUG UGAUCUGUGGGAUUGUGUACUGGAUGAGAAGGCACACACAGAAAGCCCCUAAACGC AUACGGUUGCCACACAUUCGUUGAUGA177 7.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCUACGACAAGUACGCCAGCCAGAAUACCAAUAAGGAG UACGUGUGCGACUUCACCGACCAGCUGAAGCCUACAGAGAGCGGCCCUAAAGUGAA GAAGUGCGAGGUGAAGGUGAACGAGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCU CUGAAGGGCAGCGUGGAGAAACUGUACGACAAUAUCGAGUACGUGCCUAAGAAGU CCCCUUACGUGGUGCUGACCAAGGAGGAGACCAAGCUGAAGGAGAAGCUGCUGAG CAAGCUGAUCUACGGCCUGCUGAUUAGCCCUACCGUGAACGAGAAGGAGAAUAAUU UCAAGGAGGGCGUGAUCGAGUUCACCCUGCCUCCUGUGGUGCACAAGGCCACCGU GUUCUACUUCAUCUGCGACAAUAGCAAGACCGAGGACGACAAUAAGAAGGGCAAUA GAGGCAUCGUGGAGGUGUACGUGGAGCCUUACGGCGGCGGCAGCGGCGGAGGAGG AAGCGGAGGCGGACUGAUCGCCGGAGCCGUGGGAGGCAGCCUGCUGGCCGCCCUG GUGAUCUGCGGCAUCGUGUACUGGAUGAGAAGACACACCCAGAAGGCCCCUAAGAG AAUCAGACUGCCUCACAUCAGAUGAUGA178 8.02 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAACCAACAAAGAAUACGUGUGCGACUUCACCGACCAG CUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAGUGAAAGUGAACGA GCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGCGUGGAAAAGCUG UACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGUGCUGACCAAAGA GGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACGGCCUGCUGAUUA GCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUUAUCGAGUUCACC CUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUUGCGACAACAGCAA GACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAAGUGUACGUGGAAC CCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGACUGAUUGCAGGUGC AGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAUCGUGUAUUGGAUG AGAAGGCACACCCAGAAGGCCCCUAAGAGAAUCAGACUGCCCCACAUCAGAUGAUGASEQ ID RNA Sequence (RNA)NO: Construct179 8.03 AUGGGCGGGGCAGCCGCUAGACUGGGAGCGGUGAUUCUCUUUGUGGUCAUAGUG GGCCUUCACGGAGUCCGAGGUACCAAUAAGGAGUACGUGUGCGACUUUACCGAUC AGCUGAAGCCCACAGAAUCCGGGCCAAAGGUGAAGAAGUGCGAAGUGAAAGUCAAC GAGCCUCUGAUCAAGGUCAAGAUCAUCUGUCCGCUGAAAGGCAGUGUUGAGAAGC UUUACGACAACAUCGAGUACGUCCCCAAGAAAAGCCCCUACGUGGUUCUGACCAAG GAAGAAACCAAGCUGAAAGAGAAACUCCUGUCUAAACUCAUCUACGGACUGCUGAU UAGCCCCACUGUCAACGAGAAAGAGAACAAUUUCAAGGAAGGCGUUAUUGAGUUUA CUCUGCCACCUGUGGUGCAUAAGGCUACCGUUUUCUACUUCAUCUGCGAUAACAGC AAGACAGAGGACGAUAACAAGAAGGGGAAUAGGGGUAUCGUCGAAGUGUACGUUG AACCAUACGGAGGCGGAUCAGGUGGUGGAGGCAGUGGAGGCGGCUUGAUUGCCGG AGCUGUGGGUGGCAGCCUCUUGGCAGCCCUUGUGAUCUGUGGGAUUGUGUACUG GAUGAGAAGGCACACACAGAAAGCCCCUAAACGCAUACGGUUGCCACACAUUCGUU GAUGA180 8.04 AUGGGCGGAGCCGCCGCCAGACUGGGCGCCGUGAUCCUGUUCGUGGUGAUCGUGG GCCUGCACGGCGUGAGAGGCACCAAUAAGGAGUACGUGUGCGACUUCACCGACCAG CUGAAGCCUACAGAGAGCGGCCCUAAAGUGAAGAAGUGCGAGGUGAAGGUGAACG AGCCUCUGAUCAAGGUGAAGAUCAUCUGCCCUCUGAAGGGCAGCGUGGAGAAACU GUACGACAAUAUCGAGUACGUGCCUAAGAAGUCCCCUUACGUGGUGCUGACCAAGG AGGAGACCAAGCUGAAGGAGAAGCUGCUGAGCAAGCUGAUCUACGGCCUGCUGAU UAGCCCUACCGUGAACGAGAAGGAGAAUAAUUUCAAGGAGGGCGUGAUCGAGUUC ACCCUGCCUCCUGUGGUGCACAAGGCCACCGUGUUCUACUUCAUCUGCGACAAUAG CAAGACCGAGGACGACAAUAAGAAGGGCAAUAGAGGCAUCGUGGAGGUGUACGUG GAGCCUUACGGCGGCGGCAGCGGCGGAGGAGGAAGCGGAGGCGGACUGAUCGCCG GAGCCGUGGGAGGCAGCCUGCUGGCCGCCCUGGUGAUCUGCGGCAUCGUGUACUG GAUGAGAAGACACACCCAGAAGGCCCCUAAGAGAAUCAGACUGCCUCACAUCAGAU GAUGA181 9 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGU GCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACG GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGAC UGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAU CGUGUAUUGGAUGAGAAGAAAGCGCAGAAAGGCCCCUAAGAGAAUCAGACUGCCCC ACAUCAGAUGAUGA182 10 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGU GCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACG GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGAC UGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAU CGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAAUCAGACUGCCCC ACAUCAGAGAGGACGAUCAGCCAAGCUCUCACCAGCCACUGUUCUACUGAUGA183 11 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGUGCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACGSEQ ID RNA Sequence (RNA)NO: Construct GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGAC UGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAU CGUGUAUUGGAUGAGAAGGCACACCCAGAAGGCCCCUAAGAGAUGAUGA184 12 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGU GCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACG GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGAC UGAUUGCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAU CGUGUAUUGGAUGAGAAGAAAGCGCAGAAAGGCCCCUAAGAGAUGAUGACUCGAG CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAG UCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCA CCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUA GCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAA GUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCU GGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAGAAGAGCUCCAACCGGUGUGGUAGCUCCGCCGUUUAACAUCGCCCUUCCCAA CAGUUGCGCAGCCUGAAUGGCGAAUGGAGAUCCAAUUUUUAAGUGUAUAAUGUGU UAAACUACUGAUUCUAAUUGUUUGUGUAUUUUAGAUUCACAGUCCCAAGGCUCAU UUCAGGCCCCUCAGUCCUCACAGUCUGUUCAUGAUCAUAAUCAGCCAUACCACAUU UGUAGAGGUUUUACUUGCUUUAAAAAACCUCCCACACCUCCCCCUGAACCUGAAAC AUAAAAUGAAUGCAAUUGUUGUUGUUAACUUGUUUAUUGCAGCUUAUAAUGGUUA CAAAUAAAGCAAUAGCAUCACAAAUUUCACAAAUAAAGCAUUUUUUUCACUGCAUU CUAGUUGUGGUUUGUCCAAACUCAUCAAUGUAUCUUAACGCGUAAAUUGUAAGCG UUAAUAUUUUGUUAAAAUUCGCGUUAAAUUUUUGUUAAAUCAGCUCAUUUUUUAA CCAAUAGGCCGAAAUCGGCAAAAUCCCUUAUAAAUCAAAAGAAUAGACCGAGAUAG GGUUGAGUGUUGUUCCAGUUUGGAACAAGAGUCCACUAUUAAAGAACGUGGACUC CAACGUCAAAGGGCGAAAAACCGUCUAUCAGGGCGAUGGCCCACUACGUGAACCAU CACCCUAAUCAAGUUUUUUGGGGUCGAGGUGCCGUAAAGCACUAAAUCGGAACCCU AAAGGGAGCCCCCGAUUUAGAGCUUGACGGGGAAAGCCGGCGAACGUGGCGAGAA AGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCUAGGGCGCUGGCAAGUGUAGCGGU CACGCUGCGCGUAACCACCACACCCGCCGCGCUUAAUGCGCCGCUACAGGGCGCGU CAGGUGGCACUUUUCGGGGAAAUGUGCGCGGAACCCCUAUUUGUUUAUUUUUCUA AAUACAUUCAAAUAUGUAUCCGCUCAUGAGACAAUAACCCUGAUAAAUGCUUCAAU AAUAUUGAAAAAGGAAGAAUCCUGAGGCGGAAAGAACCAGCUGUGGAAUGUGUGU CAGUUAGGGUGUGGAAAGUCCCCAGGCUCCCCAGCAGGCAGAAGUAUGCAAAGCAU GCAUCUCAAUUAGUCAGCAACCAGGUGUGGAAAGUCCCCAGGCUCCCCAGCAGGCA GAAGUAUGCAAAGCAUGCAUCUCAAUUAGUCAGCAACCAUAGUCCCGCCCCUAACU CCGCCCAUCCCGCCCCUAACUCCGCCCAGUUCCGCCCAUUCUCCGCCCCAUGGCUG ACUAAUUUUUUUUAUUUAUGCAGAGGCCGAGGCCGCCUCGGCCUCUGAGCUAUUC CAGAAGUAGUGAGGAGGCUUUUUUGGAGGCCUAGGCUUUUGCAAAGAUCGAUCAA GAGACAGGAUGAGGAUCGUUUCGCAUGAUUGAACAAGAUGGAUUGCACGCAGGUU CUCCGGCCGCUUGGGUGGAGAGGCUAUUCGGCUAUGACUGGGCACAACAGACAAU CGGCUGCUCUGAUGCCGCCGUGUUCCGGCUGUCAGCGCAGGGGCGCCCGGUUCUU UUUGUCAAGACCGACCUGUCCGGUGCCCUGAAUGAACUGCAAGACGAGGCAGCGC GGCUAUCGUGGCUGGCCACGACGGGCGUUCCUUGCGCAGCUGUGCUCGACGUUGU CACUGAAGCGGGAAGGGACUGGCUGCUAUUGGGCGAAGUGCCGGGGCAGGAUCUC CUGUCAUCUCACCUUGCUCCUGCCGAGAAAGUAUCCAUCAUGGCUGAUGCAAUGCG GCGGCUGCAUACGCUUGAUCCGGCUACCUGCCCAUUCGACCACCAAGCGAAACAUC GCAUCGAGCGAGCACGUACUCGGAUGGAAGCCGGUCUUGUCGAUCAGGAUGAUCU GGACGAAGAACAUCAGGGGCUCGCGCCAGCCGAACUGUUCGCCAGGCUCAAGGCGAGCAUGCCCGACGGCGAGGAUCUCGUCGUGACCCAUGGCGAUGCCUGCUUGCCGAASEQ ID RNA Sequence (RNA)NO: ConstructUAUCAUGGUGGAAAAUGGCCGCUUUUCUGGAUUCAUCGACUGUGGCCGGCUGGGU GUGGCGGACCGCUAUCAGGACAUAGCGUUGGCUACCCGUGAUAUUGCUGAAGAAC UUGGCGGCGAAUGGGCUGACCGCUUCCUCGUGCUUUACGGUAUCGCCGCUCCCGA UUCGCAGCGCAUCGCCUUCUAUCGCCUUCUUGACGAGUUCUUCUGAGCGGGACUC UGGGGUUCGAAAUGACCGACCAAGCGACGCCCAACCUGCCAUCACGAGAUUUCGAU UCCACCGCCGCCUUCUAUGAAAGGUUGGGCUUCGGAAUCGUUUUCCGGGACGCCG GCUGGAUGAUCCUCCAGCGCGGGGAUCUCAUGCUGGAGUUCUUCGCCCACCCUAG GGGGAGGCUAACUGAAACACGGAAGGAGACAAUACCGGAAGGAACCCGCGCUAUGA CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUCAUAAACGC GGGGUUCGGUCCCAGGGCUGGCACUCUGUCGAUACCCCACCGAGACCCCAUUGGG GCCAAUACGCCCGCGUUUCUUCCUUUUCCCCACCCCACCCCCCAAGUUCGGGUGAA GGCCCAGGGCUCGCAGCCAACGUCGGGGCGGCAGGCCCUGCCAUAGCCUCAGGUU ACUCAUAUAUACUUUAGAUUGAUUUAAAACUUCAUUUUUAAUUUAAAAGGAUCUAG GUGAAGAUCCUUUUUGAUAAUCUCAUGACCAAAAUCCCUUAACGUGAGUUUUCGU UCCACUGAGCGUCAGACCCCGUAGAAAAGAUCAAAGGAUCUUCUUGAGAUCCUUUU UUUCUGCGCGUAAUCUGCUGCUUGCAAACAAAAAAACCACCGCUACCAGCGGUGGU UUGUUUGCCGGAUCAAGAGCUACCAACUCUUUUUCCGAAGGUAACUGGCUUCAGC AGAGCGCAGAUACCAAAUACUGUUCUUCUAGUGUAGCCGUAGUUAGGCCACCACUU CAAGAACUCUGUAGCACCGCCUACAUACCUCGCUCUGCUAAUCCUGUUACCAGUGG CUGCUGCCAGUGGCGAUAAGUCGUGUCUUACCGGGUUGGACUCAAGACGAUAGUU ACCGGAUAAGGCGCAGCGGUCGGGCUGAACGGGGGGUUCGUGCACACAGCCCAGC UUGGAGCGAACGACCUACACCGAACUGAGAUACCUACAGCGUGAGCUAUGAGAAAG CGCCACGCUUCCCGAAGGGAGAAAGGCGGACAGGUAUCCGGUAAGCGGCAGGGUC GGAACAGGAGAGCGCACGAGGGAGCUUCCAGGGGGAAACGCCUGGUAUCUUUAUA GUCCUGUCGGGUUUCGCCACCUCUGACUUGAGCGUCGAUUUUUGUGAUGCUCGUC AGGGGGGCGGAGCCUAUGGAAAAACGCCAGCAACGCGGCCUUUUUACGGUUCCUG GCCUUUUGCUGGCCUUUUGCUCACAUGUUCUUUCCUGCGUUAUCCCCUGAUUCUG UGGAUAACCGUAUUACCGCCAUGCAUUAGUUAUUAAUUAAUACGACUCACUAUAAG AAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGG CGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUG CACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCCGAGGA CAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGACUUCA CCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAGUGAAA GUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGCGUGG AAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGUGCUG ACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACGGCCU GCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUUAUCG AGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUUGCGAC AACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAAGUGUA CGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGCGGACUGAUU GCAGGUGCAGUUGGAGGAUCACUGCUGGCCGCACUGGUCAUUUGCGGCAUCGUGU AUUGGAUGAGAAGAAAGCGCAGAAAGGCCCCUAAGAGAUGAUGA185 13 AUGGGCGGAGCAGCAGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUG GACUGCACGGCGUCAGAGGAUACGAGACAACCGAGAGCGGCGAUACAGCCGUGUCC GAGGACAGCUACGAUAAGUACGCCAGCCAGAACACCAACAAAGAAUACGUGUGCGA CUUCACCGACCAGCUGAAGCCUACAGAAAGCGGCCCUAAAGUGAAAAAGUGCGAAG UGAAAGUGAACGAGCCCCUGAUCAAAGUGAAGAUCAUUUGCCCUCUGAAGGGCAGC GUGGAAAAGCUGUACGACAAUAUCGAGUACGUGCCCAAGAAAAGCCCUUACGUGGU GCUGACCAAAGAGGAAACAAAGCUGAAAGAGAAGCUGCUGAGCAAGCUGAUCUACG GCCUGCUGAUUAGCCCUACCGUGAACGAGAAAGAGAACAACUUCAAAGAGGGCGUU AUCGAGUUCACCCUGCCUCCAGUGGUGCACAAGGCCACCGUGUUCUACUUCAUUU GCGACAACAGCAAGACCGAGGACGACAACAAGAAGGGCAAUAGAGGCAUCGUGGAA GUGUACGUGGAACCCUACGGCGGAGGAUCAGGUGGCGGAGGAAGCGGAGGACUGG CCAUCUAUUCUACAGUGGCCAGCAGCCUGGUGCUCCUGGUGUCUCUGGGAGCCAUCAGCUUUUGGAUGUGCAGCAACGGCAGCCUGCAGUGCAGAAUCUGUAUCUGAUGA

[0373] In some embodiments, a Pfs230 construct described herein has a nucleic acid sequence provided in Table 8. In some embodiments, a Pfs230 construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence of Table 8.III. PolyribonucleotidesA. Exemplary Polyribonucleotides Features

[0374] Polyribonucleotides described herein encode one or more Plasmodium polypeptide constructs described herein. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5'UTR of interest and / or a 3' UTR of interest. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5' cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription.1. 5' Cap

[0375] A structural feature of mRNAs is cap structure at five-prime end (5'). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5 ' to 5 '-triphosphate bridge resulting in capO structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2’-hydroxy-group (2'-OH) e.g., the 2’-hydroxyl group may be methylated to form 2’-0-Me) of the first and subsequent nucleotides producing "capl" and "cap2" five-prime ends, respectively). Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543-550, which is incorporated herein by reference in its entirety, reported that capO-mRNA cannot be translated as efficiently as capl-mRNA in which the role of 2’-O-Me in the penultimate position at the mRNA 5' end is determinant. Lack of the 2’-O-met has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Zust et al. (2011) Nature Immunology, 12:137-143, each of which is incorporated herein by reference in its entirety.

[0376] RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5'-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nucleobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0377] The term "5’-cap" as used herein refers to a structure found on the 5’-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5’- to 5’-triphosphate linkage (also referred to as Gppp or G(5’)ppp(5’)). In some embodiments, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions {e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'OMeG). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3' O methylation at a ribose (m7(3'OMeG)). It will be understood that the notation used in the above paragraph, e.g., "(m27'3'0)G" or "m7(3'OMeG)", applies to other structures described herein.

[0378] In some embodiments, providing an RNA with a 5’-cap disclosed herein may be achieved by in vitro transcription, in which a 5’-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and / or translation rate of an RNA, and / or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0379] In some embodiments, a utilized 5' caps is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A etai., and Fig. 1 of Decroly E etal, each of which is incorporated herein by reference in its entirety.See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E eta / ., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.

[0380] In some embodiments, an RNA described herein comprises a capO structure. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G). In some embodiments, such a capO structure is connected to an RNA via a 5’- to 5’-triphosphate linkage and is also referred to herein as (m7)Gppp. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 2'-position of the ribose of guanosine. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 3'-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((rm7'2'-°)G). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m27'3'0)G).

[0381] In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2' or 3' position of the ribose, and a 2'0 methylated first nucleotide in an RNA ((m2' °)Ni). In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3' position of the ribose, and a 2'0 methylated first nucleotide in an RNA ((m2'°)Ni). In some embodiments, a capl structure is connected to an RNA via a 5’- to 5’-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2' °)Ni) or (m27'3'0)Gppp(2' °)Ni), wherein Ni is as defined and described herein. In some embodiments, a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m7)Gppp(2' °)NipN2 or (m27'3'0)Gppp(2' °)NipN2, wherein each of Ni and N2 is as defined and described herein.

[0382] In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2' or 3' position of the ribose, and a 2'0 methylated first and second nucleotides in an RNA ((m2'°)Nip(m2'°)N2). In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3' position of the ribose, and a 2'0 methylated first and second nucleotide in an RNA. In some embodiments, a cap2 structure is connected to an RNA via a 5’- to 5 ’-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'0)Nip(2'0)N2) or (rm7'3 -°)Gppp(2 O)Nip(2 O)N2), wherein each of Ni and N2 is as defined and described herein.

[0383] In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure comprising Ni, wherein Ni is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*Ni, wherein Ni is as defined above and herein, and G* comprises a structure of formula (I):(I)or a salt thereof,wherein each R2and R3is -OH or -OCH3; and X is 0 or S.

[0384] In some embodiments, R2is -OH. In some embodiments, R2is -OCH3. In some embodiments, R3is -OH. In some embodiments, R3is -OCH3. In some embodiments, R2is -OH and R3is -OH. In some embodiments, R2is -OH and R3is -CH3. In some embodiments, R2is -CH3 and R3is -OH. In some embodiments, R2is -CH3 and R3is -CH3.

[0385] In some embodiments, X is 0. In some embodiments, X is S.

[0386] In some embodiments, the 5' cap is a dinucleotide capO structure e.g., (m7)GpppNi, (rm7'2'-°)GpppNi, (m27'3' °)GpppNi, (m7)GppSpNi, (m27'2' °)GppSpNi, or (m27'3' °)GppSpNi), wherein Ni is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide capO structure (e.g,, (m7)GpppNi, (rm7'2'-°)GpppNi, (m27'3'°)GpppNi, (m7)GppSpNi, (m27'2'°)GppSpNi, or (m27'3'°)GppSpNi), wherein Ni is G. In some embodiments, the 5' cap is a dinucleotide capO structure (e.g,, (m7)GpppNi, (m27'2'°)GpppNi, (m27'3'°)GpppNi, (m7)GppSpNi, (m27'2' °)GppSpNi, or (m27'3' °)GppSpNi), wherein Ni is A, U, or C. In some embodiments, the 5' cap is a dinucleotide capl structure e.g., (m7)Gppp(m2'°)Ni, (m27'2'°)Gppp(m2'°)Ni, (m27'3'°)Gppp(m2'°)Ni, (m7)GppSp(m2' °)Ni, (m27'2' °)GppSp(m2' °)Ni, or (m27'3' °)GppSp(m2' °)Ni), wherein Ni is as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of (m7)GpppG (" EcapO"), (m7)Gppp(m2' °)G (" Ecapl"), (m27'3'0)GpppG (" ARCA" or " DI"), and (m27-2'°)GppSpG ("beta-S-ARCA"). In some embodiments, the 5' cap is (m7)GpppG (" EcapO"), having a structure:OH OHor a salt thereof.

[0387] In some embodiments, the 5' cap is (m7)Gppp(m2'°)G (" Ecapl"), having a structure:OH OHor a salt thereof.

[0388] In some embodiments, the 5' cap is (m27'3'°)GpppG (" ARCA" or " DI"), having a structure:or a salt thereof.

[0389] In some embodiments, the 5' cap is (m27'2'°)GppSpG ("beta-S-ARCA"), having a structure:or a salt thereof.

[0390] In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure comprising N1PN2, wherein Ni and N2 are as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*NipN2, wherein Ni and N2 are as defined above and herein, and G* comprises a structure of formula (I):(I)or a salt thereof, wherein R2, R3, and X are as defined and described herein.

[0391] In some embodiments, the 5' cap is a trinucleotide capO structure (e.g. (m7)GpppNipN2, (m27-2°)GpppNipN2, or (m27'3'°)GpppNipN2), wherein Ni and N2 are as defined and described herein). In some embodiments, the 5' cap is a trinucleotide capl structure e.g., (m7)Gppp(m2' °)NipN2, (m27-2'0)Gppp(m2'°)NipN2, (m27'3' °)Gppp(m2' °)NipN2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2'°)Nip(m2'°)N2, (m27'2'0)Gppp(m2'°)Nip(m2'°)N2, (m27-30)Gppp(m2' °)Nip(m2' °)N2), wherein Ni and N2 are as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of (m27-3'0)Gppp(m2' °)ApG (" CleanCap AG", " CC413"), (m27'3' °)Gppp(m2'_°)GpG (" CleanCap GG"), (m7)Gppp(m2' °)ApG, (m7)Gppp(m2' °)GpG, (m27-3'0)Gppp(m25'2'0)ApG, and (m7)Gppp(m2 -°)ApU.

[0392] In some embodiments, the 5' cap is (m27-3'0)Gppp(m2' °)ApG (" CleanCap AG", " CC413"), having a structure:or a salt thereof.

[0393] In some embodiments, the 5' cap is (m27'3'0)Gppp(m2' °)GpG (" CleanCap GG"), having a structure:OHor a salt thereof.

[0394] In some embodiments, the 5' cap is (m7)Gppp(m2'°)ApG, having a structure:or a salt thereof.

[0395] In some embodiments, the 5' cap is (m7)Gppp(m2'°)GpG, having a structure:OH OHor a salt thereof.

[0396] In some embodiments, the 5' cap is (m27-3'0)Gppp(m25'2'0)ApG, having a structure:or a salt thereof.

[0397] In some embodiments, the 5' cap is (m7)Gppp(m2'°)ApU, having a structure:OH OHor a salt thereof.

[0398] In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising N1PN2PN3, wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap G*NipN2pN3, wherein Ni, N2, and N3 are as defined above and herein, and G* comprises a structure of formula (I):or a salt thereof, wherein R2, R3, and X are as defined and described herein.

[0399] In some embodiments, the 5' cap is a tetranucleotide capO structure (e.g. (m7)GpppNipN2pN3, (m27-2°)GpppNipN2pN3, or (m27-3'°)GpppNiN2pN3), wherein Ni, N2, and N3 are as defined and described herein). In someembodiments, the 5' cap is a tetranucleotide Capl structure e.g., (m7)Gppp(m2'°)NipN2pN3, (m27'2'°)Gppp(m2'_°)NipN2pN3, (m27'3' °)Gppp(m2' °)NipN2N3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide Cap2 structure e.g., (m7)Gppp(m2'°)Nip(m2'°)N2pN3, (m27-20)Gppp(m2'°)Nip(m2'°)N2pN3, (m27'3'0)Gppp(m2'°)Nip(m2'°)N2pN3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of (m27'3'°)Gppp(m2'_°)Ap(m2'°)GpG, (m27'3'0)Gppp(m2'°)Gp(m2'°)GpC, (m7)Gppp(m2'°)Ap(m2'°)UpA, and (m7)Gppp(m2'°)Ap(m2'_°)GpG.

[0400] In some embodiments, the 5' cap is (m27'3'0)Gppp(m2'°)Ap(m2'°)GpG, having a structure:O OHor a salt thereof.

[0401] In some embodiments, the 5' cap is (m27'3'0)Gppp(m2'°)Gp(m2'°)GpC, having a structure:or a salt thereof.

[0402] In some embodiments, the 5' cap is (m7)Gppp(m2'°)Ap(m2'°)UpA, having a structure:OH OHor a salt thereof.

[0403] In some embodiments, the 5' cap is (m7)Gppp(m2'°)Ap(m2'°)GpG, having a structure:OH OHO OHor a salt thereof.2. Cap Proximal Sequences

[0404] In some embodiments, a 5' UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0405] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +1 (Ni) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +2(N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide.

[0406] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence {e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity {e.g., a capl or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added {e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27'3' °Gppp(mi2' °)ApG cap is utilized, +1 (i.e., Ni) and +2 (i.e. N2) are the (mi2'°)A and G residues of the cap, and +3, +4, and +5 are added by polymerase {e.g., T7 polymerase).

[0407] In some embodiments, the 5' cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5' cap, where Ni is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5' cap is a trinucleotide cap structure {e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5' cap, wherein Ni and N2 are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5' cap is a tetranucleotide cap structure e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5' cap, wherein Ni, N2, and N3 are any nucleotide, e.g., A, C, G or U.

[0408] In some embodiments, e.g., where the 5' cap is a dinucleotide cap structure, a cap proximal sequence comprises Ni of a the 5' cap, and N2, N3, N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5' cap is a trinucleotide cap structure, a cap proximal sequence comprises Ni and N2 of a the 5' cap, and N3, N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5' cap is a tetranucleotide cap structure, a cap proximal sequence comprises Ni, N2, and N3 of a the 5' cap, and N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0409] In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is G. In some embodiments, Ni is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, Ns is A. In some embodiments, Ns is C. In some embodiments, Ns is G. In some embodiments, Ns is U. It will be understood that, each of the embodiments described above and herein (e.g., for Ni through Ns) may be taken singly or in combination and / or may be combined with other embodiments of variables described above and herein (e.g., 5' caps).

[0410] In some embodiments, a cap proximal sequence comprises Ai and G2 of the Capl structure, and a sequence comprising: A3A4U5 (SEQ ID NO: 186) at positions +3, +4 and +5 respectively of the polyribonucleotide.3. 5'UTR

[0411] In some embodiments, a nucleic acid {e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5’-UTR. In some embodiments, 5'-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements {e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5' UTR comprises multiple different sequence elements.

[0412] The term "untranslated region" or " UTR" is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5’ (upstream) of an open reading frame (5’-UTR) and / or 3’ (downstream) of an open reading frame (3’-UTR). As used herein, the terms "five prime untranslated region" or "5’ UTR" refer to a sequence of a polyribonucleotide between the 5’ end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, "5’ UTR" refers to a sequence of a polyribonucleotide that begins at the 5’ end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG)of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap.

[0413] Exemplary 5' UTRs include a human alpha globin (hAg) 5'UTR or a fragment thereof, a TEV 5' UTR or a fragment thereof, a HSP705' UTR or a fragment thereof, or a c-Jun 5' UTR or a fragment thereof.

[0414] In some embodiments, an RNA disclosed herein comprises a hAg 5' UTR or a fragment thereof.

[0415] In some embodiments, an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a 5' UTR with the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 187). In some embodiments, an RNA disclosed herein comprises a 5' UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 187).

[0416] In some embodiments, an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a 5' UTR with the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 188)(hAg-Kozak / 5’UTR). In some embodiments, an RNA disclosed herein comprises a 5' UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 188)(hAg-Kozak / 5'UTR).4. PolyA Tai!

[0417] In some embodiments, a polynucleotide e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, e.g., as described herein. In some embodiments, a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.

[0418] As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0419] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly(A) sequence (Holtkamp eta / ., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).

[0420] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term " A nucleotide" or " A" refers to adenylate.

[0421] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0422] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al, which is incorporated herein by reference in its entirety, may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. co / / and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0423] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.

[0424] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0425] In some embodiments, a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.

[0426] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 189), or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 189). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 191).

[0427] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 190), or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 190).

[0428] In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCAUAUGAC (SEQ ID NO: 192).5. 3'UTR

[0429] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3'-UTR. As used herein, the terms "three prime untranslated region," "3' untranslated region," or "3' UTR" refer to a sequence of an RNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term "3'-UTR" does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

[0430] In some embodiments, an RNA disclosed herein comprises a 3' UTR comprising an F element and / or an I element. In some embodiments, a 3' UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a Xhol site.

[0431] In some embodiments, an RNA construct comprises an F element. In some embodiments, a F element sequence is a 3'-UTR of amino-terminal enhancer of split (AES).

[0432] In some embodiments, an RNA disclosed herein comprises a 3' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a3' UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGT ATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAAC GCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 193). In some embodiments, an RNA disclosed herein comprises a 3' UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGT ATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAAC GCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 193).

[0433] In some embodiments, an RNA disclosed herein comprises a 3' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a 3' UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAG GUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAA AACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 194).

[0434] In some embodiments, an RNA disclosed herein comprises a 3' UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAG GUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAA AACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 194).

[0435] In some embodiments, a 3'UTR is an FI element as described in W02017 / 060314, which is herein incorporated by reference in its entirety.B. RNA Formats

[0436] At least three distinct formats useful for RNA compositions e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing RNA (uRNA), nucleoside-modified RNA (modRNA), and self-amplifying RNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3’ end. An ORF of an uRNA and modRNA vectors encode an antibody agent or portion thereof. An saRNA has multiple ORFs.

[0437] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0438] The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

[0439] The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

[0440] UTP (uridine 5'-triphosphate) has the following structure:oOH OH

[0441] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0442] " Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0443] Another exemplary modified nucleoside is Nl-methyl-pseudouridine (mli ), which has the structure:

[0444] Nl-methyl-pseudo-UTP has the following structure:0 0 0. II II II0— P— 0— P— 0— P— 00 o 0

[0445] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:

[0446] In some embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0447] In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

[0448] In some embodiments, the modified nucleoside is independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ip). In some embodiments, the modified nucleoside comprises Nl-methyl-pseudouridine (mlip). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ip) and Nl-methyl-pseudouridine (mlip). In some embodiments, the modified nucleosides comprise pseudouridine (ip) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise Nl-methyl-pseudouridine (mlip) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).

[0449] In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4ip), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3ip), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-metho...

Claims

CLAIMS1. A polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more antigenic portions of Plasmodium Pfs230, and a membrane-anchoring region, wherein antigenic portions of Plasmodium Pfs230 have an amino acid sequence selected from any one of SEQ ID NOs: 2 to 14 and 504 to 509 or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2 to 14 and 504 to 509.

2. The polyribonucleotide of claim 1, wherein the antigenic portion of Plasmodium Pfs230 comprises an amino acid substitution at one or more N-linked glycosylation sites, wherein the amino acid substitution prevents glycosylation at the one or more, preferably all N-linked glycosylation sites, wherein optionally the Plasmodium Pfs230 antigenic portion comprises an amino acid substitution at one or more of positions 585, 821, 829, 889, 961, 1079, and 1089, or any combination thereof, as numbered according to SEQ ID NO: 1, wherein the amino acid substitution optionally comprises an NX[T / S] to QX[T / S] substitution.

3. The polyribonucleotide of claim 1 or 2 wherein the membrane-anchoring region comprises an amino acid sequence selected from any one of SEQ ID NOs: 96 to 106, wherein preferably the transmembrane region consists of an amino acid sequence according to SEQ ID NO: 96.

4. The polyribonucleotide of any one of claims 1-3, wherein the polypeptide comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 119 to 131 or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 119 to 131.

5. The polyribonucleotide of any one of claims 1-5, having a nucleic acid sequence selected from any one of SEQ ID NOs: 132 to 185 or a nucleic acid sequence at least 90% identical to any one of SEQ ID NOs: 132 to 158.

6. An RNA construct comprising in 5' to 3' order:(i) a 5' cap;(ii) a 5' UTR;(iii) a polyribonucleotide of any one of claims 1-5;(iv) a 3' UTR; and(v) a polyA tail sequence.

7. The RNA construct of claim 6, wherein(i) the 5’ UTR consists of a nucleic acid sequence according to SEQ ID NO: 187;(ii) the 3’ UTR consists of a nucleic acid sequence according to SEQ ID NO: 194;(iii) the polyA tail sequence consists of a nucleic acid sequence according to SEQ ID NO: 190; and / or (iv) the polyribonucleotide includes modified uridines in place of all uridines, wherein preferably the modified uridines are each Nl-methyl-pseudouridine.

8. A pharmaceutical composition comprising one or more polyribonucleotides of any one of claims 1-5 or RNA constructs of claim 6 or 7.

9. The pharmaceutical composition of claim 8, comprising:(i) a second polyribonucleotide encoding a second Plasmodium polypeptide construct comprising a CSP antigen, a T cell antigen or an RH5 antigen; wherein optionally the pharmaceutical composition further comprises (ii) a third polyribonucleotide encoding a third Plasmodium polypeptide construct comprising a CSP antigen, a T cell antigen or an RH5 antigen.

10. The pharmaceutical composition of claim 8 or 9, wherein the pharmaceutical composition further comprises(i) lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the polyribonucleotides or RNA constructs are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes; and(ii) at least one pharmaceutically acceptable excipient, optionally comprising a cryoprotectant and / or an aqueous buffered solution, wherein further optionally the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPC>4, and KH2PO4.

11. A combination comprising:(i) a first pharmaceutical composition comprising a first polyribonucleotide of any of claims 1-5 or an RNA construct of claim 6 or 7; and(ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second Plasmodium polypeptide construct comprising a CSP antigen, a T cell antigen or an RH5 antigen; wherein optionally the combination further comprises(iii) a third pharmaceutical composition comprising a third polyribonucleotide, wherein the third polyribonucleotide encodes a third Plasmodium polypeptide construct comprising a CSP antigen, a T cell antigen or an RH5 antigen.

12. The pharmaceutical composition of claim 9 or the combination of claim 11, wherein the second Plasmodium polypeptide construct and / or the third Plasmodium polypeptide construct:(i) comprises a CSP antigen and wherein optionally the second and / or third Plasmodium polypeptide construct comprises an amino acid sequence selected from 203, 206, 208, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 264, 269, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 305-321, 334-345,(ii) comprises a T cell antigen and wherein optionally the second and / or third Plasmodium polypeptide construct comprises an amino acid sequence selected from 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, 409, 412, 415, 418, 421, 424, 427; or(iii) comprises a Rh5 antigen and wherein optionally the second and / or third Plasmodium polypeptide construct comprises an amino acid sequence selected from 429, 431, 433, 435, 437, 439, 441, 443, 445, 447-503 and 510-525.

13. A method for treating or preventing a malaria infection, the method comprising administering a polyribonucleotide of any one of claims 1-5, an RNA construct of claim 6 or 7, a pharmaceutical composition of any one of claims 8-10 or 12 or a combination of claim 11 or 12 to a subject.

14. Use of the polyribonucleotide of any one of claims 1-5, the RNA construct of claim 6 or 7, the pharmaceutical composition of any one of claims 8-10 or 12 or the combination of claim 11 or 12 in the treatment or prevention of a malaria infection.

15. Use of the polyribonucleotide of any one of claims 1-5, the RNA construct of claim 6 or 7, the pharmaceutical composition of any one of claims 8-10 or 12 or the combination of claim 11 or 12 for the manufacture of a medicament for the treatment or prevention of a malaria infection.

16. The polyribonucleotide of any one of claims 1-5, the RNA construct of claim 6 or 7, the pharmaceutical composition of any one of claims 8-10 or 12 or the combination of claim 11 or 12 for use in a method for treating or preventing a malaria infection.

17. A polypeptide encoded by a polyribonucleotide of any one of claims 1-5.

18. A host cell comprising a polyribonucleotide of any one of claims 1-5 or an RNA construct of claim 6 or 7.

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