Plasmodium falciparum malaria immunogens

Abstract

Non-naturally occurring circumsporozoite protein (CSP) antigens are provided, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 1-38, or an antigenic fragment thereof, as are fusion proteins thereof, nanoparticles thereof, and methods for their use in treating or limiting development of a Plasmodium falciparum infection.

Description

[0001]PLASMODIUM FALCIPARUM MALARIA IMMUNOGENS Sequence Listing Statement A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on July 28, 2025 having the file name “25- 0961-WO.xml” and is 63,715 bytes in size. Background The World Health Organization (WHO) reported that malaria caused an estimated 249 million cases and 608,000 deaths in 2022, with a majority of these deaths being caused by Plasmodium falciparum in children under 5 years of age. Public health efforts have reduced these numbers over the last two decades through use of interventions such as insecticide-treated bed nets and widespread seasonal malaria chemoprevention (SMC), but these successes are threatened by the emergence of growing insecticide and drug resistance. This necessitates the need for the development of additional interventions such as highly efficacious vaccines to mitigate this significant public health burden. Summary In a first aspect, the disclosure provides non-naturally occurring circumsporozoite protein (CSP) antigen, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or an antigenic fragment thereof. In one embodiment, the epitope domain is conserved relative to the reference sequence. In one embodiment, the disclosure provides fusion proteins, comprising (a) the non-naturally occurring CSP antigen of any embodiment; (b) one or more functional domains; and (c) optionally, an amino acid linker connecting the CSP antigen and the one or more functional domains. In one embodiment, the one or more functional domains comprises a protein nanoparticle component. In one such embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:39, wherein optional resides may be present or may be deleted, optionally wherein the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO:41-44, wherein optional resides may be present, may be substituted, or may be deleted. In another embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:45, wherein optional resides may be present or may be deleted. In a further embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of selected from SEQ ID NO:46 , optionally comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:47, wherein optional resides may be present, may be substituted, or may be deleted. In another embodiment, the disclosure comprises nanoparticles comprising multiple copies of the antigen or fusion protein of any embodiment herein. In another aspect, the disclosure provides nucleic acids encoding the non-naturally occurring CSP antigen or fusion protein of any embodiment herein. In various embodiments, the nucleic acid may comprise RNA, such as mRNA; the RNA may comprise nucleoside- modified RNA, including but not limited to N1-methylpseudouridine-5’-triphosphate containing RNA; the RNA may comprise self-amplifying mRNA; and / or the nucleic acid may encode a poly A tail (DNA) or comprise a poly A tail (RNA), and / or the nucleic acid may encode a 5’ UTR and / or a 3’ UTR (DNA) or comprise a 5’ UTR and / or a 3’ UTR (RNA). The disclosure also provides expression vectors comprising the nucleic acid of any embodiment herein operatively linked to a suitable control element, such as a promoter; host cells comprising the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid or expression vector of any embodiment herein, and pharmaceutical composition or vaccine, comprising the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, or host cell of embodiment herein, and a pharmaceutically acceptable carrier. In another aspect, the disclosure provides methods to treat or limit development of a Plasmodium falciparum infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the infection of the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, host cell, vaccine, and / or pharmaceutical composition of any embodiment herein. In one embodiment, the method treats or limits development of malaria in the subject. Description of the Figures Figure 1. Design of CIS43 epitope germline-targeting immunogens. (a) Pipeline overview of designing a scaffold to support the CIS43 epitope (PDB ID: 6B5M). (b) Input structures used for RFdiffusionTM. (c) Scatter plots of all AlphaFold2TMpredicted MPNN-FR designs from full de novo and fixed sets. Dashed lines indicate cutoffs used for filtering. Figure 2. Biochemical characterization of CIS43 epitope immunogens. (a) SDS- PAGE of IMAC eluates. (b) Overlaid SEC chromatograms of all 50 designs. (c) BLI AUC for 29 designs at 100 nM concentration tested for binding against ProATMtips loaded with either iGL-CIS43 or CIS42. Dashed lines indicate mean AUC values. (d) BLI AUC for 29 designs at 100 nM concentration tested for binding against ProATMtips loaded with either iGL-CIS43 or CIS43. Figure 3. Design and characterization of immunogen-fused nanoparticles. (a) AlphaFold2TMpredicted models for 14-glCIS43-I53-50 (top) and 14-glCIS43-T33_dn10 (bottom). Images are not to scale. (b) BLI curves of nanoparticle immunogens against iGL- CIS43 and CIS42. Immunogen was titrated from 100 nM immunogen, 2-fold down to 1.56 nM. (c) SPR sensorgrams of nanoparticle immunogens across a concentration range of 100 to 0.09 nM against iGL-CIS43 and CIS42. Figure 4. Characterization of designed immunogens targeting epitopes from mAb L9 and 2541. (a) BLI AUCs for L9 immunogens tested against on-target L9 and a potentially cross-reactive mAb 224. (b) Negative-stain EM volume reconstruction of 19-ifL9 in complex with two L9 Fabs. (c) BLI AUCs for 2541 immunogens tested against on-target 2541, cross- reactive 1210, and off-target 395 and 580. Figure 5. ELISA screening. (a, b) ELISA curves for designs plated and tested against iGL-CIS43 or CIS42 IgG titrated from 4 to 0.004 g / mL. (c) ELISA AUC plotted for all designs. Figure 6. Affinity determination of down-selected designs. (a) BLI curves to determine affinity of down-selected designs against iGL-CIS43 or, (b) CIS42. Figure 7. Characterization of 14-glCIS43 nanoparticle immunogens. (a) SDS-PAGE of SEC purified nanoparticle immunogens. (b) SEC chromatograms for 14-glCIS43- T33_dn10 and 14-glCIS43-I53-50. (c) Negative-stain electron micrographs for 14-glCIS43- T33_dn10 (92k magnification) and 14-glCIS43-I53-50 (57k magnification). Detailed Description All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney.1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp.109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319–322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX). As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides and fusion proteins disclosed herein, 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and / or the C- terminus of the polypeptide or fusion protein while retaining activity. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. In a first aspect, the disclosure provides non-naturally occurring circumsporozoite protein (CSP) antigens comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or antigenic fragments thereof. As described herein, these antigens are highly stable, present specific conformations of flexible CSP repeat epitopes, bind to their target antibody with equal or greater affinity as compared to their native epitopes, and show reduced or no binding to less protective antibodies, and thus limit eliciting of off-target antibodies. These immunogens can be used as effective vaccines to induce highly potent and protective anti-CSP repeat antibody responses. The amino acid sequences for the non-naturally occurring CSP antigens are providedin Table 1. Underlined residues are the epitope domain, and bolded amino acids indicateresidue differences from the native epitope as discussed herein. Table 1 In one embodiment, the epitope domain is conserved relative to the reference sequence. As noted above, the epitope domain are the underlined residues in Table 1. In various embodiment, the CSP antigen comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or an antigenic fragment thereof. In one embodiment, the CSP antigens comprise an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-9, or an antigenic fragment thereof. The CSP antigens of SEQ ID NO:1-9 were modified relative to antibody glCIS43 (native epitope: NPDPNANPNVDPN (SEQ ID NO: 59)). In another embodiment, the CSP antigens comprise an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:10- 25, or an antigenic fragment thereof. The CSP antigens of SEQ ID NO:10-25 were modified relative to antibody L9; (native epitope: NPNVDPNANPNVD (SEQ ID NO: 60)). In a further embodiment, the CSP antigens comprise an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:26- 38, or an antigenic fragment thereof. The CSP antigens of SEQ ID NO:26-38 were modifiedrelative to mAb2541 native epitope: NPNANPNA (SEQ ID NO: 61) or DPNANPNA (SEQID NO: 62)). In all embodiments herein, the antigenic fragment comprises the epitope domain, i.e.: underlined residues as shown in Table 1. In some embodiments, the antigenic fragment comprises at least 60 amino acids of the reference sequence, or comprises at least 70 amino acids of the reference sequence, and the epitope domain is conserved (i.e., identical) relative to the reference sequence. In some embodiments, an antigenic fragment of the CSP antigen of any one of SEQ ID NO:1-9 is at least 70 amino acids in length, including the epitope domain. In other embodiments, an antigenic fragment of the CSP antigen of any one of SEQ ID NO:10-25 is at least 60 amino acids in length, including the epitope domain. In some embodiments, the non-naturally occurring CSP antigen comprises a signal peptide at the amino-terminus to facilitate secretion in eukaryotic cells. Any signal peptide may be used as appropriate for an intended use. In various embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:53-58. SEQ ID NO:53: MDRAKLLLLLLLLLLPQAQA SEQ ID NO:54: MWWRLWWLLLLLLLLWPMVWA SEQ ID NO:55: MGWSCIILFLVATATGVHS SEQ ID NO:56: MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA SEQ ID NO:57: MKLCILLAVVAFVGLSLG SEQ ID NO:58: MKTIIALSYIFCLVLG In another embodiment of the CSP antigens of any embodiment disclosed herein, fusion proteins are provided, comprising: (a) the non-naturally occurring CSP antigen of any embodiment; (b) one or more functional domains; and (c) optionally, an amino acid linker connecting the CSP antigen and the one or more functional domains. Any one or more functional domains may be used as appropriate for an intended purpose, including but not limited to a detectable polypeptide (such as green fluorescent protein and the like), a moiety to enhance half-life of the antigen in the blood, a domain to permit isolation of the antigen, and a protein nanoparticle component. In one embodiment, the one or more functional domains comprises a protein nanoparticle component. In this embodiment, the antigens are fused to a protein nanoparticle component, permitting display of multiple copies of the antigens on the resulting nanoparticle, permitting enhanced antigen valency that promotes stronger immune responses upon immunization with the antigen- bearing nanoparticles. Any protein nanoparticle component may be used as suitable for an intended nanoparticle system. The CSP antigen and the protein nanoparticle component may be in any orientation in the fusion protein as suitable for the particular protein nanoparticle component. In one embodiment, the protein nanoparticle component is C-terminal to the non-naturally occurring CSP antigen. In another embodiment, the protein nanoparticle component comprises a trimer-forming polypeptide. In a further embodiment, the protein nanoparticle component comprises a tetrahedral-forming polypeptide. In a first embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:39, wherein optional resides may be present or may be deleted. >I53-50A.OG-cys-mut (M)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKE KGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMK LGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKG TPDEVREKAKAFVEKIRGATE (SEQ ID NO:39) Residues in parentheses are optional Residues in parentheses are optional In various embodiments of this first embodiment, the fusion protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO:41-44, wherein optional resides may be present, may be substituted, or may be deleted. >14-glCIS43-I53-50A.OG-cys-mut SERRDKIRKLRIELGEAIEEVQEKYADDPRLKDYPDLNANPNVDPGIETLIDIAKDL VKFLKEAGAPKELIERAEKIKKELEEIK(GGSGGSGS)EKAAKAEEAARKMEELFKKHKIVA VLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQA RKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGP QFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKI RGATE (SEQ ID NO:41) Residues in parentheses are optional >19-ifL9-I53-50A.OG-cys-mut KKEEEEKEKLAKEVAEKLRKRGIKINPNVSPTINPNVDASLYDDLTLEQTVLAWEEGKVSKE DAYRAALELKKRE(GGSGGSGS)EKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAV AVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPH LDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKF VPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE (SEQ ID NO:42) Residues in parentheses are optional. >21-ifL9-I53-50A.OG-cys-mut EKSFGQISNEINNLIQQAIAEGKSHYFNPNVNPLSVNPNVDPLLQKDVIESNILALS KGDPELAKKLQELLDKAEELKKK(GGSGGSGS)EKAAKAEEAARKMEELFKKHKIVAVLRAN SVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVE SGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKA MKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE (SEQ ID NO:43) Residues in parentheses are optional. >28-ifL9-I53-50A.OG-cys-mut EEKLKELEEKLKEYKKKIEELGGTLEINPNVNPNGLNPNVSESEQGVDVVVNHYALQ AKKLEEEIKKLKEEK(GGSGGSGS)EKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEK AVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVS PHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNV KFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE (SEQ ID NO:44) Residues in parentheses are optional. In these first embodiments, the optional amino acid linker is shown in parentheses and bold font. The linker can be present or deleted, and can also be substituted with any other linker as deemed appropriate for an intended use. In a second embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:45, wherein optional resides may be present or may be deleted. >I53_dn5B (MEE)AELAYLLGELAYKLGEYRIAIRAYRIALKRDPNNAEAWYNLGNAYYKQGRYREAIEY YQKALELDPNNAEAWYNLGNAYYERGEYEEAIEYYRKALRLDPNNADAMQNLLNAKMREE (SEQ ID NO:45) Residues in parentheses are optional In a third embodiment, the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of selected from SEQ ID NO:46. >T33_dn10A* EEAELAYLLGELAYKLGEYRIAIRAYRIALKRDPNNAEAWYNLGNAYYKQGDYDEAI EYYQKALELDPNNAEAWYNLGNAYYKQGDYDEAIEYYEKALELDPENLEALQNLLNAMDKQG (SEQ ID NO: 46) In one such embodiment of the third embodiment of fusion proteins, the fusion protein comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:47, wherein optional resides may be present, may be substituted, or may be deleted. >14-glCIS43_T33_dn10A SERRDKIRKLRIELGEAIEEVQEKYADDPRLKDYPDLNANPNVDPGIETLIDIAKDL VKFLKEAGAPKELIERAEKIKKELEEIK(GGSGGSGS)EEAELAYLLGELAYKLGEYRIAIR AYRIALKRDPNNAEAWYNLGNAYYKQGDYDEAIEYYQKALELDPNNAEAWYNLGNAYYKQGD YDEAIEYYEKALELDPENLEALQNLLNAMDKQGGS (SEQ ID NO:47) Residues in parentheses are optional In this third embodiment of fusion proteins, the optional amino acid linker is shown in parentheses and bold font. The linker can be present or deleted, and may also be substituted with any other linker as deemed appropriate for an intended use. In another embodiment, the disclosure provides nanoparticles comprising multiple copies (2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 60, or more) of the fusion protein of any embodiment of the disclosure. The fusion proteins may all comprise an identical CSP antigen, or may comprise different CSP antigens of the present disclosure (i.e. “mosaic” nanoparticles). In one embodiment, the nanoparticles comprise: (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:48, and, (b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to the first embodiment of fusion proteins detailed above, wherein optional resides may be present, may be substituted, or may be deleted wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen. I53-50B.4PT1 MNQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHA RTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSK AHTLLFLALFAVKGMEAARACVEILAAREKIAAGSLE (SEQ ID NO:48) In another embodiment, the nanoparticles comprise (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:49-51, wherein optional residues may be present or may be deleted, and, (b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to the second embodiment of fusion proteins detailed above; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen. I53_dn5A* (MG)KYDGSKLRIGILHARWNAEIILALVLGALKRLQEFGVKRENIIIETVPGSFEL PYGSKLFVEKQKRLGKPLDAIIPIGVLIKGSTMHFEYICDSTTHQLMKLNFELGIPVIFGVL TCLTDEQAEARAGLIEGKMHNHGEDWGAAAVEMATKFN (SEQ ID NO:49) Residues in parentheses are optional; >I53_dn5A.1 (MG)KYDGSKLRIGILHARGNAEIILALVLGALKRLQEFGVKRENIIIETVPGSFEL PYGSKLFVEKQKRLGKPLDAIIPIGVLIRGSTPHFDYIADSTTHQLMKLNFELGIPVIFGVI TADTDEQAEARAGLIEGKMHNHGEDWGAAAVEMATKFN (SEQ ID NO:50) Residues in parentheses are optional; and >I53_dn5A.2 (MG)KYDGSKLRIGILHARGNAEIILELVLGALKRLQEFGVKRENIIIETVPGSFEL PYGSKLFVEKQKRLGKPLDAIIPIGVLIRGSTAHFDYIADSTTHQLMKLNFELGIPVIFGVL TTESDEQAEERAGTKAGNHGEDWGAAAVEMATKFN (SEQ ID NO:51) Residues in parentheses are optional. In a further embodiment, the nanoparticles comprise: (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52 and, (b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to the third embodiment of fusion proteins detailed above; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen. >T33_dn10B* IEEVVAEMIDILAESSKKSIEELARAADNKTTEKAVAEAIEEIARLATAAIQLIEALAKNLA SEEFMARAISAIAELAKKAIEAIYRLADNHTTDTFMARAIAAIANLAVTAILAIAALASNHT TEEFMARAISAIAELAKKAIEAIYRLADNHTTDKFMAAAIEAIALLATLAILAIALLASNHT TEKFMARAIMAIAILAAKAIEAIYRLADNHTSPTYIEKAIEAIEKIARKAIKAIEMLAKNIT TEEYKEKAKKIIDIIRKLAKMAIKKLEDNRT (SEQ ID NO: 52) In all of these embodiments, a plurality (2, 3, 4, 5, 6, or more) of first polypeptides self-assemble to form a first assembly, and a plurality (2, 3, 4, 5, 6, or more) of second polypeptides self-assemble to form a second assembly. A plurality of these first and second assemblies then self-assemble non-covalently via the designed interfaces to produce thenanostructures. The number of first polypeptides in the first assemblies may be the same ordifferent than the number of second polypeptides in the second assemblies. In one exemplary embodiment, the first assembly comprises trimers of the first polypeptides, and the second assembly comprises pentamers of the second polypeptides. In another aspect, the disclosure provides nucleic acids encoding the non-naturally occurring CSP antigen or fusion protein of any embodiment or combination of embodiments herein. The nucleic acid sequence may comprise RNA (such as mRNA) or DNA. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the proteins of the disclosure. In one embodiment, the nucleic acid comprises mRNA. The mRNA may be modified as appropriate, for example, for use as a vaccine. In one embodiment, the RNA comprises nucleoside-modified RNA, including but not limited to N1-methylpseudouridine-5’- triphosphate containing RNA. In another embodiment, the mRNA comprises self-amplifying mRNA. In a further embodiment, the nucleic acid encodes a poly A tail (DNA) or comprises a poly A tail (RNA). In another embodiment, the nucleic acid encodes a 5’ UTR and / or a 3’ UTR (DNA) or comprises a 5’ UTR and / or a 3’ UTR (RNA). The disclosure also provides expression vectors comprising the nucleic acid of any embodiment herein operatively linked to a suitable control element, such as a promoter. "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. "Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence is still considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited to plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). In one aspect, the present disclosure provides cells comprising the non-naturally occurring CSP antigen, the fusion protein, the nanoparticle, the nucleic acid, and / or the expression vector of any embodiment or combination of embodiments of the disclosure, wherein the cells can be either prokaryotic or eukaryotic, such as mammalian cells. In one embodiment, the cells may be transiently or stably transfected with the nucleic acids or expression vectors of the disclosure. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art. A method of producing a polypeptide according to the disclosure is an additional part of the disclosure. The method comprises the steps of (a) culturing a host according to this aspect of the disclosure under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The disclosure also provides pharmaceutical compositions and vaccines, comprising the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, or host cell of any preceding claim and a pharmaceutically acceptable carrier. The compositions / vaccines may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, like glycine. In yet other embodiments, the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate- 85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition additionally includes a stabilizer, e.g., a molecule which substantially prevents or reduces chemical and / or physical instability of the nanostructure, in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride. In some embodiments, the pharmaceutical composition or vaccine comprises a nucleic acid encoding a polypeptide or fusion protein of any embodiment herein. In some such embodiments, the pharmaceutically acceptable carrier comprises a cationic lipid such as a liposome, or a cationic protein such as protamine. This embodiment is particularly useful with nucleic acid / mRNA vaccines of the disclosure. The compositions / vaccines may further comprise one or more other agents suitable for an intended use, including but not limited to adjuvants to stimulate the immune system generally and improve immune responses overall. Any suitable adjuvant can be used. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Exemplary adjuvants include, but are not limited to, Adju-PhosTM, AdjumerTM, albumin-heparin microparticles, Algal Glucan, Algammulin, Alum, Antigen Formulation, AS-2 adjuvant, autologous dendritic cells, autologous PBMC, AvridineTM, B7-2, BAK, BAY R1005, Bupivacaine, Bupivacaine-HCl, BWZL, Calcitriol, Calcium Phosphate Gel, CCR5 peptides, CFA, Cholera holotoxin (CT) and Cholera toxin B subunit (CTB), Cholera toxin A1-subunit-Protein A D-fragment fusion protein, CpG, CRL1005, Cytokine-containing Liposomes, D-Murapalmitine, DDA, DHEA, Diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / Alum Complex, Fowlpox, Freund's Complete Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN-gamma in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL- 2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, ImiquimodTM, ImmTherTM, Immunoliposomes Containing Antibodies to Costimulatory Molecules, Interferon-gamma, Interleukin-1 beta, Interleukin-12, Interleukin-2, Interleukin-7, ISCOM(s)TM, Iscoprep 7.0.3TM, Keyhole Limpet Hemocyanin, Lipid-based Adjuvant, Liposomes, Loxoribine, LT(R192G), LT-OA or LT Oral Adjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL.TM., MPL-SE, MTP-PE, MTP-PE Liposomes, Murametide, Murapalmitine, NAGO, nCT native Cholera Toxin, Non-Ionic Surfactant Vesicles, non-toxic mutant E112K of Cholera Toxin mCT-E112K, p- Hydroxybenzoique acid methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptomer- NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic L121, PMMA, PODDSTM, Poly rA: Poly rU, Polysorbate 80, Protein Cochleates, QS-21, Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribilike adjuvant system (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Span 85, Specol, Squalane 1, Squalene 2, Stearyl Tyrosine, Tetanus toxoid (TT), TheramideTM, Threonyl muramyl dipeptide (TMDP), Ty Particles, and Walter Reed Liposomes. Selection of an adjuvant depends on the subject to be treated. Preferably, a pharmaceutically acceptable adjuvant is used. In another aspect, the disclosure provides methods to treat or limit development of a Plasmodium falciparum infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the infection of the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, host cell, vaccine, and / or pharmaceutical composition of any embodiment or combination of embodiments herein. In one embodiment, the subject is not infected with Plasmodium falciparum, wherein the administering elicits an immune response against Plasmodium falciparum in the subject that limits development of a Plasmodium falciparum infection in the subject. As used herein, "limiting development" includes, but is not limited to accomplishing one or more of the following: (a) generating an immune response (antibody and / or cell-based) to Plasmodium falciparum in the subject; (b) generating neutralizing antibodies against Plasmodium falciparum in the subject (b) limiting build-up of Plasmodium falciparum titer in the subject after exposure to Plasmodium falciparum; and / or (c) limiting or preventing development of Plasmodium falciparum infection symptoms after infection. Exemplary symptoms of Plasmodium falciparum infection include, but are not limited to, fever, chills, headache, muscle aches. respiratory distress, anemia, jaundice, and malaria. In another embodiment, the subject is infected with Plasmodium falciparum, wherein the administering elicits an immune response against Plasmodium falciparum in the subject that treats a Plasmodium falciparum infection in the subject. As used herein, "treat" or "treating" includes, but is not limited to accomplishing one or more of the following: (a) reducing Plasmodium falciparum titer in the subject; (b) limiting any increase of Plasmodium falciparum titer in the subject; (c) reducing the severity of Plasmodium falciparum symptoms; (d) limiting or preventing development of Plasmodium falciparum symptoms after infection; (e) inhibiting worsening of Plasmodium falciparum symptoms; (f) limiting or preventing recurrence of Plasmodium falciparum symptoms in subjects that were previously symptomatic for Plasmodium falciparum infection; and / or (e) improving survival. In one embodiment, the method treats or limits development of malaria in the subject. The administering may comprise any regimen as deemed appropriate by attending medical personnel. In one embodiment, the administering comprises administering a first dose and a second dose, wherein the second dose is administered about 2 weeks to about 12 weeks, or about 4 weeks to about 12 weeks after the first dose is administered. As used herein, an “effective amount” refers to an amount of the immunogenic composition that is effective for treating and / or limiting Plasmodium falciparum infection. The antigens, fusion proteins, nanoparticles, nucleic acids, expression vector, cell composition, and or pharmaceutical composition, or vaccine of any embodiment herein are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. Polypeptide compositions may also be administered via microspheres, liposomes, immune-stimulating complexes (ISCOMs), or other microparticulate delivery systems or sustained release formulations introduced into suitable tissues (such as blood). Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range may, for instance, be 0.1 g / kg-100 mg / kg body weight of the polypeptide or nanoparticle thereof. The composition can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by attending medical personnel. In one embodiment, the administering comprises administering a first dose and a second dose of the immunogenic composition, wherein the second dose is administered about 2 weeks to about 12 weeks, or about 4 weeks to about 12 weeks after the first does is administered. In various further embodiments, the second dose is administered about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose. In another embodiment, three doses may be administered, with a second dose administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose, and the third dose administered about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 weeks after the second dose. In one embodiment, the immune response comprises generation of neutralizing antibodies against Plasmodium falciparum. The subject may be any subject that can benefit from the methods of the disclosure, including but not limited to a human subject. Examples After the bloodmeal of an infected Anopheles mosquito, the P. falciparum sporozoite stage must travel through the skin and blood into the liver to establish an infection, which then progresses to its erythrocytic stage and infects red blood cells (RBCs), and is where symptomatic malaria illness occurs. As such, many vaccine efforts aim to intervene at the sporozoite stage to provide sterilizing immunity and break this life cycle chain. The primary target of pre-erythrocytic vaccines is the circumsporozoite protein (CSP), which is the most abundant protein on the surface of sporozoites and is important for its development, motility, and infection of hepatocytes. CSP is an intrinsically disordered protein composed of three domains: an N-terminal domain with a conserved KLKQP (Region I) sequence found at its C-terminal end, a central tetrapeptide repeat domain starting with a single junctional sequence (NPDP) and consisting of downstream major (NANP) and minor (NVDP) repeats, and a structured C-terminal domain that adopts an alpha-thrombospondin repeat fold with a GPI anchor. The world’s most advanced malaria vaccines, RTS,S / AS01 and R21 / Matrix-M, display only major repeats and the C-terminal domain on HepBsAg virus-like particles (VLPs), and have been recommended by the WHO for use in children and infants in Africa for the prevention of malaria with efficacy ranging from 36-77%. Though these vaccines are likely to further reduce malaria burden, there are concerns with their antibody durability and quality. Solved structures of monoclonal antibodies (mAbs) isolated from individuals immunized with RTS,S or attenuated sporozoite vaccines have revealed that protective mAbs primarily target the CSP major, minor, and junctional repeats, typically in type I beta-turn or pseudo310 turn conformations. Clinical trials involving passive immunization of two of these mAbs, the NPDP-targeting CIS43LS and NVDP-targeting L9LS, have shown significant efficacy against malaria infection in the field and provide a proof-of-concept that antibodies targeting these two epitopes can provide protection at relatively low antibody titers. Based on these results, numerous groups have developed preclinical vaccine candidates that include these epitopes in hopes that they can improve the efficacy of CSP vaccines. However, current transgenic parasite challenge mouse models have not shown a significant improvement in protection as compared to R21 or RTS,S-like vaccines. One possible reason for this is that CSP repeats are inherently flexible, and thus simple genetic fusion of these known epitopes may not be enough to generate antibodies that target protective epitope conformations. For example, protective mAb CIS43 targets the junctional NPDPNANPNVDP (SEQ ID NO: 63) sequence, but non-protective mAb CIS42 binds the same sequence in a different conformation, so it is likely that immunization with this sequence would generate a polyclonal response that induces both types of antibodies. Thus, one strategy would be to “lock” the CIS43 epitope in a stable conformation and thus disallow binding of CIS42. Here we design immunogens that display stabilized protective CSP repeat epitopes of CIS43, L9, 224, and 2541. These immunogens are highly stable, bind to their target antibody with equal or greater affinity as compared to their native epitopes, and show reduced or no binding to less protective antibodies. These immunogens can be used as effective vaccines to induce highly potent and protective anti-CSP repeat antibody responses. Results Design and characterization of CSP repeat immunogens: We hypothesized that we could use RFDiffusionTMto generate a backbone that would buttress the CIS43 epitope (NPDPNANPNVDPN (SEQ ID NO: 59)) and stabilize its conformation, thus disallowing the generation of off-target antibodies such as CIS42 (Figure 1a). We first used the crystal structure of CIS43 in complex with its epitope and used three inputs: the native peptide and two “hydrophobic” inputs where residues 3, 5, or 7 were replaced with leucines to promote the packing of secondary structure on the side of the epitope that faced away from the antibody paratope (Figure 1b). RFdiffusionTMwas run using these three inputs in complex with a CIS43 antibody model that was mutated to its inferred-germline sequence (Kratochvil, 2021) and we tested multiple parameter combinations in order to generate a significant number of prospective backbones. A total of 14102 backbones were generated and were filtered on 1) 0.3Å RMSD of the target motif in the diffused backbone to the reference structure, and 2) zero antibody backbone clashes with the newly diffused immunogen. 1880 total designs from the native peptide input and 2838 from the hydrophobic peptide inputs passed these filters and were used for subsequent sequence design using a ProteinMPNNTMand RosettaTMFastRelaxTMprotocol with two subsets for design: 1) “full de novo,” whereby design was allowed on the entire diffused immunogen, including the epitope; and 2) “fixed,” where the entire epitope was fixed, except for “hydrophobic” or masked inputs which allowed design at mutated positions (e.g., residues 3, 5, or 7 of the epitope). The generated sequences were then predicted with an AlphaFold2TMinitial guess protocol and filtered on >80 pLDDT of the immunogen and <10 pAE of interaction with CIS43 (Figure 1c) for a total of 44 designs from the full de novo set (0.46% passing) and 6 designs from the fixed set (0.06% passing). Passing designs were expressed in BL21(DE3) E. coli and purified by immobilized metal affinity chromatography (IMAC). All designs contained a significant amount of purified protein at the expected molecular weight (MW) in their IMAC eluate as assessed by SDS-PAGE, except for MDL90-glCIS43 (Figure 2a). We further purified IMAC eluates by size exclusion chromatography (SEC) and found 29 of 50 designs (58%) were monodisperse with a significant peak at their expected retention volumes (Figure 2b). SEC fractions were collected and were pure as assessed by SDS-PAGE (Figure 2b), and 280 nm absorbance was measured to determine concentrations for binding experiments. We performed biolayer interferometry (BLI) using ProteinATMtips to assess if our CIS43 immunogens could bind to IgG of inferred-germline of CIS43, while having little or no binding to CIS42. A soluble and stable full-length CSP, SAmut CSP, and a construct with a truncated repeat region that contained primarily junctional and minor repeat epitopes, SAmut 5 / 3, were used as controls. As an initial screen, loaded IgG tips were dipped into 100 nM of immunogen or control to determine if there was reactivity to either mAb. Our controls behaved as expected with a binding shift observed for both SAmut CSP and SAmut 5 / 3 against either iGL-CIS43 or CIS42. Significant binding shifts, analyzed as an area under the curve (AUC) greater than the mean iGL-CIS43 AUC of all immunogens, were observed in 10 of the 29 designs tested (Figure 2c). Of these 10 designs (designs 14, 32, 37, 20, 24, 6, 23, 22, 31, 27), only two had CIS42 AUCs greater than the mean CIS42 AUC indicating detectable off-target binding at this immunogen concentration (Figure 2c). This same screen was performed against mature CIS43 in comparison to iGL-CIS43 and we found that for all constructs, mature CIS43 bound with a higher shift as compared to iGL-CIS43 at 100 nM of immunogen (Figure 2d). Enzyme linked immunosorbent assay (ELISA) using iGL-CIS43 and CIS42 mAbs tested for binding against plated immunogens also confirmed the results and general trends of the BLI screen, showing potent binding to iGL-CIS43, though a higher level of CIS42 reactivity was observed for certain designs (32, 31, 22) as compared to the BLI experiment (Figure 5). Based on this data, we down-selected seven designs that had high-specificity for iGL- CIS43 (14, 20, 22, 23, 24, 31, 32) and one design (37) that had appreciable binding to CIS42 for affinity measurements by BLI across a concentration range of 100 nM to 1.56 nM. iGL- CIS43 affinities for all immunogens tested were close to or better than the reported affinities for NPDP19 or Peptide21 (27 and 635 nM, respectively; Table 1, Figure 6). Background level binding for CIS42 was observed at this concentration range for all but design 37, consistent with the previous screening experiments. Altogether these results show that CIS43 epitope-specific immunogens designed with RFdiffusionTMand MPNN-FR can bind their target antibody with high specificity. Table 1 Design and characterization of immunogen-fused nanoparticles: We decided to move forward with design 14, hereafter referred to as 14-glCIS43, for genetic fusion on two-component nanoparticle platforms: the tetrahedral T33-dn10 and icosahedral I53-50.14-glCIS43 was selected because it was highly specific toward iGL- CIS43 and had no reactivity to CIS42 in both the BLI and ELISA experiments (Figure 2c, Figure 6).14-glCIS43 was genetically fused via an 8GS linker to trimeric components T33- dn10A and I53-50A (Figure 3a). These constructs were expressed in E. coli, purified by IMAC and SEC, their purity was verified by SDS-PAGE, and concentrations obtained by UV-vis spectroscopy (Figure 7). Nanoparticles for 14-glCIS43-T33-dn10 were assembled by mixing 14-glCIS43-T33-dn10A and T33-dn10B in a 1:1 molar ratio.14-glCIS43-I53-50 nanoparticles were assembled by mixing 14-glCIS43-I53-50A and I53-50B.4PT1 in a 1.1:1 molar ratio. Assembled nanoparticles were purified by SEC to remove residual components and both had peaks at their expected retention volumes (Figure 7). We performed UV-vis spectroscopy to measure the concentrations of the assembled nanoparticles. We then tried a BLI titration series of each nanoparticle against iGL-CIS43 and CIS42 to see if these multivalent nanoparticles would change the binding profile of 14-glCIS43. Interestingly, 14-glCIS43-T33-dn10 and 14- glCIS43-I53-50 nanoparticles bound potently to iGL-CIS43 IgG as expected, but a significant binding shift against CIS42 was observed for 14-glCIS43-I53-50 (Figure 3b). This could be due to avidity effects as the T33-dn10 nanoparticles display 12 copies of 14-glCIS43, and I53-50 displays 60 copies of 14-glCIS43. We next tried surface plasmon resonance (SPR) on these nanoparticles and found that 14-glCIS43-I53-50 had strong binding to iGL-CIS43 and had weak-to-no binding for CIS42 at the same concentration range (Figure 3c). These results confirmed that 14-glCIS43 retained its binding specificity when presented on multivalent nanoparticles. Design and characterization of de novo immunogens targeting other CSP repeat epitopes: We designed additional immunogens to scaffold epitopes from potent CSP repeat mAbs L9 and 2541 (both encoded by IGHV3-33 and IGKV1-5) using the same RFdiffusionTMand MPNN-FRTMpipeline used for CIS43 (Figure 1). After purification we tested the binding of these designs by BLI and found that a significant number of designs were able to bind their target antibody, while having diminished or no binding to other CSP repeat mAbs (Figure 4 a,c). One of the designs for the mAb L9 epitope, 19-ifL9, was examined by negative-stain electron microscopy in complex with L9 antigen-binding fragments (Fabs) to determine if it recapitulated the same Fab binding angles as the original L9 epitope (Figure 4b). Volume reconstructions showed that this was indeed the case, with two L9 Fabs binding at an angle reminiscent of the original structure. These data show that other CSP repeat epitopes can be scaffolded using deep-learning enabled protein design, and successfully bind their target epitopes with high specificity as compared to other off-target CSP repeat mAbs. These immunogens would likely activate and recruit their target B cell lineages if used in immunization experiments.

Claims

We claim:

1. A non-naturally occurring circumsporozoite protein (CSP) antigen, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or an antigenic fragment thereof.

2. The CSP antigen of claim 1, wherein the epitope domain is conserved relative to the reference sequence.

3. The CSP antigen of claim 1 or 2, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or an antigenic fragment thereof.

4. The CSP antigen of claim 1 or 2, comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-38, or an antigenic fragment thereof.

5. The CSP antigen of any one of claims 1-4, comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-9, or an antigenic fragment thereof.

6. The CSP antigen of any one of claims 1-4, comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:10-25, or an antigenic fragment thereof. The CSP antigen of any one of claims 1-4, comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:26-38, or an antigenic fragment thereof.

8. The CSP antigen of any one of claims 1-7, comprising an antigenic fragment of the polypeptide, wherein the antigenic fragment comprises at least 60 amino acids of the reference sequence, or comprises at least 70 amino acids of the reference sequence, and wherein the epitope domain is conserved relative to the reference sequence.

9. The non-naturally occurring CSP antigen of any one of claims 1-8, comprising a signal peptide at the amino-terminus.

10. The non-naturally occurring CSP antigen of claim 9, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:53-58.

11. A fusion protein, comprising: (a) the non-naturally occurring CSP antigen of any one of claims 1-10; (b) one or more functional domains; and (c) optionally, an amino acid linker connecting the CSP antigen and the one or more functional domains.

12. The fusion protein of claim 11, wherein the one or more functional domains comprises a protein nanoparticle component.

13. The fusion protein of claim 12, wherein the protein nanoparticle component is C- terminal to the non-naturally occurring CSP antigen.

14. The fusion protein of claim 12 or 13, wherein the protein nanoparticle component comprises a trimer-forming polypeptide or a tetrahedral-forming polypeptide.

15. The fusion protein of any one of claims 12-14, wherein the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:39, wherein optional resides may be present or may be deleted.

16. The fusion protein of claim 15, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO:41-44, wherein optional resides may be present, may be substituted, or may be deleted.

17. The fusion protein of any one of claims 12-14, wherein the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%,94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:45, wherein optional resides may be present or may be deleted.

18. The fusion protein of any one of claims 12-14, wherein the protein nanoparticle component comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of selected from SEQ ID NO:

46.

19. The fusion protein of claim 18, comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:47, wherein optional resides may be present, may be substituted, or may be deleted.

20. A nanoparticle comprising multiple copies of the antigen or fusion protein of any one of claims 1-19.

21. The nanoparticle of claim 20, comprising (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:48, and, (b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to claim 15 or 16; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen.

22. The nanoparticle of claim 20, comprising (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:49-51, wherein optional residues may be present or may be deleted, and,(b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to claim 17; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen.

23. The nanoparticle of claim 20, comprising (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52 and, (b) a plurality of second assemblies, each second assembly comprising a plurality of fusion proteins according to claim 19; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanoparticle; and wherein the nanoparticle displays on its surface an immunogenic portion of non- naturally occurring CSP antigen.

24. A nucleic acid encoding the non-naturally occurring CSP antigen or fusion protein of any one of claims 1-23.

25. The nucleic acid of claim 24, wherein the nucleic acid comprises RNA.

26. The nucleic acid of claim 25, wherein the RNA comprises mRNA.

27. The nucleic acid of claim 25 or 26, wherein the RNA comprises nucleoside-modified RNA, including but not limited to N1-methylpseudouridine-5’-triphosphate containing RNA.

28. The nucleic acid of any one of claims 25-27, wherein the RNA comprises self- amplifying mRNA.

29. The nucleic acid of any one of claims 24-28, wherein the nucleic acid encodes a poly A tail (DNA) or comprises a poly A tail (RNA).

30. The nucleic acid of any one of claims 24-29, wherein the nucleic acid encodes a 5’ UTR and / or a 3’ UTR (DNA) or comprises a 5’ UTR and / or a 3’ UTR (RNA).

31. An expression vector comprising the nucleic acid of any one of claims 24-30 operatively linked to a suitable control element, such as a promoter.

32. A host cell comprising the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid or expression vector of any one of claims 1-31.

33. A pharmaceutical composition or vaccine, comprising the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, or host cell of any one of claims 1-32, and a pharmaceutically acceptable carrier.

34. The pharmaceutical composition or vaccine of claim 33, wherein the pharmaceutically acceptable carrier comprises a cationic lipid such as a liposome, or a cationic protein such as protamine.

35. The pharmaceutical composition or vaccine of any preceding claim, further comprising an adjuvant.

36. A method to treat or limit development of a Plasmodium falciparum infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the infection of the non-naturally occurring CSP antigen, fusion protein, nanoparticle, nucleic acid, expression vector, host cell, vaccine, and / or pharmaceutical composition of any one of claims 1-35.

37. The method of claim 36, wherein the subject is not infected with Plasmodium falciparum, wherein the administering elicits an immune response against Plasmodium falciparum in the subject that limits development of a Plasmodium falciparum infection in the subject.

38. The method of claim 36 or 37, wherein the administering comprises administering a first dose and a second dose, wherein the second dose is administered about 2 weeks to about 12 weeks, or about 4 weeks to about 12 weeks the first dose is administered.

39. The method of claim any one of claims 36-38, wherein the immune response comprises generation of neutralizing antibodies against Plasmodium falciparum.

40. The method of claim 36 or 38-39, wherein the subject is infected with Plasmodium falciparum, wherein the administering elicits an immune response against Plasmodium falciparum in the subject that treats Plasmodium falciparum infection in the subject.

41. The method of any one of claims 36-40, wherein the method treats or limits development of malaria in the subject.

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