Nanoparticles comprising the junctional epitope of plasmodium falciparum circumsporozoite protein (PFCSP) and their use

Abstract

Nanoparticles including a fusion protein having multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide, and methods of producing such nanoparticles, are provided. Also provided are immunogenic compositions including the nanoparticles, and methods of inducing an immune response to Plasmodium falciparum in a subject.

Description

[0001] 4239-112675-02

[0002] NANOPARTICLES COMPRISING THE JUNCTIONAL EPITOPE OF PLASMODIUM FALCIPARUM CIRCUMSPOROZOITE PROTEIN (PFCSP) AND THEIR USE

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 789,868, filed April 16, 2025, which is incorporated by reference in its entirety.

[0005] FIELD

[0006] This disclosure relates to malaria vaccines, immunogenic compositions, and their use.

[0007] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0008] The Sequence Listing is submitted as an XML file named “Sequence.xml,” created on March 13, 2026, 91,232 bytes, which is incorporated by reference herein.

[0009] BACKGROUND

[0010] Malaria is one of the world’s deadliest infectious diseases. In humans, it is caused by five species of the Plasmodium parasite: P. falciparum, P. vivax, P. ovale, P. knowlesi and P. malariae. However, P. falciparum causes the most severe form of malaria disease, leading to the death of about 600,000 people annually, most of whom are young children.

[0011] Each of the Plasmodium species that infect humans is transmitted through the bite of an infected female Anopheles mosquito, which introduces Plasmodium sporozoites into the bloodstream of the human host. The sporozoites rapidly reach the liver where they are sequestered by hepatocytes and undergo asexual expansion. One week later, the infected hepatocytes rupture and release mature parasites, the merozoites. These then begin the erythrocytic phase of malaria by attaching to and invading red blood cells, or erythrocytes. The invasion of the erythrocytes by tire malarial parasites leads to malarial pathogenesis and clinical infection.

[0012] Plasmodium falciparum circumsporozoite protein (PfCSP) is the most abundant protein on tire surface of sporozoites and is a major target for liver stage malaria vaccines and antibodies. While CSP is a promising target for malaria vaccines, efficacy remains a challenge. RTS,S / AS01, an approved malaria vaccine, is a recombinant protein-based vaccine that includes a large portion of CSP (consisting of 19 NANP repeats and tire C-terminal region). However, RTS,S / AS01 provides only modest protection (30-50%) from clinical malaria in endemic areas, and protection has been shown to wane rapidly following immunization. Over 100 other vaccine candidates have been evaluated in clinical trials, yet only one candidate, R21, with an efficacy of 76%, has been able to meet the 75% efficacy goal set by the World Health Organization. Thus, new malaria vaccines, particularly with improved efficacy, are needed.4239-112675-02

[0013] SUMMARY

[0014] Nanoparticles including a fusion protein including multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide are provided herein. In some aspects, the fusion protein includes two to five copies of the PfCSP peptide, for example, 2 to 3 copies. In some aspects, tlie PfCSP peptide includes a PfCSP junctional epitope, for example, NPDP19 (SEQ ID NO: 1). The fusion proteins can further include a T cell epitope, for example, PADRE, P2, or TpD, to enhance an immune response in a subject. The fusion protein can be conjugated to a nanoparticle (e.g., gold nanoparticle, gold shell nanoparticle, carbon nanoparticle, or liposome), or can include a protein nanoparticle subunit, for example, a protein nanoparticle subunit that is capable of self-assembly into corresponding protein nanoparticles. In some aspects, the protein nanoparticle subunit is a lumazine synthase, ferritin, encapsulin, sulfur oxygenase reductase (SOR), pyruvate dehydrogenase, or BP26 subunit. In a non-limiting example, tire protein nanoparticle subunit is a lumazine synthase subunit. The protein nanoparticle subunit, PfCSP peptide, and / or T cell epitope can be directly linked, or indirectly linked, for example, by peptide linkers (e.g., a glycine-serine linker). In some aspects, the fusion protein includes from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, and the multiple copies of the PfCSP peptide (e.g., SEQ ID NO: 8 or 9).

[0015] Also disclosed are the fusion proteins of the nanoparticles disclosed herein, nucleic acid molecules encoding the fusion proteins, and vectors (e.g., expression vectors) comprising such nucleic acid molecules. Further disclosed are methods of making protein nanoparticles, the methods including, for example, expressing a nucleic acid molecule encoding a fusion protein disclosed herein in a host cell under conditions sufficient for the expressed fusion proteins to self-assemble into nanoparticles. The methods can further include purifying the nanoparticles from the host cell culture. In some aspects, tire methods include expressing a nucleic acid molecule encoding a fusion protein disclosed herein in a host cell, purifying the expressed fusion proteins, and incubating the expressed fusion proteins under conditions sufficient for self-assembly into nanoparticles, thereby producing nanoparticles.

[0016] Immunogenic compositions including a nanoparticle disclosed herein and a pharmaceutically-acceptable carrier are provided. Such immunogenic compositions can elicit an immune response in a subject to Plasmodium falciparum. Also provided are methods of eliciting an immune response in a subject to Plasmodium falciparum, the methods including, for example, administering to tire subject an effective amount of a nanoparticle or immunogenic composition disclosed herein. In some aspects, the methods are prophylactic. In some aspects, the methods induce a protective immune response to Plasmodium falciparum in the subject.

[0017] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.4239-112675-02

[0018] BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1M show design and characterization of nanoparticle immunogens incorporating tlie CIS43 epitope. FIG. 1A shows a schematic of plasmodium falciparum circumsporozoite protein (PfCSP) highlighting the epitope recognized by CIS43 (NPDPNANPNVDPN; SEQ ID NO: 32). FIG. IB shows tlie structure of CIS43 bound to peptide 21 of PfCSP. FIG. 1C shows yield of constructs initially selected for expression. FIG. ID shows SDS-PAGE gels of die constructs shown in FIG. 1C. FIGS. 1E-1H show serology data. FIG. II shows sequence information for additional constructs that were designed and expressed in view of FIG. 1H. FIG. 1J is a schematic of LuS-2T (construct 20) and LuS-3T (construct 21). FIG. IK shows protein analysis for LuS-2T and LuS-3T, as well as expression yield for constructs 5, 20, and 21. FIG. IL shows representative negative EM stain images for LuS-2T and LuS-3T, which show properly assembled nanoparticle immunogens. FIG. IM provides antigenicity of nanoparticle immunogens to junction, minor, and major-directed antibodies assessed by biolayer interferometry (BLI).

[0019] FIGS.2A-2E show malaria protection by vaccination with tandem-displayed peptides including the CIS43 epitope on a LuS nanoparticle. FIG. 2A shows the immunization schema comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. FIGS. 2B-2D show serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. In FIGS. 2B-2D, A=naive; B=R21(1 pg); C=NPDP19-KLH (10 pg); D=NPDP19-LuS-PADRE (1 pg); E= NPDP19-LuS-PADRE (10 pg); F= (NPDP19)2-LuS-PADRE (10 pg). FIG. 2E shows liver burden assessment of immunized mice at day 2 post sporozoite challenge. Horizontal lines denote the median flux, error bars show 95% confidence interval; N=10 mice per group. The percent reduction was calculated based on geometric mean of each group (* p < 0.05; ** p < 0.01 as calculated by two-tailed Mann- Whitney test). The data show enhanced protection with tandem repeats at a low dose.

[0020] FIGS.3A-3E show the tandem LuS-displayed CIS43 epitope vaccine provides protection equivalent to R21 at 1 pg dose. FIG. 3A shows the immunization schema, comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. FIGS. 3B-3D show serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. In FIGS.

[0021] 3B-3D, A=naive; B=R21(1 pg); C=(NPDP19)2-LuS-PADRE (0.2 pg); D=(NPDP19)2-LuS-PADRE (1 pg); E= (NPDP19)2-LuS-PADRE (5 pg); F= PADRE-LuS-(NPDP19)2(1 pg); G= (NPDP19)3-LuS-PADRE (1 pg); H= PADRE-LuS-(NPDP19)3 (1 pg). FIG. 3E shows liver burden assessment of immunized mice at day 2 post sporozoite challenge. Horizontal lines denote the median flux, error bars show 95% confidence interval; N=10 mice per group. The percent reduction was calculated based on geometric mean of each group (* p < 0.05; ** p < 0.01 as calculated by two-tailed Mann-4239-112675-02

[0022] Whitney test). The data show enhanced protection with tandem repeats displayed at C-terminus of LuS and a dose response, with lower dose being more protective than higher dose.

[0023] FIGS.4A-4F show that the tandem LuS-displayed CIS43 epitope vaccine provides increased protection relative to R21 at 0.25 pg dose. FIG.4A shows the immunization schema, comprising of 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. FIGS. 4B-4D show serum antibody titers at weeks 2 and 6 assessed by biolayer interferometry (BLI) against biotinylated PfCSP, NANP9 (major repeat), and NPDP19 (junction). The average titer of each group is listed. In FIGS.

[0024] 4B-4D, A=naive; B=R21(0.25 pg); C= PADRE-LuS-(NPDP19)2(0.25 pg); D= PADRE-LuS-(NPDP19)3(0.25 pg); E=R21+ PADRE-LuS-(NPDP19)3(0.25 pg + 0.25 pg); F=R21 (1 pg); G= PADRE-LuS-(NPDP19)2(1 pg); H= PADRE-LuS-(NPDP19)3(1 pg); 1= R21+PADRE-LuS-(NPDP19)3(1 pg + 1 pg). FIGS. 4E and 4F show liver burden assessment of immunized mice at day 2 post sporozoite challenge. Horizontal lines denote the median flux, error bars show 95% confidence interval. In FIG. 4E, A=naive; B= max burden; C= R2H0.25 pg); D= PADRE-LuS-2T (0.25 pg); E= PADRE-LuS-3T (0.25 pg); F=R21 + PADRE-LuS-3T (0.25 pg + 0.25 pg); G= R21 (1 pg); H= PADRE-LuS-2T (1 pg); 1= PADRE-LuS-3T (1 pg); J= R21 + PADRE-LuS-3T (1.0 pg + 1.0 pg). FIG. 4F (left) shows a repeat experiment for R21 and PADRE-LuS-3T-NPDP19 at 0.25 pg, N=10 mice per group (A=naive; B= max burden; C= R21 (0.25 pg); D= PADRE-LuS-3T (0.25 pg)). The right panel shows combined results of the two experiments (N=20 mice per group). Percent reduction was calculated based on geometric mean of each group (* p < 0.05; ** p < 0.01 as calculated by two-tailed Mann-Whitney test). The data show significantly better protection from PADRE-LuS-(NPDP19)3than R21 at 0.25 pg dose.

[0025] FIGS.5A-5B show in vivo protective efficacy of PADRE-LuS-3T formulated in SMNP adjuvant. FIG. 5A shows the immunization schema comprising 2 immunizations 4 weeks apart followed by sporozoite challenge at week 8. FIG. 5B shows liver burden assessment of immunized mice at day 2 post Pb-PfCSP-Luc sporozoite challenge. Horizontal lines denote the median flux; error bars show 95% confidence interval; N=10 mice per group. The percent reduction was calculated based on geometric mean of each group.

[0026] SEQUENCES

[0027] The nucleic and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0028] SEQ ID NO: 1 is the amino acid sequence of PfCSP junctional epitope NPDP19.

[0029] KQPADGNPDPNANPNVDPN SEQ ID NO: 2 is an exemplary amino acid sequence of a pan HLA-DR epitope (PADRE). AKFVAAWTLKAAA4239-112675-02

[0030] SEQ ID NO: 3 is an exemplary amino acid sequence of a lumazine synthase subunit.

[0031] MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLR SEQ ID NO: 4 is an exemplary amino acid sequence of a fusion protein including two copies of a PfCSP peptide. The linker sequence is shown in lowercase.

[0032] KQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPN

[0033] SEQ ID NO: 5 is an exemplary ainino acid sequence of a fusion protein including three copies of a PfCSP peptide. Linker sequences are shown in lowercase.

[0034] KQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPN VDPN SEQ ID NO: 6 is an exemplary amino acid sequence of a fusion protein including two copies of a PfCSP peptide fused to a lumazine synthase subunit. Linker sequences are shown in lowercase. MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGN PDPNANPNVDPN SEQ ID NO: 7 is an exemplary amino acid sequence of a fusion protein including three copies of a PfCSP peptide fused to a lumazine synthase subunit. Linker sequences are shown in lowercase.

[0035] MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGN PDPNANPNVDPNggsggKQPADGNPDPNANPNVDPN

[0036] SEQ ID NO: 8 is an exemplary amino acid sequence of a fusion protein including two copies of a PfCSP peptide fused to a lumazine synthase subunit and PADRE (LuS-2T; construct 20). Linker sequences are shown in lowercase.

[0037] AKFVAAWTLKAAASLVRgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRH GGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSL ELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGggsggKQPADGNPD PNANPNVDPNggsggKQPADGNPDPNANPNVDPN

[0038] SEQ ID NO: 9 is an exemplary amino acid sequence of a fusion protein including three copies of a PfCSP peptide fused to a lumazine synthase subunit and PADRE (LuS-3T; construct 21). Linker sequences are shown in lowercase.

[0039] AKFVAAWTLKAAASLVRgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRH GGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSL ELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGggsggKQPADGNPD PNANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPN SEQ ID NO: 10 is an exemplary amino acid sequence of a precursor fusion protein that includes a signal peptide (LuS-2T precursor). Linker sequences are shown in lowercase and the signal peptide is underlined.4239-112675-02

[0040] NHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGAT PHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANL FKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPN SEQ ID NO: 11 is an exemplary amino acid sequence of a precursor fusion protein that includes a signal peptide (LuS-3T precursor). Linker sequences are shown in lowercase and the signal peptide is underlined.

[0041]

[0042] FNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGA TPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMAN LFKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggKQPA DGNPDPNANPNVDPN SEQ ID NOS: 12-27 are additional exemplary amino acid sequences of fusion protein subunits of protein nanoparticles.

[0043] SEQ ID NOS: 28-30 are skipped sequences.

[0044] SEQ ID NO: 31 is an exemplary amino acid sequence of a glycine-serine linker. GGSGG SEQ ID NO: 32 is an exemplary amino acid sequence of a Plasmodium falciparum circumsporozoite protein epitope recognized by the CIS43 antibody. NPDPNANPNVDPN

[0045] SEQ ID NO: 33 is an exemplary amino acid sequence of a signal peptide.

[0046] MDSKGSSQKGSRLLLLLVVSNLLLPQGV SEQ ID NO: 34 is an exemplary nucleic acid sequence encoding a fusion protein subunit of a protein nanoparticle (LuS-2T; construct 20). This sequence includes a signal peptide.

[0047] ATGGATTCTAAGGGCTCTAGCCAAAAAGGCAGCCGTTTGTTGTTGTTGTTAGTTGTTTCTA ATCTGTTGTTGCCGCAGGGTGTTGTTGGTGCCAAATTTGTTGCTGCCTGGACCTTGAAAG CTGCTGCTTCTTTGGTTCGCGGCAGCGGTAGCGCTATGCAGATCTATGAAGGCAAATTGA CCGCCGAAGGTTTGCGCTTTGGCATTGTTGCTTCTCGCTTTAATCATGCCTTGGTTGATCG CTTGGTTGAAGGCGCCATTGATGCTATTGTTCGCCATGGCGGCCGTGAAGAAGATATTAC CTTGGTTCGCGTTCCTGGCTCTTGGGAAATTCCTGTTGCCGCTGGCGAATTGGCCCGCAA AGAAAATATTTCTGCCGTTATTGCCATTGGTGTTTTGATTCGTGGCGCCACCCCGCATTTT GATTATATTGCTAGCGAAGTTTCTAAGGGCTTGGCTGATTTGAGCTTGGAATTGCGTAAA CCTATTACCTTTGGTGTTATTACCGCCGATACCTTGGAACAAGCCATTGAACGTGCTGGT ACCAAACATGGCAATAAGGGCTGGGAAGCCGCTTTGTCTGCCATTGAAATGGCCAATTT GTTTAAATCTCTGCGTGGTGGTGGCAGCGGTGGCAAACAACCGGCCGATGGTAATCCGG ACCCTAATGCCAATCCGAATGTTGATCCTAATGGCGGTAGCGGTGGTAAACAGCCGGCC GATGGCAATCCTGATCCTAATGCTAATCCTAATGTTGATCCGAAT SEQ ID NO: 35 is an exemplary nucleic acid sequence encoding a fusion protein subunit of a protein nanoparticle (LuS-3T; construct 21). This sequence includes a signal peptide.

[0048] ATGGATAGCAAAGGCTCTTCTCAGAAAGGCAGCCGCTTGTTGTTGTTGTTAGTTGTTTCT AATCTGTTGTTGCCTCAGGGCGTTGTTGGTGCTAAATTTGTTGCCGCTTGGACCTTGAAAG CCGCCGCCAGCTTGGTTCGCGGCAGCGGTAGCGCCATGCAAATCTATGAAGGCAAATTG ACCGCTGAAGGTTTGCGTTTTGGCATTGTTGCTAGCCGTTTTAATCATGCTTTGGTTGATC GTTTGGTTGAAGGTGCTATTGATGCCATTGTTCGCCATGGTGGCCGTGAAGAAGATATTA CCTTGGTTCGTGTTCCGGGCTCTTGGGAAATTCCTGTTGCTGCCGGCGAATTGGCCCGTA AAGAAAATATTAGCGCTGTTATTGCCATTGGCGTTTTGATTCGTGGCGCCACCCCTCATTT TGATTATATTGCTAGCGAAGTTTCTAAGGGCTTGGCTGATTTGTCTTTGGAATTGCGTAAA CCTATTACCTTTGGTGTTATTACCGCCGATACCTTGGAACAAGCCATTGAACGCGCCGGT4239-112675-02

[0049] ACCAAACATGGCAATAAGGGCTGGGAAGCTGCCTTGTCTGCTATTGAAATGGCTAATTTG TTTAAGAGCTTGCGTGGTGGTGGTAGCGGCGGCAAACAACCGGCCGATGGTAATCCGGA CCCTAATGCTAATCCTAATGTTGATCCTAATGGCGGCTCTGGCGGCAAACAGCCTGCTGA TGGCAATCCGGACCCTAACGCCAATCCTAATGTCGATCCGAATGGTGGTAGCGGTGGCA AACAGCCGGCCGATGGCAATCCTGATCCGAATGCCAATCCTAACGTTGATCCGAAT SEQ ID NOs: 36-46 are additional exemplary amino acid sequences of lumazine synthase nanoparticle subunits.

[0050] SEQ ID NOs: 47-57 are exemplary amino acid sequences of ferritin nanoparticle subunits.

[0051] SEQ ID NO: 58-61 are exemplary amino acid sequences of encapsulin nanoparticle subunits.

[0052] SEQ ID NO: 62 is an exemplary amino acid sequence of a BP26 nanoparticle subunit. QENQMTTQP ARIA VTGEGMMTASPDMAILNLSVLRQAKT AREAMT ANNE AMTKVLDAMK KAGIEDRDLQTGGIDIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLG VNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPI ARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK SEQ ID NO: 63 is an exemplary amino acid sequence of a P2 epitope. QYIKANSKFIGITEL SEQ ID NO: 64 is an exemplary amino acid sequence of a TpD epitope.

[0053] ILMQYIKANSKFIGKVSVRQSIALSSLMVAQ SEQ ID NO: 65 is an exemplary amino acid sequence of a ferritin nanoparticle subunit. DPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIF LNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS SEQ ID NOs: 66-79 are additional exemplary amino acid sequences of T cell epitopes. SEQ ID NO: 80 is an exemplary amino acid sequence of a NANP5 PfCSP peptide.

[0054] NANPNANPNANPNANPNANP SEQ ID NO: 81 is an exemplary amino acid sequence of a NANP9 PfCSP peptide.

[0055] NANPNANPNANPNANPNANPNANPNANPNANPNANP SEQ ID NO: 82 is an exemplary peptide sequence that can be added to SEQ ID NO: 32 to create tire NPDP19 peptide sequence. KQPADG

[0056] SEQ ID NOs: 83-85 are exemplary signal peptides.

[0057] SEQ ID NOs: 86-90 are exemplary peptide linkers.

[0058] DETAILED DESCRIPTION

[0059] I. Introduction

[0060] Malaria is a mosquito-borne parasitic disease that has high morbidity and mortality, primarily in infants and young children in sub-Saharan Africa. Development of a highly effective vaccine for malaria is urgently needed. This disclosure shows vaccine efficacy for constructs incorporating a junctional epitope of PfCSP. The immunogens were designed to display the junctional epitope on protein nanoparticles in a tandem fashion. It is shown that the junctional peptide presented three times in tandem on a lumazine synthase nanoparticle (LuS-3T) coupled with pan HLA DR-binding epitope (PADRE) provides superior protection than the WHO approved vaccine, R21, at 0.25 pg dose4239-112675-02

[0061] in mice. Importantly, there was enhanced protection when LuS-3T and R21 were co-administered as compared to either vaccine alone. The disclosed immunogens are useful as a prophylactic agent against malaria, for example, for subjects residing and / or traveling to geographic areas of the world where malaria is endemic and / or prevalent.

[0062] II. Summary of Terms

[0063] Unless otherwise noted, technical terms are used according to conventional usage.

[0064] Definitions of many common terms in molecular' biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a peptide” includes singular or plural peptides and can be considered equivalent to the phrase “at least one peptide.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0065] About: Unless context indicates otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to the range of 95 to 105.

[0066] Administration: The introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Administration can be local or systemic. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, inhalation, or other suitable route.

[0067] Biological Sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection (for example, P. falciparum infection) in subjects, including, but not limited to, cells, tissues, and bodily fluids, for example, blood, derivatives and fractions of blood (e.g., serum), cerebrospinal fluid, as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having a P. falciparum infection.

[0068] Circumsporozoite Protein (CSP): A major malaria parasite surface protein during the sporogonic cycle. CSP covers the surface of P. falciparum sporozoites, which are transmitted from4239-112675-02

[0069] the mosquito salivary gland to host hepatocytes. CSP includes an immunodominant central repeat region, which contains -35-38 copies of an NANP motif and up to four copies of an NVDP motif, flanked by N- and C-terminal regions. CSP plays a role in facilitating parasite invasion of hepatocytes, and antibodies that target CSP have been found to block infection. The term “PfCSP” refers to CSP of P. falciparum.

[0070] CIS43 Antibody: A monoclonal antibody that specifically binds to an epitope of PfCSP and can neutralize P. falciparum infection. The CIS43 antibody and methods for its production are described, for example, in US Patent Publication No. 2019-0375831 Al.

[0071] Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. The linkage can be by chemical or recombinant means. A peptide linker (short peptide sequence) can optionally be included between the two molecules. Because conjugates can be prepared from two molecules with separate functionalities, they are also sometimes referred to as “chimeric molecules.”

[0072] Conservative Variant: A sequence variant including conservative substitutions. A “conservative” substitution is a substitution of an original amino acid with a different amino acid that has similar properties as the original amino acid. Conservative substitutions, as used herein, do not substantially affect or decrease a function of a protein, such as the ability of the protein to elicit an immune response in a subject. A conservative variant can include, for example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference sequence and retain a desired function, for example, eliciting an immune response and the production of antibodies that bind PfCSP.

[0073] The following six groups are examples of amino acids that are generally considered to be conservative substitutions for one another:

[0074] 1) Alanine (A), Serine (S), Threonine (T);

[0075] 2) Aspartic acid (D), Glutamic acid (E);

[0076] 3) Asparagine (N), Glutamine (Q);

[0077] 4) Arginine (R), Lysine (K);

[0078] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0079] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0080] A non-conservative substitution is the substitution of an original amino acid with an amino acid that has different properties Oran the original amino acid, for example, substituting a non-polar amino acid with a charged, polar amino acid. Non-conservative substitutions can, but do not necessarily, change the activity or function of a protein.

[0081] Contacting: Placing in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro.

[0082] Control: A reference standard. In some aspects, the control is a negative control, positive control, historical control (e.g., values from previously subjects having a known prognosis or4239-112675-02

[0083] outcome), or standard reference value or range of values (e.g., a group of samples that represent “baseline” or “normal” values).

[0084] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference (e.g., p- value less than 0.05, or less than 0.01). In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, or at least 500%.

[0085] Detectable Marker: A molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, for example, a peptide, to facilitate detection of the second molecule. The detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy, or other diagnostic imaging technique (for example, CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0086] Detecting: To identify the existence, presence, or fact of something.

[0087] Effective Amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit a P. falciparum infection, for example the amount necessary to inhibit or prevent P. falciparum sporozoites from invading the liver in tire subject or to measurably alter outward symptoms of the P. falciparum infection.

[0088] In some aspects, administration of an effective amount of a disclosed antibody or antigen binding fragment that binds to PfCSP can reduce or inhibit a P. falciparum infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the P. falciparum, or by an increase in tire survival time of infected subjects, or reduction in symptoms associated with P. falciparum infection) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable P. falciparum infection), as compared to a suitable control (e.g., an individual or population of individuals not administered a disclosed antibody).

[0089] The effective amount of an agent that is administered to a subject to inhibit P. falciparum infection will vary depending upon a number of factors associated with that subject, for example the4239-112675-02

[0090] age, overall health, condition, and / or weight of the subject. An effective amount can be determined by varying the dosage and measuring a resulting response, for example, measuring an immune response to PfCSP. Effective amounts can also be determined through various in vitro, in vivo or in situ immunoassays.

[0091] An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attain an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

[0092] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. Antibodies specifically bind particular antigenic epitopes on polypeptides. SEQ ID NO: 1 provides an exemplary PfCSP junctional epitope.

[0093] Expression: Transcription or translation of a nucleic acid sequence. For example, a nucleic acid sequence (e.g., a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become mRNA. An encoding nucleic acid sequence (e.g., a gene) may also be expressed when its mRNA is translated into a corresponding amino acid sequence, for example a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules, for example mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after drey are produced.

[0094] Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when die expression control sequences control and regulate the transcription and, as appropriate, hanslation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.4239-112675-02

[0095] Expression Vector: A vector including expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the nucleotide sequence to be expressed.

[0096] A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of transformed cells.

[0097] Fusion Protein: A single polypeptide chain including the sequence of two or more heterologous proteins, often linked by a peptide linker. Reference to a first protein “fused” to a second protein indicates that the first and second proteins are contained within a single contiguous polypeptide chain. The first and second protein may be directly linked (for example, the C-terminus of the first protein is linked to the N-terminus of the second protein by a peptide bond), or indirectly linked (for example, the C-terminus of the first protein is directly linked to the N-terminus of a peptide linker by a peptide bond, and the C-terminus of the peptide linker is directly linked to the N-terminus of the second protein by a peptide bond).

[0098] Heterologous: Originating from a different genetic source.

[0099] Host Cell: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used to the extent that the progeny are functionally equivalent to the parental cell.

[0100] Immune Response: A response of a cell of the immune system, for example, a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigen-specific response”). In one aspect, an immune response is a T cell response, for example a CD4+ response or a CD8+ response. In another aspect, the response is a B cell response, and results in die production of specific antibodies. “Priming an immune response” refers to treatment of a subject with a “prime” immunogen to induce an immune response that is subsequently “boosted” widi a boost immunogen. Together, the prime and boost immunizations produce the desired immune response in the subject. In some aspects, an immune response is enhanced by co-administering an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant.

[0101] Immunogen: A protein or a portion thereof that is capable of inducing an immune response in a mammal, for example, a human subject at risk of infection with a pathogen (e.g., P. falciparum).4239-112675-02

[0102] Inhibiting a Disease or Condition: Reducing or preventing the full development of a disease or condition in a subject, for example, reducing or preventing the full development of a P. falciparum infection or malaria in a subject who is at risk of a P. falciparum infection.

[0103] Inhibiting a disease or condition refers to a prophylactic intervention that is administered before the disease or condition has begun to develop, for example a treatment initiated in a subject at risk of P. falciparum infection, but not presently infected by P. falciparum. Inhibiting a disease or condition encompasses a reduction in detectable infection of the subject when the subject is subsequently exposed to the target pathogenic agent (e.g., P. falciparum), as well as amelioration of any signs, symptoms, or severity of disease (e.g., malaria) when the subject is subsequently infected with the pathogenic agent (e.g., P. falciparum). Amelioration (or “ameliorating”), with reference to inhibiting a disease or condition refers to any observable beneficial effect of the prophylactic intervention intended to inhibit the disease or condition. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease or condition in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease or condition, a slower progression of the disease or condition, an improvement in the overall health or well-being of the subject, a reduction in infection, or by other parameters that are specific to the particular disease or condition.

[0104] Isolated: A biological component (e.g., a nucleic acid, peptide, protein or protein complex, for example a nanoparticle) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example, other chromosomal and extra-chromosomal DNA, RNA, lipids, metabolites, and / or proteins. Isolated nucleic acids, peptides and proteins include nucleic acids and proteins that are purified using conventional purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein (for example a fusion protein or nanoparticle disclosed herein), can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0105] Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link heterologous proteins (e.g., a PfCSP peptide and a protein nanoparticle subunit) into a contiguous polypeptide. Non-limiting examples of peptide linkers include glycineserine linkers.

[0106] The terms “conjugating,” “joining," “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker or other molecule to a polypeptide. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.4239-112675-02

[0107] Malaria: Malaria is a parasitic infection of humans by the Plasmodium species P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Humans become infected following the bite of an infected mosquito, tire host of die malarial parasite. Malaria rarely occurs in humans following a blood transfusion or subsequent to needle-sharing. Clinical manifestations of malarial infection which may occur include blackwater fever, cerebral malaria, respiratory failure, hepatic necrosis, occlusion of myocardial capillaries, and deadr.

[0108] Infection begins when malaria sporozoites gain access to or are directly injected into the bloodstream of a host by a mosquito. After injection, they migrate to the liver and multiply in hepatocytes for one week. The sporozoites substantially expand in the liver and differentiate to merozoites which are released from the liver into the blood stream, where they infect erythrocytes. When the merozoite matures in the red blood cell, it is known as a trophozoite and, when fully developed, as a schizont. A schizont is the stage when nuclear division occurs to form individual merozoites which are released to invade other red cells. Malaria clinical symptoms appear during the blood-stage. After several schizogonic cycles, some parasites, instead of becoming schizonts through asexual reproduction, develop into large uninucleate parasites, known as gametocytes. These gametocytes are the sexual blood cell stage forms of the parasite.

[0109] Sexual development of the malaria parasites involves the female macrogametocyte and the male microgametocyte. If a mosquito feeds on the blood of an infected host, it can ingest gametocytes within the blood. Fertilization and sexual recombination of the parasite occurs in the mosquito's gut. The fertilized parasite, which is known as a zygote, then develops into an ookinete. The ookinete penetrates the midgut wall of the mosquito and develops into an oocyst, within which many small sporozoites form. When the oocyst ruptures, the sporozoites migrate to the salivary gland of the mosquito via the hemolymph. Once in the saliva of the mosquito, the parasite can be injected into a host, repeating tire life cycle.

[0110] Nanoparticlc: A small particle that typically ranges between 1 to 100 nanometers in size. In medicine, nanoparticles can be used as vehicles to a therapeutic or immunogenic agent. In some instances, a therapeutic or immunogenic agent is conjugated to the nanoparticle and / or is enclosed within the nanoparticles. Exemplary classes of nanoparticles include, but are not limited to, protein nanoparticles, liposomes, ceramic nanoparticles, metallic particles (e.g., iron oxide or gold nanoparticles), gold shell nanoparticles, and carbon nanoparticles.

[0111] Nucleic Acid Molecule: A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA or RNA. The nucleic acid can be double stranded (ds) or single stranded (ss). When single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (e.g., A, T / U, C, and G), and can include analogs of natural nucleotides, e.g., labeled nucleotides.4239-112675-02

[0112] “cDNA” refers to an artificial DNA sequence that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0113] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, for example a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0114] Operably Linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0115] Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers of use in the compositions and methods disclosed herein are conventional. Remington’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020), describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

[0116] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids for example, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain mirror¬ amounts of non-toxic auxiliary substances, for example wetting or emulsifying agents, added preservatives (e.g., non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form, for example a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage4239-112675-02

[0117] or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

[0118] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When tire amino acids are alpha-amino acids, either die L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and includes modified sequences. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end (C-terminal). In some aspects, the polypeptide is a disclosed fusion protein.

[0119] Purified: The enrichment of a target agent in a sample relative to a starting environment. The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nanoparticle preparation is one in which the nanoparticle is more enriched than the nanoparticle was in a prior environment. In one aspect, a preparation is purified such that the purified nanoparticle represents at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc.) of the total content of the preparation.

[0120] Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid molecule that has been introduced into a host cell, for example a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of directing expressing of a protein product encoded by the nucleic acid in the host cell.

[0121] Protein Nanoparticle: A multi-subunit protein-based nanoparticle, typically globular in shape. Non-limiting examples of protein nanoparticles include lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306: 1099-1114, 2001), ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Set., 12:5406-5421, 2011), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct, and Mol. Biol., 15:939-947, 2008), sulfur oxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006), and pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 96: 1240-1245, 1999). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that are capable of assembling into globular protein complexes (protein nanoparticles) that in some cases consists of 24, 60, 24, 60, and 60 protein subunits, respectively. A self-assembling protein nanoparticle is a multi-subunit protein-based4239-112675-02

[0122] nanoparticle formed from subunit monomers that self-assemble under suitable conditions to form the nanoparticle.

[0123] A monomeric subunit of ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase can be linked to one or more heterologous amino acid sequences to facilitate selfassembly of nanoparticles that include the heterologous sequences. In some aspects, a ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase subunit is fused to a PfCSP peptide disclosed herein and / or T cell epitope. In some aspects, the subunits that a nanoparticle is comprised of are identical.

[0124] Sequence Identity: The degree of similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of die identity between die sequences. Sequence identity can be measured in terms of percentage identity ; the higher the percentage, the more identical the sequences.

[0125] Methods of alignment of sequences for comparison are known. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.

[0126] 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(1):237-244, 1988; Higgins and Sharp, Bioinformatics, 5(2): 151-3, 1989; Corpet, Nucleic Acids Res.

[0127] 16(22): 10881-10890, 1988; Huang et al. Bioinformatics, 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including die National Center for Biological Information (NCBI) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

[0128] Generally, once two sequences are aligned, the number of matches is determined by counting die number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches eidier by die length of die sequence set forth in die identified sequence, or by an articulated lengdi (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying die resulting value by 100. Unless otherwise specified, percentage identity between sequences is determined by pairwise alignment over die full-length of the reference sequence using an appropriate global alignment algorithm, such as the Needleman-Wunsch algorithm as implemented in EMBOSS program stretcher (see, e.g., bioinformatics.nl / cgi-bin / emboss / stretcher) for longer sequences such as genomic genome sequences and as implemented in EMBOSS program needle (see, e.g., bioinformatics.nl / cgi-bin / emboss / needle) for shorter sequences (e.g., protein sequences and nucleic acid sequences less than lOkb), typically4239-112675-02

[0129] with default gap penalties and scoring matrix. Alternative alignment programs and parameters may also be used.

[0130] Signal Peptide: A short amino acid sequence (e.g., approximately 18-30 amino acids in length) that directs newly synthesized secretory or membrane proteins to and through membranes, for example, the endoplasmic reticulum membrane. Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases after the polypeptide has crossed the membrane. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). Signal peptide sequences include, for example and without limitation, MDSKGSSQKGSRLLLLLVVSNLLLPQGV (SEQ ID NO: 33);

[0131] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 83);

[0132] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVA (SEQ ID NO: 84); or MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA (SEQ ID NO: 85).

[0133] Subject: A living multi-cellular vertebrate organism, a category that includes humans, nonhuman primates, and other mammals. In some aspects, the subject is a human. In some aspects, the subject is uninfected and at risk of P. falciparum infection.

[0134] Transformed: A transformed cell is a cell into which a heterologous nucleic acid molecule has been introduced by a molecular biology technique. As used herein, the term transformed encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including, for example and without limitation, transduction or transfection with viral vectors, transformation with plasmid vectors, or introduction of DNA by electroporation, lipofection, or particle gun acceleration.

[0135] Under Conditions Sufficient For: A phrase that is used to describe an environment that permits a desired activity, for example, the assembly of fusion proteins into corresponding nanoparticles.

[0136] Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the iinniune response is a protective immune response.

[0137] Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example P. falciparum CSP. Vaccines include an immunogenic agent, which can include, for example, polynucleotides (e.g., nucleic acids encoding an antigenic epitope), proteins (e.g., proteins including an antigenic epitope), viruses, cells, or one or more cellular constituents. Vaccines can further include adjuvants to help elicit an immune response. In some aspects, a vaccine includes a nanoparticle disclosed herein.

[0138] Vector: A nucleic acid vehicle to cany a heterologous nucleic acid of interest into a host cell, for example, to facilitate cloning, replication, and / or expression of the heterologous nucleic acid. Exemplary vectors include but are not limited to plasmids, viral vectors, cosmids, and artificial chromosomes. In some aspects, the heterologous nucleic acid is operably linked to a promoter or4239-112675-02

[0139] other expression control sequence. Recombinant DNA vectors are vectors including recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, for example an origin of replication. A vector can also include one or more selectable marker genes, tags, or other genetic elements. Viral vectors include recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some aspects, a vector includes a nucleic acid molecule encoding a disclosed fusion protein.

[0140] III. Nanoparticles and Fusion Proteins

[0141] Disclosed are nanoparticles including a fusion protein, wherein the fusion protein includes multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide. The PfCSP peptide includes a sequence of consecutive amino acid residues of PfCSP. In some aspects, the PfCSP peptide is no more than 55 amino acids in length, for example, 19 to 21 amino acids in length. In several aspects, the PfCSP peptide includes or consists of SEQ ID NO: 1. The multiple copies of the PfCSP peptide are identical in sequence and fused together by peptide linkers to form a contiguous polypeptide. In some examples, the peptide linkers are glycine-serine linkers, however, any suitable peptide linker can be used.

[0142] The fusion protein can be attached to any nanoparticle suitable for use in a vaccine, for example, a protein nanoparticle, metallic nanoparticle (e.g., iron oxide or gold nanoparticles), gold shell nanoparticle, carbon nanoparticle, or liposome. Conventional attachment methods can be used. In some aspects, the fusion protein is attached to the nanoparticle, for example, by a peptide linker or a covalent attachment. In some aspects, an avidin / strepta vidin-bio tin system or spytag-spycatcher system is used to link a fusion protein disclosed herein to a nanoparticle. In some aspects, the fusion proteins disclosed herein further include a protein nanoparticle subunit. The protein nanoparticle subunit can be a self-assembling protein nanoparticle subunit, and thereby facilitate assembly of the fusion proteins into corresponding protein nanoparticles.

[0143] In some aspects, the fusion proteins further include a T cell epitope (e.g., pan HLA-DR epitope (PADRE), a P2 epitope, or TpD epitope). The PfCSP peptides and / or tire T-cell epitope can be linked to the protein nanoparticle subunit directly or indirectly, for example, by a peptide linker.

[0144] The nanoparticles disclosed herein can be used to generate an immune response to Plasmodium falciparum in a subject, for example, to inhibit or prevent Plasmodium falciparum infection or development of malaria in tine subject. Additional details and description regarding tine disclosed nanoparticles, fusion proteins, and components thereof are provided below.

[0145] A. PfCSP Peptides

[0146] The fusion proteins disclosed herein include multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide. The circumsporozoite protein (CSP) is a major malaria parasite surface protein during the sporogonic cycle. PfCSP is the most abundant protein on the4239-112675-02

[0147] surface of sporozoites and therefore is a potential target for liver stage malaria vaccines and antibodies. Several PfCSP epitopes have been described, for example, CIS43 (NPDPNANPNVDPN; SEQ ID NO: 32), NANP5 (SEQ ID NO: 80), and NANP9 (SEQ ID NO: 81). In some aspects, die fusion proteins disclosed herein include one or more PfCSP junctional epitopes, which are epitopes diat include a portion of die N-terminus and die repeat motif of PfCSP (see, FIG. 1A). An exemplary PfCSP junctional epitope is NPDP19, which is provided herein as SEQ ID NO: 1 (KQPADGNPDPNANPNVDPN). In some aspects, the PfCSP junctional epitope includes at least 13 contiguous amino acids of SEQ ID NO: 1 (e.g., NPDPNANPNVDPN; SEQ ID NO: 32).

[0148] The multiple copies of the PfCSP peptide refers to repeats of the same PfCSP peptide sequence. In some aspects, a fusion protein disclosed herein includes at least 2 copies of die PfCSP peptide, for example, at least 3 copies, at least 4, at least 5 copies, etc. In some aspects, a fusion protein disclosed herein includes 1 to 5 copies of the PfCSP peptide, for example, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, or 4 to 5 copies. In a non-limiting example, a fusion protein disclosed herein includes 2 to 3 copies of the PfCSP peptide (e.g., SEQ ID NO: 1 or 32).

[0149] In some aspects, the PfCSP peptide includes or consists of 13 to 55 consecutive amino acid residues of PfCSP, for example, 13 to 50, 13 to 45, 13 to 40, 13 to 35, 13 to 30, 13 to 25, 13 to 22, 13 to 21, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 22, 15 to 21 , 17 to 55, 17 to 50, 17 to 45, 17 to 40, 17 to 35, 17 to 30, 17 to 25, 17 to 22, 17 to 21, 19 to 55, 19 to 50, 19 to 45, 19 to 40, 19 to 35, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 21, or 19 to 20 amino acid residues, wherein the consecutive amino acid residues include SEQ ID NO: 32. In a non-limiting example, the PfCSP peptide includes or consists of 13 to 25 consecutive amino acid residues of PfCSP, wherein the consecutive amino acid residues include SEQ ID NO: 32. In a further non-limiting example, the PfCSP peptide includes or consists of 13 to 21 consecutive amino acid residues of PfCSP, wherein the consecutive amino acid residues include SEQ ID NO: 32.

[0150] In some aspects, the PfCSP peptide includes or consists of 19 to 55 consecutive amino acid residues of PfCSP, for example, 19 to 50, 19 to 45, 19 to 40, 19 to 35, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 21, or 19 to 20 amino acid residues, wherein the consecutive amino acid residues include SEQ ID NO: 1. In a non- limiting example, die PfCSP peptide includes or consists of 19 to 25 consecutive amino acid residues of PfCSP, wherein the consecutive amino acid residues include SEQ ID NO: 1. In a further non-limiting example, die PfCSP peptide includes or consists of 19 to 21 consecutive amino acid residues of PfCSP, wherein the consecutive amino acid residues include SEQ ID NO: 1.

[0151] In some aspects, the PfCSP peptide includes or consists of a NPDP19 epitope. In some aspects, the PfCSP peptide includes or consists of an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. In a non-limiting example, the PfCSP peptide includes or consists of an amino acid sequence at least 94% identical to4239-112675-02

[0152] SEQ ID NO: 1. In a further non-limiting example, the PfCSP peptide includes or consists of the amino acid sequence set forth as SEQ ID NO: 1.

[0153] In some aspects, the PfCSP peptide includes or consists of a truncated NPDP19 epitope. In some aspects, tire PfCSP peptide includes or consists of an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 32. In a nonlimiting example, the PfCSP peptide includes or consists of an amino acid sequence at least 92% identical to SEQ ID NO: 32. In a further non-limiting example, the PfCSP peptide includes or consists of tire amino acid sequence set forth as SEQ ID NO: 32.

[0154] The multiple copies of the PfCSP peptide are fused by peptide linkers. The peptide linkers can be any suitable peptide linker, for example and without limitation, GGSGG (SEQ ID NO: 31), GSGSA (SEQ ID NO: 86), GGGGSn(SEQ ID NO: 87), KESGSVSSEQLAQFRSLD (SEQ ID NO: 88), EGKSSGSGSESKS (SEQ ID NO: 89), or SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 90). In some aspects, the peptide linker is GGSGG (SEQ ID NO: 31) or GSGSA (SEQ ID NO: 86). The peptide linkers of use in the fusion proteins disclosed herein are conventional, and additional examples have been described, for example, in Chen et al., “Fusion Protein Linkers: Property, Design and Functionality,” Adv Drug Deliv Rev. 65(10): 1357-1369, 2012.

[0155] In a non-limiting example, the multiple copies of the PfCSP peptide include or consist of 2 copies of the PfCSP peptide (e.g., two repeats of SEQ ID NO: 1). In some aspects, the multiple copies of the PfCSP peptide comprises or consists of SEQ ID NO: 4. In a further non-limiting example, the multiple copies of the PfCSP peptide include or consist of 3 copies of the PfCSP peptide (e.g., three repeats of SEQ ID NO: 1). In some aspects, the multiple copies of the PfCSP peptide comprises or consists of SEQ ID NO: 5. In several aspects, the multiple copies of the PfCSP peptides are included in the fusion protein as a contiguous tandem array fused by peptide linkers.

[0156] B. Nanoparticle Subunits4239-112675-02

[0157] The fusion proteins disclosed herein can further include a subunit of a protein nanoparticle. In some aspects, the protein nanoparticle subunit is a self-assembling protein nanoparticle subunit, which is a monomer of a self-assembling protein nanoparticle, or a fragment of the monomer that at least retains the portion of the monomer required for self-assembly into nanoparticles.

[0158] Examples of protein nanoparticle subunits that can be included in a fusion protein disclosed herein include, but are not limited to: lumazine synthase subunit, ferritin subunit, encapsulin subunit, sulfur oxygenase reductase (SOR) subunit (see, e.g., Urich et al., Science, 311:996-1000, 2006), pyruvate dehydrogenase subunit nanoparticles (see, e.g., Izard et al., PNAS 96: 1240-1245, 1999), and BP26 subunit (see, e.g., SEQ ID NO: 62). In some aspects, the protein nanoparticle subunit includes or consists of a lumazine synthase, ferritin, or BP26 subunit. In some aspects, the protein nanoparticle subunit includes or consists of a lumazine synthase or ferritin subunit.

[0159] In some aspects, a fusion protein disclosed herein includes a lumazine synthase subunit to construct a self-assembling lumazine synthase nanoparticle including the multiple copies of the PfCSP peptide. Lumazine synthase is an enzyme that catalyzes the penultimate step in the biosynthesis of riboflavin, which is generally known as vitamin B2. Lumazine synthase nanoparticles are icosahedral cages composed of 60 lumazine synthase subunits, however, it is possible to create larger cages with 180 subunits.

[0160] Lumazine synthase subunits can be obtained or derived from any suitable organism, for example, a bacterial species (e.g., Aquifex aeolicus). Non-limiting examples of suitable lumazine synthase subunits are provided herein, for example:

[0161] MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 3) MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERA GTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 36) QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAG ELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAG TKHGNKCWEAALSAIEMANLFKSLR (SEQ ID NO: 37) QIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAG ELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGT KHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 38) QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAG ELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAG TKHGNKCWEAALSAIEMANLFKSLR (SEQ ID NO: 39)

[0162] Also encompassed by the disclosure are lumazine synthase subunits including one or more modifications, for example to enhance a desired property or function of a resulting lumazine synthase4239-112675-02

[0163] nanoparticle. The modifications can be conservative or non-conservative amino acid substitutions, provided that the substitutions do not inhibit or prevent self-assembly of the nanoparticles.

[0164] In a non-limiting example, one or more cysteine substitutions (e.g., 2-10, 2-6, or 2-4 substitutions) are introduced to create non-native disulfide bond(s) that stabilize lumazine synthase nanoparticles formed from self-assembled subunits. In some aspects, the non-native disulfide bond(s) are introduced with L121C-K131C, L121CG-K131C, L121GC-K131C, K7C-R40C, I3C-L50C, I82C-K131CG, E5C-R52C, or E95C-A101C substitutions, or combinations thereof (e.g., I3C-L50C and I82C-K131CG; E5C-R52C and I82C-K131CG; or E95C-A101C and I82C-K131CG). The residue numbering is with reference to the lumazine synthase subunit set forth as SEQ ID NO: 36. Nonlimiting examples of lumazine synthase subunits non-native disulfide bond(s) include:

[0165] QIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAG ELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTCEQAIERAG TCHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 40) QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAG ELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTCGEQAIERA GTCHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 41) QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAG ELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTGCEQAIERA GTCHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 42) QIYEGCLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVCHGGREEDITLVRVPGSWEIPVAAG ELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGT KHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 43) QCYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITCVRVPGSWEIPVAAG ELARKEDIDAVIAIGVLCRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAG TCGHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 44) QIYCGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVCVPGSWEIPVAAG ELARKEDIDAVIAIGVLCRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAG TCGHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 45) QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAG ELARKEDIDAVIAIGVLCRGATPHFDYIASCVSKGLCDLSLELRKPITFGVITADTLEQAIERAG TCGHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 46)

[0166] In some aspects, a fusion protein disclosed herein includes a lumazine synthase subunit including an amino acid sequence at least 80% (e.g., 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%) identical to an amino acid sequence set forth as any one of SEQ ID NOs: 3 or 36-46. In a non-4239-112675-02

[0167] limiting example, a fusion protein disclosed herein includes a lumazine synthase subunit at least 90% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 3 or 36-46. In a further nonlimiting example, a fusion protein disclosed herein includes a lumazine synthase subunit at least 95% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 3 or 36-46. In another nonlimiting example, a fusion protein disclosed herein includes a lumazine synthase subunit including or consisting of an amino acid sequence set forth as any one of SEQ ID NOs: 3 or 36-46.

[0168] In some aspects, a fusion protein disclosed herein includes a lumazine synthase subunit at least 80% (e.g., 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%) identical to the amino acid sequence set forth as SEQ ID NO: 3. In a non-limiting example, a fusion protein disclosed herein includes a lumazine synthase subunit at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 3. In a further non-limiting example, a fusion protein disclosed herein includes a lumazine synthase subunit at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 3. In another non-limiting example, a fusion protein disclosed herein includes a lumazine synthase subunit including or consisting of the amino acid sequence set forth as SEQ ID NO: 3.

[0169] The lumazine synthase subunits useful for the compositions and methods disclosed herein include at least a portion of monomeric lumazine synthase that is required for assembly of a corresponding nanoparticle.

[0170] In some aspects, a fusion protein disclosed herein includes a ferritin subunit to construct a self-assembling ferritin nanoparticle including the multiple copies of the PfCSP peptide. Ferritin is a globular protein that is found in all animals, bacteria, and plants, that acts primarily to control the rate and location of polynuclear Fedll^Oj formation through the transportation of hydrated iron ions and protons to and from a mineralized core. Although ferritins from different species vary in amino acid sequence, they share similarity in architecture. Ferritin nanoparticles are formed from 24 copies of monomeric ferritin subunits.

[0171] Ferritin subunits can be obtained or derived from any suitable organism, for example, a bacterium, mammal, or insect. In some aspects, die ferritin subunit is obtained or derived from a bacterium. Non-limiting examples of suitable ferritin subunits are provided herein, for example: DPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIF LNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori- SEQ ID NO: 65) DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori', SEQ ID NO: 47) DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPHHKFHGLTHIFHKAYHHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHE EEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori' SEQ ID NO: 48)4239-112675-02

[0172] DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori SEQ ID NO: 49) DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori SEQ ID NO: 50) DIIKLLNEQVNKEMDSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (H. pylori; SEQ ID NO: 51) DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIG (H. pylori; SEQ ID NO: 52) DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNEN (H. pylori; SEQ ID NO: 53)

[0173] Insect ferritin protein nanoparticles are also of use in the compositions and methods disclosed herein. Insect ferritin and, production and use in nanoparticles, has been described, for example, in PCT Pub. No. WO 2018 / 005558. Unlike bacterial ferritin, insect ferritin includes twelve copies of two different subunits (termed heavy and light chains; 24 subunits total). The insect ferritin heavy chains trimerize and the insect ferritin light chains trimerize, thereby forming four trimers of heavy chains and four trimers of light chains, which self-assemble into the globular nanoparticle. In several aspects, the insect ferritin heavy and light chains are from the Lepidoptera order of insects, for example, ferritin heavy and light chains from Trichoplusia (e.g., Trichoplusia ni), or ferritin heavy and light chains from Manduca. Exemplary ferritin heavy and light chain amino acid sequences for Trichoplusia ni and Manduca proteins are provided below:

[0174] Trichoplusia ni ferritin heavy chain RSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLL MRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHL VDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV (SEQ ID NO: 54) Trichoplusia ni ferritin light chain EYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDII KHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNS QHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

[0175] (SEQ ID NO: 55)4239-112675-02

[0176] Manduca ferritin heavy chain RSCRDSMRRQIQMEVGASLQYLAMGAHFSKDKINRPGFAKLFFDAAGEEREHAMKLIEYLL MRGELTNDVTSLIQVRAPQRNKWEGGVDALEHALKMESDVTKSIRTVIKACEDDPEFNDYH LVDYLTGEFLEEQYKGQRDLAGKASTLKKMLDRNSALGEFIFDKKLMGMDI (SEQ ID NO: 56)

[0177] Manduca ferritin light chain EYGHHGNVAKEMQAYAALHLERSYEYLLSSSYFNNYQTNRAGFSKLFRKLSDDAWEKTIDL IKHITMRGDEMNFAQRSTQKSVDRKNYTVELHELESLAKALDTQKELAERAFFIHREATRNS QHLHDPEVAQYLEEEFIEDHAKTIRNLAGHTTDLKRFVSGDNGQDLSLALYVFDEYLQKTV

[0178] (SEQ ID NO: 57)

[0179] In some aspects, a fusion protein disclosed herein includes a ferritin subunit including an amino acid sequence at least 80% (e.g., 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%) identical to an amino acid sequence set forth as any one of SEQ ID NOs: 47-57 or 65. In a nonlimiting example, a fusion protein disclosed herein includes a ferritin subunit at least 90% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 47-57 or 65. In a further non-limiting example, a fusion protein disclosed herein includes a ferritin subunit at least 95% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 47-57 or 65. In another non-limiting example, a fusion protein disclosed herein includes a ferriti n subunit including or consisting of an amino acid sequence set forth as any one of SEQ ID NOs: 47-57 or 65.

[0180] In some aspects, a fusion protein disclosed herein includes a ferritin subunit including an amino acid sequence at least 80% (e.g., 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%) identical to the amino acid sequence set forth as SEQ ID NO: 65. In a non-limiting example, a fusion protein disclosed herein includes a ferritin subunit at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 65. In a further non-limiting example, a fusion protein disclosed herein includes a ferritin subunit at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 65. In another non-limiting example, a fusion protein disclosed herein includes a ferritin subunit including or consisting of the amino acid sequence set forth as SEQ ID NO: 65.

[0181] Also encompassed by this disclosure are ferritin subunits including one or more modifications, for example to enhance a desired property or function of a resulting ferritin nanoparticle. The modifications can be conservative or non-conservative amino acid substitutions, provided that the substitutions do not inhibit or prevent self-assembly of the nanoparticles. In some aspects, the modifications include one or more cysteine substitutions (e.g., 2-10, 2-6, or 2-4 substitutions) to introduce non-native disulfide bond(s) that stabilize the ferritin nanoparticle formed from the self-assembled subunits. Examples of stabilized ferritin nanoparticles have been described, for example, in US 12,053,519 (US 17 / 277,694).4239-112675-02

[0182] The ferritin subunits useful for the compositions and methods disclosed herein include at least a portion of monomeric ferritin that is required for assembly of a corresponding nanoparticle.

[0183] In some aspects, a fusion protein disclosed herein includes a BP26 subunit to construct a selfassembling BP26 nanoparticle including the multiple copies of the PfCSP peptide. BP26 is a protein from die Brucella bacterium diat is known to self-assemble into stable, nano-sized structures, which are sometimes referred to as nanobarrels (or NanoB) due to their barrel-like structure with a hollow center. BP26 nanoparticles are typically formed from 16 monomeric BP26 subunits.

[0184] A non-limiting example of a suitable BP26 subunit is provided herein as SEQ ID NO: 62. In some aspects, a fusion protein disclosed herein includes a BP26 subunit including an amino acid sequence at least 80% (e.g., 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%) identical to the amino acid sequence of SEQ ID NO: 62. In a non-limiting example, a fusion protein disclosed herein includes a BP26 subunit at least 90% identical to the amino acid sequence of SEQ ID NO: 62. In a further non-limiting example, a fusion protein disclosed herein includes a BP26 subunit at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 62. In another non-limiting example, a fusion protein disclosed herein includes a BP26 subunit including or consisting of the amino acid sequence set forth as SEQ ID NO: 62.

[0185] Also encompassed by this disclosure are BP26 subunits including one or more modifications, for example to enhance a desired property or function of a resulting BP26 nanoparticle. The modifications can be conservative or non-conservative amino acid substitutions, provided that the substitutions do not inhibit or prevent self-assembly of the nanoparticles. In some aspects, the modifications include one or more cysteine substitutions (e.g., 2-10, 2-6, or 2-4 substitutions) to introduce non-native disulfide bond(s) that stabilize the BP26 nanoparticle formed from the selfassembled subunits.

[0186] The BP26 subunits useful for the compositions and methods disclosed herein include at least a portion of monomeric BP26 that is required for assembly of a corresponding nanoparticle.

[0187] In some aspects, a fusion protein disclosed herein includes an encapsulin subunit to construct a self-assembling encapsulin nanoparticle including tire multiple copies of the PfCSP peptide.

[0188] Encapsulin proteins are a conserved family of bacterial protein, which can be found, for example, in Thermotoga maritime, Pyrococcus furiosus, Rhodococcus erythropolis, Myxococcus xanthus.

[0189] Encapsulins are also known as linocin-like proteins, and they form large protein assemblies that function as a minimal compartment to package enzymes. Encapsulin nanoparticles are made up of monomeric subunits, which are polypeptides having a molecule weight of approximately 30 kDa. Following production, the monomeric subunits self-assemble into tire globular- encapsulin assembly including 60, or in some cases, 180 monomeric subunits. Methods of constructing encapsulin nanoparticles have been described, for example, in Sutter et al. (Nature Struct, and Mol. Biol. , 15:939-947, 2008).4239-112675-02

[0190] A non-limiting example of a suitable encapsulin subunit is provided herein:

[0191] MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDE NEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCE KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYP LEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQV VNPEALILLKF (SEQ ID NO: 58)

[0192] Also encompassed by this disclosure are encapsulin subunits including one or more modifications, for example to enhance a desired property or function of a resulting encapsulin nanoparticle. The modifications can be conservative or non-conservative amino acid substitutions, provided that the substitutions do not inhibit or prevent self-assembly of the nanoparticles. In some aspects, the modifications include one or more cysteine substitutions (e.g., 2-10, 2-6, or 2-4) to introduce non-native disulfide bond(s) that stabilize the encapsulin nanoparticle formed from the selfassembled subunits. Examples of stabilized encapsulin subunits include, for example:

[0193] MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDE NEVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCE KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYP LEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQV VNPEALILLKF (SEQ ID NO: 59) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDE NEVVKWGLRKSLPLIELRATFTLDLWELDNLErGcPNVDLSSLEETVRKVAEFEDEVIFRGCE KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYP LEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQV VNPEALILLKF (SEQ ID NO: 60) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLgEVEVLSDEN EVVKWGLRKSLPLIELRATFTLDLWELDNLErGkPNVDLSSLEETVRKVAEFEDEVIFRGCEKS GVKGLLSFEERKIECGSTPcDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEcGHYPLEK RVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNP EALILLKF (SEQ ID NO: 61)

[0194] In some aspects, a fusion protein disclosed herein includes an encapsulin subunit including an amino acid sequence at least 80% (e.g., 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%) identical to amino acid sequence set forth as any one of SEQ ID NOs: 58-61. In a non-limiting example, a fusion protein disclosed herein includes an encapsulin subunit at least 90% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 58-61. In a further non-limiting example, a fusion protein disclosed herein includes an encapsulin subunit at least 95% identical to an amino acid sequence set forth as any one of SEQ ID NOs: 58-61. In another non-limiting example, a fusion protein disclosed herein includes an encapsulin subunit including or consisting of an amino acid sequence set forth as any one of SEQ ID NOs: 58-61.4239-112675-02

[0195] The encapsuliii subunits useful for the compositions and methods disclosed herein include at least a portion of monomeric encapsulin that is required for self-assembly of a corresponding nanoparticle.

[0196] In some aspects, the fusion protein includes two copies of the PfCSP epitope fused to a protein nanoparticle subunit (e.g., SEQ ID NO: 6). In some aspects, the fusion protein includes three copies of die PfCSP epitope fused to a protein nanoparticle subunit (e.g., SEQ ID NO: 7). In nonlimiting examples, the fusion protein comprises or consists of SEQ ID NO: 6 or SEQ ID NO: 7.

[0197] C. T-Cell Epitopes

[0198] The fusion proteins disclosed herein can further include a T-cell epitope. T cell epitopes are immunogenic peptide sequences that elicit a T-cell response. Without being bound by any particular theory, the presence of a T-cell epitope on a nanoparticle may improve elicitation of an immune response of a subject, for example, by eliciting long-lived pathogen-specific memory T cells that can quickly activate protective effector functions.

[0199] Any suitable T-cell epitope sequence can be included on the fusion protein, for example, and without limitation, a pan DR epitope (PADRE; SEQ ID NO: 2), P2 epitope (SEQ ID NO: 63), TpD epitope ( ILMQYIKANSKFIGKVSVRQSIALSSLMVAQ; SEQ ID NO: 64), HIV envelope epitope (e.g., AENLWVTVYYGVPVW (SEQ ID NO: 66) or TEKLWVTVYYGVPVW (SEQ ID NO: 67)), p458m epitope (NEDQKIGIEIIKRALKI; SEQ ID NO: 68), P30 epitope (FNNFTVSFWLRVPKVSASHLE; SEQ ID NO: 69), diphtheria toxin epitope (e.g., PVFAGANYAAWAVNVAQVI (DTD271-290; SEQ ID NO: 70), HHNTEEIVAQSIALSSLMV (DTD321-340; SEQ ID NO: 71), QSIALSSLMVAQAIPLVGEL (DTD331-350; SEQ ID NO: 72), VDIGFAAYNFVESIINLFQV (DTD351-370; SEQ ID NO: 73), QGESGHDIKITAENTPLPIA (DTD411-430; SEQ ID NO: 74), or GVLLPTIPGKLDVNKSKTHI (DTD431^150; SEQ ID NO: 75)), or tetanus toxin epitope (e.g., NSVDDALINSTKIYSYFPSV (TT 580-599; SEQ ID NO: 76), QYIKANSKFIGITEL (TT 830-844; SEQ ID NO: 77), PGINGKAIHLVNNESSE (TT 916-932; SEQ ID NO: 78), FNNFTVSFWLRVPKVSASHLE (TT 947-967; SEQ ID NO: 79)).

[0200] In some aspects, the T-cell epitope includes or consists of an amino acid sequence set forth as any one of SEQ ID NOs: 2, or 63-64. In a non-limiting example, die T-cell epitope is a pan DR epitope (PADRE) including or consisting of the amino acid sequence set forth as SEQ ID NO: 2. In a furdier non-limiting example, the T-cell epitope is a P2 epitope including or consisting of the amino acid sequence set forth as SEQ ID NO: 63. In a further non-limiting example, die T-cell epitope is a TpD epitope including or consisting of an amino acid sequence set forth as SEQ ID NO: 64.

[0201] The heterologous T-cell epitope can be located at any suitable position of the fusion protein, including (but not limited to) the N-terminus, the C-terminus, or at a position between a protein nanoparticle subunit and the multiple copies of the PfCSP peptide. In some aspects, the T cell epitope is N-terminal to the multiple copies of the PfCSP peptide. In some aspects, the heterologous T-cell4239-112675-02

[0202] epitope is linked to a protein nanoparticle subunit or the multiple copies of the PfCSP peptide by one or more peptide linkers (e.g., GGSGG (SEQ ID NO: 31), GSGSA (SEQ ID NO: 86), GGGGSn(SEQ ID NO: 87), KESGSVSSEQLAQFRSLD (SEQ ID NO: 88), EGKSSGSGSESKS (SEQ ID NO: 89), or SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 90)).

[0203] D. Fusion Protein Configurations

[0204] For illustrative purposes, several non-limiting exemplary fusion protein configurations are provided. In some aspects, the fusion protein includes from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, and the multiple copies of tire PfCSP peptide (e.g., two or three copies of tire PfCSP peptide). In a non-limiting example, the fusion protein includes from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, a first copy of the PfCSP peptide, and a second copy of the PfCSP peptide. In a further non-limiting example, the fusion protein includes from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, a first copy of the PfCSP peptide, a second copy of the PfCSP peptide; and a third copy of the PfCSP peptide. The first, second, and third copies of the PfCSP peptide are copies of the same amino acid sequence. In some aspects, the first, second, and third PfCSP peptides all include SEQ ID NO: 32, or include or consist of SEQ ID NO: 1.

[0205] In some aspects, the fusion protein includes or consists of an amino acid sequence at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 8, 9, or 13-26. In a non-limiting example, the fusion protein includes or consists of an amino acid sequence at least 90% identical to SEQ ID NO: 8, 9, or 13-26. In a further non-limiting example, the fusion protein includes or consists of an amino acid sequence at least 95% identical to SEQ ID NO: 8, 9, or 13-26. In another non-limiting example, the fusion protein includes or consists of an amino acid sequence at least 98% identical to SEQ ID NO: 8, 9, or 13-26. In some aspects, the fusion protein includes or consists of SEQ ID NO: 8, 9, or 13-26.

[0206] In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of SEQ ID NOs: 8, 9, or 13-26. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 8, 9, or 13-26. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 8, 9, or 13-26. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 98% identical to any one of SEQ ID NOs: 8, 9, or 13-26. In some aspects, the fusion protein includes or consists of any one of SEQ ID NOs: 8, 9, or 13-26.

[0207] In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and includes or consists of an amino acid sequence at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,4239-112675-02

[0208] 99% or 100%) identical to SEQ ID NO: 8 or SEQ ID NO: 9. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 9. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 95% identical to SEQ ID NO: 8 or SEQ ID NO: 9. In some aspects, the fusion protein includes SEQ ID NO: 1 or 32, and the fusion protein includes or consists of an amino acid sequence at least 98% identical to SEQ ID NO: 8 or SEQ ID NO: 9.

[0209] Individual components of the fusion protein (e.g., T cell epitope, protein nanoparticle subunit, or the PfCSP peptide) can be directly linked, or indirectly linked by peptide linkers (see, e.g., SEQ ID NO: 31 or 86-90). In some aspects, the peptide linker is a glycine serine linker (e.g., SEQ ID NO: 31). The fusion protein can further include spacer sequences, which are sequences added to facilitate cloning (e.g., adding restriction enzyme sites or additional sequence to maintain proper reading frame). Spacers are typically short sequences, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In some aspects, a spacer is 1-9 amino acids, for example, 1 to 3 or 1 to 5 amino acids.

[0210] The fusion proteins disclosed herein can further include a signal peptide, which is a sequence that directs newly synthesized secretory or membrane proteins to and through target membranes. Signal peptides are typically located at the N-terminus of a polypeptide (e.g., a fusion protein disclosed herein). Fusion proteins including a signal peptide are typically considered a precursor molecule or immature form of the fusion protein because signal peptides are typically removed by signal peptidases after the polypeptide have crossed a target membrane. Thus, signal peptides are not included in the mature fusion protein or nanoparticles thereof. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). An exemplary signal peptide sequence is set forth as MDSKGSSQKGSRLLLLLVVSNLLLPQGV (SEQ ID NO: 33), however, one of ordinary skill in the art can select an appropriate signal peptide based on the relevant application or expression system. Further examples of suitable signal peptide sequences include, but are not limited to, MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 83);

[0211] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVA (SEQ ID NO: 84); or MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA (SEQ ID NO: 85).

[0212] In some aspects, the fusion proteins disclosed herein include a signal peptide including or consisting of any one of SEQ ID NOs: 33 or 83-85, or a sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical thereto linked to the N-terminal end of the fusion protein. In a non-limiting example, the fusion proteins disclosed herein include a signal peptide including or consisting of any one of SEQ ID NOs: 33 or 83-85, and the signal peptide is fused to the N-terminal end of the fusion protein. In a further non-limiting example, the fusion proteins disclosed herein include a signal peptide including or consisting of SEQ ID NO: 33 or 83,4239-112675-02

[0213] and the signal peptide is fused to the N-terminal end of the fusion protein. In some aspects, the signal peptide is N-terminal relative to a T cell epitope and / or the multiple copies of the PfCSP peptide.

[0214] E. Additional Description

[0215] The fusion proteins disclosed herein can further include an additional Plasmodium falciparum peptide, for example, a second, third, fourth, fifth, etc., Plasmodium falciparum peptide that is different relative to the PfCSP peptide that is included in multiple copies. As a non-limiting example, a fusion protein disclosed herein can include a Plasmodium falciparum peptide sequence including a NANP5 or NANP9 epitope sequence in addition to multiple copies of a PfCSP peptide including or consisting of SEQ ID NO: 1. In some aspects, the additional Plasmodium falciparum peptide does not include a CSP junctional epitope.

[0216] The additional Plasmodium falciparum peptide can include or consist of a peptide sequence including the NANP5 epitope (e.g., SEQ ID NO: 80). In a non-limiting example, the additional Plasmodium falciparum peptide includes 20-200 consecutive residues of PfCSP, wherein the 20-200 consecutive residues include SEQ ID NO: 80. In a further non-limiting example, the additional Plasmodium falciparum peptide includes 20-100 consecutive residues of PfCSP, wherein the 20-100 consecutive residues include SEQ ID NO: 80. In another non-limiting example, the additional Plasmodium falciparum peptide includes 20-40 consecutive residues of PfCSP, wherein the 20-40 consecutive residues include SEQ ID NO: 80. In some aspects, the additional Plasmodium falciparum peptide includes or consists of the amino acid sequence set forth as SEQ ID NO: 80.

[0217] The additional Plasmodium falciparum peptide can include or consist of a peptide sequence including the NANP9 epitope (e.g., SEQ ID NO: 81). In a non-limiting example, the additional Plasmodium falciparum peptide includes 36-200 consecutive residues of PfCSP, wherein the 36-200 consecutive residues include SEQ ID NO: 81. In a further non-limiting example, the additional Plasmodium falciparum peptide includes 36-100 consecutive residues of PfCSP, wherein the 36-100 consecutive residues include SEQ ID NO: 81. In another non-limiting example, the additional Plasmodium falciparum peptide includes 36-72 consecutive residues of PfCSP, wherein the 36-72 consecutive residues include SEQ ID NO: 81. In some aspects, the additional Plasmodium falciparum peptide includes or consists of the amino acid sequence set forth as SEQ ID NO: 81.

[0218] The fusion proteins (or nanoparticles thereof) disclosed herein can further include other- heterologous proteins or peptides provided that such sequences do not prevent or inhibit nanoparticle formation or assembly. In some aspects, the heterologous protein is a carrier protein, which is any carrier protein suitable for use in a vaccine. Examples of suitable carrier proteins are those that can increase the immunogenicity of the protein nanoparticle and / or help elicit antibodies that are diagnostically, analytically, and / or therapeutically beneficial. Specific, non-limiting examples of suitable carrier proteins include, but are not limited to, natural, semi-synthetic or synthetic polypeptides or proteins from bacteria or viruses.4239-112675-02

[0219] Bacterial products that are useful as carrier proteins include bacterial toxins, for example those that are single polypeptide chains (or a fragment thereof) that mediate toxic effects, inflammatory responses, stress, shock, chronic sequelae, or mortality in a susceptible host. Specific, non-limiting examples of such bacterial toxins include, but are not limited to: single polypeptide chains of B. anthracis PA (for example, as encoded by bases 143779 to 146073 of GENBANK® Accession No. NC 007322); B. anthracis LF (for example, as encoded by the complement of bases 149357 to 151786 of GENBANK® Accession No. NC 007322); bacterial toxins and toxoids, for example tetanus toxin / toxoid (for example, as described in U.S. Patent Nos. 5,601,826 and 6,696,065); diphtheria toxin / toxoid (for example, as described in U.S. Patent Nos. 4,709,017 and 6,696,065), for example tetanus toxin heavy chain C fragment; P. aeruginosa exotoxin / toxoid (for example, as described in U.S. Patent Nos. 4,428,931, 4,488,991 and 5,602,095); pertussis toxin / toxoid (for example, as described in U.S. Patent Nos.4,997,915, 6,399,076 and 6,696,065); and C. perfringens exotoxin / toxoid (for example, as described in U.S. Patent Nos. 5,817,317 and 6,403,094) C. difficile toxin B or A, or analogs or mimetics of and combinations of two or more thereof. Viral proteins, for example hepatitis B surface antigen (for example, as described in U.S. Patent Nos. 5,151,023 and 6,013,264) and core antigen (for example, as described in U.S. Patent Nos. 4,547,367 and 4,547,368) can also be used as carriers, as well as single polypeptide chains of proteins from higher organisms for example keyhole limpet hemocyanin (KLH), horseshoe crab hemocyanin, Concholepas Concholepas Hemocyanin (CCH), Ovalbumin (OVA), edestin, mammalian serum albumins (e.g., bovine serum albumin), and mammalian immunoglobulins.

[0220] In some aspects, the heterologous carrier protein is a tetanus toxoid, diphtheria toxin variant CRM197, or H influenzae protein D (HiD). CRM197 is a genetically detoxified form of diphtheria toxin; a single mutation at position 52, substituting glutamic acid for glycine, causes tire ADP-ribosyltransferase activity of the native diphtheria toxin to be lost. For additional description of exemplary protein carriers for vaccines, see Pichichero, Protein carriers of conjugate vaccines: characteristics, development, and clinical trials, Hum Vaccin Immunother., 9: 2505-2523,2013).

[0221] The fusion proteins disclosed herein (or nanoparticles thereof) can further include linked or conjugated targeting moieties, for example, a moiety that binds to a component of die immune system of a subject. In some aspects, tire targeting moiety is a TLR-1 / 2 / 6 agonist (e.g., lipopeptides and glycolipids, e.g., Pam2cys or Pam3cys lipopeptides); TLR-3 agonist (e.g., dsRNA, e.g., PolyEC, nucleotide base analogs); TLR-4 agonist (e.g., lipopolysaccharide (LPS) derivatives and small molecule analogs of pyrimidoindole); TLR5 agonist (e.g., flagellin); TLR-7 / 8 agonist (e.g., ssRNA and nucleotide base analogs, including derivatives of imidazoquinolines, hydroxy-adenine, benzonapthyridine and loxoribine); and TLR-9 agonist (e.g., unmethylated CpG); Stimulator of Interferon Genes (STING) agonist (e.g., cyclic dinucleotides, e.g., cyclic diadenylate monophosphate); C-type lectin receptor (CLR) agonist (e.g., various mono, di, tri and polymeric sugars drat can be linear or branched, e.g., mannose, Lewis-X tri-saccharides, etc.); RIG-I-like4239-112675-02

[0222] receptor (RLR) agonist; and NOD-like receptor (NLR) agonist (e.g., peptidogylcans and structural motifs from bacteria, e.g., meso-diaminopimelic acid and muramyl dipeptide); or combinations thereof. In a non-limiting example, the targeting moiety is a TLR-5 agonist (e.g., flagellin).

[0223] Targeting moieties can be linked or conjugated to a fusion protein disclosed herein (or nanoparticle thereof) by any suitable means. For example, in some aspects, the fusion protein includes a streptavidin sequence, and the targeting moiety is biotinylated, thereby linking the targeting moiety and fusion protein (or nanoparticle thereof) through biotin-streptavidin linkage. In other aspects, the biotinylated moiety is covalently linked to the fusion protein (or nanoparticle thereof), or translationally fused (e.g., by a peptide linker).

[0224] The fusion proteins disclosed herein can further include a tag, for example, a tag to measure or detection expression (e.g., fluorescent tag (e.g., GFP), or a luciferase tag), or a tag to facilitate isolation or purification (e.g., his tag) of the fusion proteins (or nanoparticles thereof). In some aspects, the tag is cleavable from the fusion protein (e.g., is removed from the fusion protein after purification or isolation). A tag can be made “cleavable,” for example, by including a sequence that is recognized by a protease that links the fusion protein and the tag. Treatment with the protease facilitates release of the tag from the fusion protein. A step of removing a tag from a fusion protein can be included in any of the methods disclosed herein.

[0225] The fusion proteins disclosed herein can further include a streptavidin sequence to facilitating linkage of biotinylated molecule (e.g., tag, label, targeting moiety, vaccine antigen, or therapeutic (e.g., a therapeutic to treat or inhibit malaria infection)) to the fusion protein (or a nanoparticle thereof). In some aspects, the streptavidin sequence can be used to facilitate purification of the fusion proteins or nanoparticles thereof. A spytag-spycatcher system can also be used, for example, to link a tag, label, targeting moiety, vaccine antigen, or therapeutic to a fusion protein disclosed herein (see, e.g., Hatlem et al., “Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins” Int J Mol Sci. 20(9):2129, 2019).

[0226] IV. Nucleic Acids and Expression

[0227] Nucleic acid molecules (polynucleotides) encoding tire fusion proteins disclosed herein are also provided. Nucleic acid molecules include, for example, DNA, cDNA, and RNA sequences that encode the fusion protein. The genetic code can be used to determine suitable sequences encoding a fusion protein disclosed herein. It is understood that due to redundancy in the genetic code, a variety of functionally equivalent nucleic acids (e.g., degenerate variants) can be constructed that differ in nucleic acid sequence but encode the same amino acid sequence. Such variants are encompassed by the disclosure.

[0228] In several aspects, the nucleic acid molecule encodes a precursor of a disclosed fusion protein, that, when expressed in cells under appropriate conditions, is processed and self-assembles into protein nanoparticles. For example, the nucleic acid molecule can encode a signal peptide4239-112675-02

[0229] sequence (e.g., a N-terminal signal peptide) for entry into the cellular secretory system that is proteolytically cleaved during processing of the fusion protein. In another example, the nucleic acid molecule encodes a tag that is removed (e.g., cleaved by a protease) prior to nanoparticle assembly. In some aspects, the nucleic acid molecule does not include a signal peptide and / or tag.

[0230] Nucleic acid molecules can be synthesized or prepared using molecular cloning techniques. Examples of appropriate cloning techniques, and instructions sufficient to direct persons of ordinary skill in the art, are known (see, e.g., Sambrook et al. (Molecular' Cloning: A Laboratory Manual, 4thed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the selfsustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are known.

[0231] Nucleic acid molecules encoding a fusion protein disclosed herein can be incorporated into a vector (e.g., an autonomously replicating plasmid or virus) or into the genomic DNA of a prokaryote or eukaryote, or may exist as an isolated molecule (e.g., a cDNA) independent of other nucleic acid sequences. The nucleotides of the nucleic acid molecules disclosed herein can include ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term “nucleic acid molecule” includes both single and double stranded forms of DNA.

[0232] In some aspects, a nucleic acid molecule encoding a fusion protein disclosed herein is operatively linked to one or more expression control sequences, for example and without limitation, promoters, enhancers, transcription terminators, start codons (i.e., ATG in front of a protein-encoding gene), splicing signals for introns, stop codons, etc.

[0233] A nucleic acid molecule disclosed herein can be comprised on a nucleic acid vector.

[0234] Exemplary nucleic acid vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. In a non-limiting example, a nucleic acid disclosed herein is comprised on a plasmid or viral vector. Vectors can include nucleic acid sequences that permit replication in a host cell, for example, an origin of replication, or expression control sequences for protein expression (e.g., promoters, enhancers, transcription terminators, start codons, stop codons, etc.). Vectors can also include one or more selectable marker genes or tags to enable the selection of cells that have been successfully transformed with the vector, or other genetic elements. pVRC8400 is a non-limiting example of a suitable vector.

[0235] In some aspects, a host cell is transformed with a nucleic acid molecule encoding a disclosed fusion protein (or a vector thereof). Transformation includes stable (foreign DNA is continuously maintained in the host) or transient (foreign DNA is maintained in the host for a period of time, but is eventually degraded or lost) transformation. The host cell may be prokaryotic or eukaryotic. Suitable hosts include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA4239-112675-02

[0236] sequences having eukaryotic or viral sequences in prokaryotes are known, and a practitioner can select an appropriate method.

[0237] Non-limiting examples of suitable host cells include bacteria, archaea, insect, fungi (for example, yeast), plant, and animal cells (e.g., mammalian cells (e.g., human)). Specific, non-limiting examples include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid or lymphoid cell lines. Techniques for the propagation of mammalian cells in culture have been described (see, e.g., Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4thEd., Humana Press). Examples of commonly used mammalian host cell lines include VERO, HeLa, CHO, WI38, BHK, and COS cell lines, although other cell lines may be used, for example cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some aspects, the host cells include HEK293 cells or derivatives thereof, for example GnTI / _cells (ATCC® No. CRL-3022), or HEK-293F cells. The term “host cell’’ also includes any progeny of the subject host cell that cany the nucleic acid molecule.

[0238] Transformation of a host cell can be carried out by conventional techniques. In some aspects, if the host is prokaryotic, for example, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCU method. Alternatively, MgCl or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

[0239] When the host is a eukaryote, such methods of transformation include chemical methods (e.g., calcium phosphate coprecipitates), mechanical methods (e.g., micro injection or electroporation), liposome methods, or viral vector methods can be used. In a non-limiting example, a eukaryotic viral vector, for example simian virus 40 (SV40) or bovine papilloma virus, can be used to transiently infect or transform eukaryotic cells to express a fusion protein disclosed herein (see, for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). A practitioner can readily select an appropriate method of transformation based on the expression system.

[0240] Modifications can be made to a nucleic acid encoding a disclosed fusion protein and / or nanoparticle subunit without diminishing its biological activity. Some modifications can be made to facilitate the cloning or expression of the fusion protein. A non-limiting example of such modifications includes the addition of nucleic acids on either terminus to create conveniently located restriction sites or sequence spacers to maintain proper reading frame.

[0241] Further provided are methods of producing protein nanoparticles, the methods including expressing a nucleic acid molecule encoding a fusion protein disclosed herein in a host cell. In some aspects, the host cell is cultured under conditions sufficient for self-assembly of the expressed fusion proteins into protein nanoparticles. Conditions sufficient for self-assembly of protein nanoparticles disclosed herein include physiological conditions in a host cell, or a human cell or serum. In some aspects, the protein nanoparticles disclosed herein spontaneously self-assemble under standard cell4239-112675-02

[0242] culture conditions. In some aspects, the conditions sufficient for self-assembly of protein nanoparticles disclosed herein include an aqueous solution or buffer at a neutral or physiological pH. In some aspects, the conditions sufficient for self-assembly of protein nanoparticles disclosed herein include a temperature of 4-60 °C (e.g., 4-40 °C, 10-40 °C, 15-40 °C, 20-40 °C, 25-40 °C, 20-37 °C, 22-37 °C, etc.) In a non-limiting example, the conditions sufficient for self-assembly of protein nanoparticles include a temperature of 35-40 °C, about 8% CO2, and at least 50% humidity (e.g., at least 70% humidity, or about 80% humidity).

[0243] Self-assembled protein nanoparticles can be isolated or purified from the cell culture. In other aspects, expressed fusion proteins are purified from the cell culture and then separately incubated under conditions sufficient for self-assembly into protein nanoparticles.

[0244] Fusion proteins can be purified from a cell culture or sample using conventional techniques, for example, precipitation, centrifugation, chromatography (affinity, ion exchange, size exclusion, immobilized metal affinity chromatography, etc.), or electrophoresis. Similarly, nanoparticles can be purified from a cell culture or sample using conventional techniques, for example, centrifugation, ultrafiltration, chromatography, precipitation, or dialysis. In some aspects, a purified fusion protein or nanoparticle is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure. Isolated or purified protein nanoparticles can be included, for example, in an immunogenic composition.

[0245] V. Immunogenic Compositions

[0246] Immunogenic compositions that include a nanoparticle disclosed herein and a pharmaceutically acceptable carrier are also provided. Immunogenic compositions can be administered to a subject by any suitable route of administration, for example, oral, intranasal, intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In a non-limiting example, an immunogenic composition disclosed herein is administered to a subject intramuscularly. Actual methods for preparing administrable compositions are described in more detail in publications such as Remington’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020). In some aspects, an immunogenic composition disclosed herein is formulated for an intradermal or intramuscular injection.

[0247] In some aspects, an immunogenic composition described herein is formulated with a pharmaceutically acceptable earner to help retain biological activity while also promoting stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced mediums, phosphate buffer saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers, for example, proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is, or can be lyophilized. Lyophilized4239-112675-02

[0248] preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

[0249] Formulated compositions, particularly liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ^1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, mediylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

[0250] The immunogenic compositions of the disclosure can contain, as pharmaceutically acceptable vehicles, substances as required to approximate physiological conditions, for example pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0251] The immunogenic composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants include, for example, toll-like receptor agonists, alum (aluminum salts), A1PO4, alhydrogel, lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP), for example, POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA) may also be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to an immunogenic composition. In non-limiting examples, the adjuvant includes or consists of SMNP (saponin / MPLA nanoparticle), ALF (Army Liposome Formulation), ALFQ (ALF containing QS21 saponin), or ALFQA (ALFQ adsorbed to aluminum hydroxide); see, e.g., Alving el al. “Army Liposome Formulation (ALF) family of vaccine adjuvants” Expert Rev Vaccines 19(3): 279-292, 2020.

[0252] In some aspects, the immunogenic composition can be provided as a sterile composition. The immunogenic composition typically contains an effective amount of a disclosed nanoparticle. In some aspects, the immunogenic composition includes 1-300 pg of a disclosed nanoparticle, for example, 1-200 pg, 1-100 pg, 1-50 pg, 1-25 pg, 1-20 pg, 1-15 pg, 1-10 pg, or 1-5 pg. In anon-limiting example, the immunogenic composition includes about 1-10 pg. In some aspects, the immunogenic composition includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg. In some aspects, the immunogenic composition includes about 1 pg. In some aspects, the immunogenic composition includes about 5 pg. In some aspects, the immunogenic composition includes about 10 pg. Actual dosage is typically determined by practitioners using clinical trials, for example, to determine the amount of nanoparticles that elicits a protective immune response in a subject without significant, adverse side effects. In some aspects, the4239-112675-02

[0253] immunogenic composition can be provided in unit dosage form for use to elicit or prime an immune response in a subject, for example, to prevent Plasmodium falciparum infection in die subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and / or a metering mechanism for administering die unit dose or multiples thereof.

[0254] VI. Methods of Inducing an Immune Response

[0255] The disclosed nanoparticles or immunogenic compositions thereof can be administered to a subject to induce an immune response to Plasmodium falciparum to prevent or inhibit a Plasmodium falciparum infection and / or the development of malaria. The immune response can be a protective immune response, for example a response that prevents or reduces subsequent infection with Plasmodium falciparum. In several aspects, an effective amount of a nanoparticle disclosed herein, or immunogenic composition thereof, can be administered to a subject in order to generate a neutralizing immune response to Plasmodium falciparum.

[0256] When inhibiting or preventing Plasmodium falciparum infection, the methods are typically used to avoid subsequent infection in a Plasmodium falciparum seronegative subject (e.g., by inducing an immune response that protects against Plasmodium fal iparum infection). In some aspects, the methods include selecting a subject that is not currently infected with Plasmodium falciparum. In some aspects, the methods include selecting a subject at risk for contracting Plasmodium falciparum infection, or a subject at risk of developing malaria, and administering a disclosed nanoparticle or immunogenic composition to the subject to elicit a protective immune response to Plasmodium falciparum in the subject.

[0257] Inhibition of Plasmodium falciparum infection can include delaying the development of malaria or decreasing the severity of malaria symptoms in a subject. In some aspects, using the methods disclosed herein increases the time of survival or survival rate of a subject or population that is subsequently infected with Plasmodium falciparum relative to a control subject or population.

[0258] Typical subjects intended for treatment with the therapeutics and methods of the present disclosure include humans, as well as non-human primates and other susceptible veterinarian subjects (e.g., dogs, cats, goats, sheep, etc.). To identify subjects for prophylaxis, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, for example various ELISA and other immunoassay methods to detect and / or characterize Plasmodium falciparum infection. These and other routine methods allow the clinician to select patients in need of therapy using the methods and pharmaceutical compositions of the present disclosure.4239-112675-02

[0259] The disclosed nanoparticles and immunogenic compositions thereof can be used in coordinate (e.g., prime-boost) immunization protocols or combinatorial formulations. In some aspects, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogenic compositions or formulations, each directed toward eliciting an anti- Plasmodium falciparum immune response, for example an immune response to Plasmodium falciparum CSP protein. Separate compositions that elicit an anti-Plasmodium falciparum immune response can be combined to create a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol.

[0260] In a non-limiting example, an immunogenic composition disclosed herein (e.g., an immunogenic composition including nanoparticles dial include multiple copies of the PfCSP peptide) is administered in combination with another malaria vaccine, for example, R21 or RTS,S / AS01. In some aspects, an immunogenic composition disclosed herein is administered in combination with a malaria vaccine targeting another stage of the malaria life cycle, for example, the blood stage. In some aspects, co-administering the immunogenic composition disclosed herein with another malaria vaccine synergistically protects the subject from Plasmodium falciparum infection. In some aspects, co-administering the immunogenic composition disclosed herein (e.g., an immunogenic composition including LuS-2T orLuS-3T nanoparticles) with the R21 vaccine synergistically protects the subject from Plasmodium falciparum infection. Co-administration encompasses administration at substantially the same time, for example, within a time period of 0 to 36 hours (e.g., 0 to 48 hours, 0 to 24 hours, 0 to 12 hours, 0 to 6 hours, 0 to 4 hours, 0 to 1 hours, 0 to 30 minutes, 0 to 15 mins, 30 seconds to 12 hours, 30 seconds to 6 hours, 30 seconds to 4 hours, 30 seconds to 1 hour, 30 seconds to 30 minutes, 30 seconds to 15 mins, 1 minute to 48 hours, 1 minute to 24 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 4 hours, 1 minute to 1 hour, etc.). Co-administration can further encompass co-administration as part of a planned vaccine schedule, for example, where a first vaccination (e.g., R21) is administered to the subject, and a second vaccination (e.g., an immunogenic composition disclosed herein) is administered to the subject as a boost that occurs, for example, up to 4 weeks, 6 weeks, 12 weeks, 3 months, 6 months, 12 months, 24 months, or 36 months after tire first vaccination. In some aspects, the boost is administered 2-6 weeks after die first vaccination. In some aspects, the boost is administered 4-6 weeks after die first vaccination. In some aspects, the boost is administered about 4 weeks after the first vaccination.

[0261] In some aspects, the synergistic effect is an increase of at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, etc.) in protection against Plasmodium falciparum infection in the subject relative to a control. In some aspects, the synergistic effect is an increase of at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, etc.) of an immune response in the subject against Plasmodium falciparum relative to a control. In some aspects, the synergistic effect is an increase of at least 10% (e.g., at least 20%, at4239-112675-02

[0262] least 30%, at least 40%, at least 50%, at least 75%, at least 100%, etc.) in the production of neutralizing antibodies against Plasmodium falciparum in the subject relative to a control. In some aspects, the control is a subject or population administered R21 alone, or the immunogenic composition disclosed herein alone.

[0263] The immunogenic composition can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, for example, one to five (e.g., 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, or 3 to 4), or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. In some aspects, the immunogenic composition disclosed herein is administered to a subject annually or bi-annually.

[0264] In a non-limiting example, a suitable immunization regimen includes at least two separate inoculations with an immunogenic composition disclosed herein, with the second inoculation being administered at least 2, 4, 6, 8, 12, 24, or 52 weeks following the first inoculation. In some aspects, the second inoculation is administered about 4 weeks, 6 weeks, 8 weeks, 12 weeks, 6 months, one year, two years, three years, five years, or longer, after the first inoculation. Periodic inoculations beyond the second may also be desirable to enhance the subject's “immune memory.” The adequacy of the immunization parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., a neutralizing immune response to Plasmodium falciparum. If such monitoring indicates that immunization is sub-optimal, subjects can be boosted with an additional dose of immunogenic composition, and the immunization parameters can be modified in a fashion expected to potentiate the immune response.

[0265] In several aspects, the immunogenic composition disclosed herein is administered to the subject simultaneously with the administration of an adjuvant. In other aspects, the immunogenic composition is administered to tire subject after the administration of an adjuvant and within a sufficient amount of time to elicit tire immune response.

[0266] Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce tire occurrence or severity of targeted disease symptoms or conditions in the subject, or that elicit a desired response in the subject (e.g., a neutralizing or protective immune response). Suitable models in this regard include, for example, murine, porcine, feline, ferret, non-human primate models. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, ordinary calculations and adjustments can be used to4239-112675-02

[0267] determine an appropriate concentration and dose to administer an effective amount of the composition (for example, amounts that are effective to elicit a desired immune response).

[0268] Dosage can be varied depending on a variety of factors, for example, mode of administration, tlie subject’s condition, age, weight, height, sex, etc. Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. The actual dosage of disclosed immunogenic composition will vary according to factors, for example, the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of tire composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic response.

[0269] A non-limiting range for an effective amount of the disclosed nanoparticles, within the methods and immunogenic compositions of the disclosure, is about 0.00001 mg / kg body weight to about 1 mg / kg body weight, for example, about 0.00001 mg / kg, about 0.00005 mg / kg, about 0.0001 mg / kg, about 0.0005 mg / kg, about 0.001 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg body weight. In some aspects, the dosage includes a set amount of a disclosed immunogenic composition, for example, from about 1-300 pg, for example, a dosage of 1-200 pg, 1-100 pg, 1-50 pg, 1-25 pg, 1-20 pg, 1-15 pg, 1-10 pg, or 1-5 pg. In a non-limiting example, the dosage of a disclosed immunogenic composition is 1-10 pg. In some aspects, the dosage includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg of a disclosed immunogenic composition per administration. In some aspects, the dosage includes about 1 pg of a disclosed immunogenic composition per administration. In some aspects, tire dosage includes about 5 pg of a disclosed immunogenic composition per administration. In some aspects, tire dosage includes about 10 pg of a disclosed immunogenic composition per administration.

[0270] The dosage and number of doses will depend on the setting, for example, in an adult or anyone primed by prior Plasmodium falciparum infection or immunization, a single dose may be sufficient. In naive subjects, in some examples, at least two doses may be given, for example, at least three doses. In some aspects, an annual boost is given, for example, along with an annual influenza vaccination.

[0271] Immunization with an immunogenic composition disclosed herein can be combined with an anti-malarial treatment, for example, artesunate, artemether-lumefantrine, atovaquone-proguanil, quinine, or mefloquine.

[0272] Plasmodium falciparum infection does not need to be completely prevented for the methods to be effective. For example, elicitation of an immune response to Plasmodium falciparum with one4239-112675-02

[0273] or more of the disclosed immunogenic compositions can reduce risk of a Plasmodium falciparum infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more, as compared to a control (e.g., risk of infection in subjects that did not receive die immunogenic composition). In additional examples, severity of a subsequent Plasmodium falciparum infection can be reduced, for example, die signs or symptoms of a subsequent Plasmodium falciparum infection can be reduced by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more as compared to a control (e.g., subjects that did not receive the immunogenic composition). Symptoms of malaria include, for example, fever, chills, headache, nausea, vomiting, diarrhea, abdominal pain, muscle or joint pain, fatigue, rapid breather, rapid heart rate, or cough. Malaria signs and symptoms typically begin within a few weeks after transmission by an infected mosquito, however, some types of malarial parasites can lie dormant in a subject for up to a year.

[0274] Following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), serum neutralization assays (SNAs), and transgenic sporozoite neutralization assay (TSNA).

[0275] Clauses

[0276] Clause 1. A nanoparticle, comprising a fusion protein comprising multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide, wherein: each PfCSP peptide in the fusion protein comprises 13 to 55 consecutive residues of PfCSP, wherein the 13 to 55 consecutive residues comprise SEQ ID NO: 32 (NPDPNANPNVDPN); and the copies of the PfCSP peptide in the fusion protein are fused by peptide linkers.

[0277] Clause 2. The nanoparticle of clause 1, wherein each PfCSP peptide in the fusion protein comprises 19 to 25 consecutive residues of PfCSP, and the 19 to 25 consecutive residues comprise SEQ ID NO: 1 (KQPADGNPDPNANPNVDPN).

[0278] Clause 3. The nanoparticle of clause 1 or clause 2, wherein each PfCSP peptide in tire fusion protein comprises 19 to 21 consecutive residues of PfCSP, and tire 19 to 21 consecutive residues comprise SEQ ID NO: 1.

[0279] Clause 4. The nanoparticle of any one of tire prior clauses, wherein each PfCSP peptide in the fusion protein consists of SEQ ID NO: 1.

[0280] Clause 5. The nanoparticle of any one of tire prior clauses, wherein the nanoparticle is a protein nanoparticle, metallic nanoparticle, gold shell nanoparticle, carbon nanoparticle, or liposome.

[0281] Clause 6. The nanoparticle of any one of the prior clauses, wherein the nanoparticle is a selfassembling protein nanoparticle and the fusion protein further comprises a subunit of the selfassembling protein nanoparticle.4239-112675-02

[0282] Clause 7. The nanoparticle of any one of the prior clauses, wherein: (i) the self-assembling protein nanoparticle is a lumazine synthase nanoparticle and tire fusion protein comprises a lumazine synthase nanoparticle subunit; (ii) the self-assembling protein nanoparticle is a ferritin nanoparticle and die fusion protein comprises a ferritin nanoparticle subunit; (iii) the self-assembling protein nanoparticle is a BP26 nanoparticle and die fusion protein comprises a BP26 nanoparticle subunit; (iv) the self-assembling protein nanoparticle is an encapsulin nanoparticle and tire fusion protein comprises an encapsulin nanoparticle subunit; (v) the self-assembling protein nanoparticle is a sulfur oxygenase reductase (SOR) nanoparticle and the fusion protein comprises a SOR nanoparticle subunit; or (vi) the self-assembling protein nanoparticle is a pyruvate dehydrogenase nanoparticle and the fusion protein comprises a pyruvate dehydrogenase nanoparticle subunit.

[0283] Clause 8. The nanoparticle of any one of the prior clauses, wherein: (i) the lumazine synthase nanoparticle subunit comprises any one of SEQ ID NOs: 3 or 36-46, or an amino acid sequence at least 90% identical thereto; (ii) the ferritin nanoparticle subunit comprises any one of SEQ ID NOs: 47-57 or 65, or an amino acid sequence at least 90% identical thereto; or (iii) the BP26 nanoparticle subunit comprises SEQ ID NO: 62, or an amino acid sequence at least 90% identical thereto.

[0284] Clause 9. The nanoparticle of any one of the prior clauses, wherein the self-assembling protein nanoparticle is the lumazine synthase nanoparticle and the lumazine synthase nanoparticle subunit comprises or consists of SEQ ID NO: 3, or an amino acid sequence at least 90% identical thereto.

[0285] Clause 10. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises two to five copies of the PfCSP peptide.

[0286] Clause 11. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises two copies of the PfCSP peptide.

[0287] Clause 12. The nanoparticle of any one of die prior clauses, wherein the fusion protein comprises three copies of the PfCSP peptide.

[0288] Clause 13. The nanoparticle of any one of the prior clauses, wherein the fusion protein further comprises a T cell epitope.

[0289] Clause 14. The nanoparticle of any one of the prior clauses, wherein the T-cell epitope is a pan HLA-DR epitope (PADRE), a P2 epitope, or TpD epitope.

[0290] Clause 15. The nanoparticle of any one of the prior clauses, wherein the T-cell epitope is PADRE, wherein PADRE comprises or consists of tire amino acid sequence set fortii as SEQ ID NO: 2, or an amino acid sequence at least 90% identical thereto.

[0291] Clause 16. The nanoparticle of any one of the prior clauses, wherein the peptide linkers comprise or consist of glycine-serine peptide linkers.

[0292] Clause 17. The nanoparticle of any one of the prior clauses, wherein the peptide linkers comprise or consist of the amino acid sequence set forth as SEQ ID NO: 31.4239-112675-02

[0293] Clause 18. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, and the multiple copies of the PfCSP peptide.

[0294] Clause 19. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises two copies of the PfCSP peptide, and the fusion protein comprises SEQ ID NO: 4 or 6.

[0295] Clause 20. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises three copies of die PfCSP peptide, and the fusion protein comprises SEQ ID NO: 5 or 7.

[0296] Clause 21. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises or consists of any one of SEQ ID NOs: 8, 9, or 13-26.

[0297] Clause 22. The nanoparticle of any one of the prior clauses, wherein the fusion protein comprises or consists of SEQ ID NO: 8 or 9.

[0298] Clause 23. A fusion protein, comprising a protein nanoparticle subunit fused to multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide,

[0299] wherein the PfCSP peptide comprises 13 to 55 consecutive residues of PfCSP, the 13 to 55 consecutive residues comprise SEQ ID NO: 32 (NPDPNANPNVDPN), and the protein nanoparticle subunit and the multiple copies of the PfCSP peptide are fused by peptide linkers.

[0300] Clause 24. The fusion protein of clause 23, further comprising a T cell epitope.

[0301] Clause 25. The fusion protein of clause 23 or 24, wherein the protein nanoparticle subunit comprises a lumazine synthase, ferritin, encapsulin, sulfur oxygenase reductase (SOR), pyruvate dehydrogenase, or BP26 subunit.

[0302] Clause 26. The fusion protein of any one of clauses 23-25, wherein the fusion protein comprises two to five copies of die PfCSP peptide.

[0303] Clause 27. The fusion protein of any one of clauses 23-26, wherein the fusion protein comprises any one of SEQ ID NOs: 4-9.

[0304] Clause 28. The fusion protein of any one of clauses 23-27, wherein the fusion protein comprises of consists of SEQ ID NO: 8 or 9.

[0305] Clause 29. A nanoparticle, comprising a multimer of die fusion protein of any one of clauses 23-28.

[0306] Clause 30. A nucleic acid molecule encoding the fusion protein of any one of clauses 1-28. Clause 31. The nucleic acid molecule of clause 30, wherein die nucleic acid molecule comprises or consists of SEQ ID NO: 34 or 35.

[0307] Clause 32. A vector comprising the nucleic acid molecule of clause 30 or 31.

[0308] Clause 33. A method of producing nanoparticles, comprising:

[0309] expressing the nucleic acid or vector of any one of the prior clauses in a host cell; and purifying assembled nanoparticles from the host cell or culture thereof, thereby producing the nanoparticles.

[0310] Clause 34. Nanoparticles produced by the method of clause 33.4239-112675-02

[0311] Clause 35. An immunogenic composition comprising the nanoparticles of any one of the prior clauses and a pharmaceutically acceptable carrier, wherein the immunogenic composition elicits an immune response in a subject to Plasmodium falciparum.

[0312] Clause 36. A method of eliciting an immune response to Plasmodium falciparum in a subject, comprising administering to die subject an effective amount of die immunogenic composition of clause 35.

[0313] Clause 37. The method of clause 36, wherein administering die immunogenic composition to the subject induces a protective immune response to Plasmodium falciparum in the subject.

[0314] Clause 38. The method of clause 36 or clause 37, wherein the immune response inhibits development of malaria in the subject.

[0315] Clause 39. Use of the immunogenic composition of clause 35 to elicit an immune response to Plasmodium falciparum in a subject.

[0316] EXAMPLES

[0317] The following examples are provided to illustrate particular features of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0318] Example 1

[0319] Materials and Methods

[0320] Protein production and purification

[0321] For protein expression, 3 ml of Turbo293 transfection reagent (Speed BioSystems) was mixed witii 50 ml Opti-MEM medium (Life Technology) and incubated at room temperature (RT) for 5 min.

[0322] 1 mg plasmid DNAs was mixed with 50 ml of Opti-MEM medium in a separate tube, and the mixture added to the Turbo293 Opti-MEM mixture. The transfection mixture was incubated for 15 min at RT tiren added to 800 ml of Expi293 cells (Life Technology) at 2.5 million cells / ml. The transfected cells were incubated overnight in a shaker incubator at 9% CO2, 37 °C, and 120 rpm. On the second day, about 100 ml of Expi293 expression medium was added. On day 6 post transfection, supernatants were harvested, filtered. Proteins were purified from the supernatant using GNA lectin affinity chromatography. GNA resin was equilibrated with 10 column volumes (CVs) of PBS (pH 7.4) followed by supernatant loading. Unbound protein was removed by washing with 20 CVs of PBS buffer. Bound protein was eluted witii 250 mM metiiyl a-d-mannopyranoside in PBS buffer (pH 7.4). All proteins were purified by size exclusion chromatography on Superdex 200 Increase 10 / 300 GL in PBS and further characterized by SDS-PAGE.

[0323] Negative-stain electron microscopy (EM)

[0324] Samples were diluted to 0.02-0.05 mg / ml with a buffer containing 10 mM HEPES, pH 7, and 150 mM NaCl. A 4.7-pl drop of the diluted sample was applied to a glow-discharged carbon-coated4239-112675-02

[0325] copper grid for approximately 15 s. The drop was removed using blotting paper, and the grid was washed three times with 4.7-pl drops of the same buffer. Adsorbed proteins were negatively stained by applying consecutively three 4.7-pl drops of 0.75% uranyl formate and removing each drop with filter paper. Micrographs were collected using SerialEM45 on an FEI Tecnai T20 electron microscope operated at 200 kV and equipped with an Eagle CCD camera or using EPU on a ThermoFisher Talos F200C electon microscope operated at 200 kV and equipped with a Ceta CCD camera.

[0326] Antigenicity characterization by Biolayer Interferometry

[0327] The antigenicity of nanoparticles was assessed by binding to junctional, minor, and major directed antibodies. Antibodies were diluted to 5 pg / mL in (PBS + 1% BSA), immobilized on AHC biosensors to a density of 0.1 nm, and dipped into varying nanoparticle concentrations. Assays were performed with agitation at 30°C. In all Octet measurements, parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in PBS. Data analysis was carried out using Octet software, version 9.0.

[0328] Experimental data were fitted globally with a 1:1 Langmuir model of binding for all the antigens.

[0329] Immunogenicity and Protection Studies

[0330] Mice

[0331] Female 6- to 8-week old B6(Cg)-Tyrc-2J / J albino mice were obtained from The Jackson Laboratory. All animals were cared for in accordance with American Association for Accreditation of Laboratory Animal Care standards in accredited facilities.

[0332] Immunizations

[0333] Unless otherwise indicated, vaccine immunogens were diluted in sterile PBS to their respective dose and mixed with 33.3 pL ALFQ, which, like AS01, includes a liposomal adjuvant formulation containing monophosphoryl lipid A and QS-21 in a final volume of 50 pL. Immunization was performed intramuscularly in the quadriceps at weeks 0 and 4. Serum sampling was performed 2 weeks following the 1stand 2ndimmunizations. Challenges were performed 4 weeks following the last immunization.

[0334] IV challenge and quantification of protection

[0335] Transgenic P. berghei (strain ANKA 676mlcl 1, MRA-868) expressing full-length P. falciparum CSP and a green fluorescent protein / luciferase fusion protein (Pb-PfCSP-GFP / Luc-SPZ) were obtained. Approximately 4 weeks following the final immunization, mice were intravenously challenged in the tail vein with 2000 freshly harvested Pb-PfCSP-GFP / Luc-SPZ in Leibovitz’s L-154239-112675-02

[0336] medium (Thermo Fisher Scientific Inc., Waltham, MA, USA). Then, 40M2 h post-challenge, mice were injected intraperitoneally with 150 pL of D-luciferin (PerkinElmer, Waltham, MA, USA; 30 mg / mL), anesthetized with isoflurane, and imaged with the IVIS® Spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA) 10 min after luciferin injection. Liver burden was quantified by analyzing a region of interest (ROI) in the upper abdominal region; the total flux (p / s) was measured using the manufacturer’s software (Living Image 4.5, PerkinElmer, Waltham, MA, USA). Percent (%) protection for each mouse was calculated based on the liver burden flux (p / s) data. Percent (%) protection = [100 - ((antibody-treated mouse flux / geometric mean flux of untreated mice) x 100)].

[0337] Serology

[0338] Immune responses were measured to full-length PfCSP or to 15-mer peptides by biolayer interferometry (BL1). Biotinylated flCSP and peptides were captured on streptavidin (SA) biosensor and dipped into 100-fold diluted serum samples from individual mice. Assays were performed with agitation at 30°C and the binding response was calculated. In all Octet measurements, parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in PBS. Data analysis was carried out using Octet software, version 9.0.

[0339] Example 2

[0340] Nanoparticle Design

[0341] The junctional peptide of PfCSP has been identified as a target epitope of the potent neutralizing antibody known as CIS43 (see, e.g., Kisalu et al 2018). The junctional epitope sequence (referred to herein as NPDP19 epitope) is disclosed herein as SEQ ID NO: 1. 18 different constructs including the junctional epitope were initially designed (constructs 1-18, see, Table 1 below).

[0342] Table 1: Sequences of exemplary constructs (without an N-terminal signal peptide).

[0343] Construct # Sequence

[0344] 1 KQPADGNPDPNANPNVDPNgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGA TPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEA ALSAIEMANLFKSLRGakfvaawtlkaaa (SEQ ID NO: 12)

[0345] 2 KQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPNgsgsaMQIYEGK LTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPV AAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVIT ADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGakfvaawtlkaaa (SEQ ID NO: 13)

[0346] 3 KQPADGNPDPNANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggKQPADG NPDPNANPNVDPNgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRL VEG AIDA IVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIA SEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMA

[0347]

[0348] NLFKSLRGakfvaawtlkaaa (SEQ ID NO: 14)4239-112675-02

[0349] 7 hlihlihhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNggsggDPMLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDG AGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEH EQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEsH GLYLADQYVKGIAKSRKSgsgQYIKANSKFIGITEL (SEQ ID NO: 15) 8 hhhhhhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNggsggADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAAL DAQnATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAA EQLKTTRNAYIQKYLggAEAAAKEAAAKEAAAKAgsgSAMQIYEGKLTAEGLRF GIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARK ENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIE RAGTKHGNKGWEAALSAIEMANLFKSLRGakfvaawtlkaaas (SEQ ID NO: 16) 9 hhhhhhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNgggADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALD AQnATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAE QLKTTRNAYIQKYLGGggAEAAAKEAAAKEAAAKAgsgDIIKLLNEQVNKEMQ SSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSI SAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQ HEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSgsgQYIKANSKFIGIT EL (SEQ ID NO: 17)

[0350] 10 hhhhhhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggDPMLSKDIIKLLNEQVN KEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPV QLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWY VAEQHEEEVLFKDILDKIELIGNEsHGLYLADQYVKGIAKSRKSgsgQYIKANSKF IGITEL (SEQ ID NO: 18)

[0351] 11 hhhhhhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNggsggKQPADGNPDPNANPNVDPNggsggADLEDNWETLNDNLKVI EKADNAAQVKDALTKMRAAALDAQnATPPKLEDKSPDSPEMKDFRHGFDILV GQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYLggAEAAAKEAAAKEA AAKAgsgSAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGRE ED1TLVRVPGSWEIPVAAGELARKENISAV1AIGVL1RGATPHFDYIASEVSKGLA DLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRG

[0352] akfvaawtlkaaas (SEQ ID NO: 19)

[0353] 12 hhhhhhsawshpqfekgglevlfqgpggKQPADGNPDPNANPNVDPNggsggKQPADGNPDP NANPNVDPNggsggKQPADGNPDPNANPNVDPNgggADLEDNWETLNDNLKVIE KADNAAQVKDALTKMRAAALDAQnATPPKLEDKSPDSPEMKDFRHGFDILVG QIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYLGGggAEAAAKEAAAKE AAAKAgsgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAE EYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIV DHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYV KGIAKSRKSgsgQYIKANSKFIGITEL (SEQ ID NO: 20)

[0354] 13 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGKQPAD GNPDPNANPNVDPNGGGSGNPDPNANPNVDPNGGGSGKQPADGNPDPNANP NVDPNGGGSGNPDPNANPNVDPNGGGSGNANPNANPNANPNANPNANPGG GSGNANPNANPNANPNANPNANPGGGSGNANPNANPNANPNANPNANPGG GSGRRAKFVAAWTLKAAA (SEQ ID NO: 21)

[0355] 14 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT

[0356] ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI

[0357]

[0358] DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG4239-112675-02

[0359] GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGGGGSG KQPADGNPDPNANPNVDPNGGGSGGGGSGNPDPNANPNVDPNGGGSGGGG SGKQPADGNPDPNANPNVDPNGGGSGGGGSGNPDPNANPNVDPNGGGSGG GGSGNANPNANPNANPNANPNANPGGGSGGGGSGNANPNANPNANPNANP NANPGGGSGGGGSGNANPNANPNANPNANPNANPGGGSGGGGSGRRAKFV AAWTLKAAA (SEQ ID NO: 22)

[0360] 15 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGGGGSG GGGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGGGGSGNPDPNANPNVD PNGGGSGGGGSGGGGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGGGG SGNPDPNANPNVDPNGGGSGGGGSGGGGSGNANPNANPNANPNANPNANP GGGSGGGGSGGGGSGNANPNANPNANPNANPNANPGGGSGGGGSGGGGS GNANPNANPNANPNANPNANPGGGSGGGGSGGGGSGRRAKFVAAWTLKAA

[0361] A (SEQ ID NO: 23)

[0362] 16 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGKQPAD GNPDPNANPNVDPNGGGSGNPDPNANPNVDPNGGGSGKQPADGNPDPNANP NVDPNGGGSGNPDPNANPNVDPNGGGSGKQPADGNPDPNANPNVDPNGGGS GNPDPNANPNVDPNGGGSGKQPADGNPDPNANPNVDPNGGGSGRRAKFVAA WTLKAAA (SEQ ID NO: 24)

[0363] 17 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGGGGSG KQPADGNPDPNANPNVDPNGGGSGGGGSGNPDPNANPNVDPNGGGSGGGG SGKQPADGNPDPNANPNVDPNGGGSGGGGSGNPDPNANPNVDPNGGGSGG GGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGNPDPNANPNVDPNGGGS GGGGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGRRAKFV AAWTLKAAA

[0364] (SEQ ID NO: 25)

[0365] 18 HHHHHHSAWSHPQFEKGGLEVLFQGPGGQENQMTTQPARIAVTGEGMMT ASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGI DIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQG GDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPP MPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKGGGSGGGGSG GGGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGGGGSGNPDPNANPNVD PNGGGSGGGGSGGGGSGKQPADGNPDPNANPNVDPNGGGSGGGGSGGGG SGNPDPNANPNVDPNGGGSGGGGSGGGGSGKQPADGNPDPNANPNVDPNG GGSGGGGSGGGGSGNPDPNANPNVDPNGGGSGGGGSGGGGSGKQPADGN PDPNANPNVDPNGGGSGGGGSGGGGSGRRAKFV AAWTLKAAA (SEQ ID NO: 26)

[0366] 19 AKFVAAWTLKAAASLVRgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVE GAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATP HFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL SAIEMANLFKSLRGggsggKQPADGNPDPNANPNVDPN (SEQ ID NO: 27) 20 AKFVAAWTLKAAASLVRgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVE

[0367]

[0368] GAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATP4239-112675-02

[0369] HFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL SAIEMANLFKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNA NPNVDPN (SEQ ID NO: 8)

[0370] 21 AKFVAAWTLKAAASLVRgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVE GAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATP HFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL SAIEMANLFKSLRGggsggKQPADGNPDPNANPNVDPNggsggKQPADGNPDPNA

[0371]

[0372] NPNVDPNggsggKQPADGNPDPNANPNVDPN (SEQ ID NO: 9)

[0373] Five diverse constructs (construct # 1, 2, 7, 14, and 17) were selected for protein expression and analysis (see, FIGS. 1C and ID). Biolayer interferometry (BLI) responses and liver burden were further evaluated for each construct (see, FIGS. 1E-1H). Based on initial data, which suggested that construct # 2 was tire most promising, variations of construct #2 were designed and expressed (construct# 19, 20, and 21; see FIG. II). The variations of construct #2 differed in the number of repeats of the NPDP19 peptide (tire CIS43 epitope with KQPADG (SEQ ID NO: 82) added at the N- terminus) and the location of PADRE (N or C terminal).

[0374] Schematics of construct 20, also referred to as LuS-2T, and construct 21, also referred to as LuS-3T, are shown in FIG. 1J. Lumazine synthase 2-tandem (LuS-2T) comprised a N-terminal PADRE to enhance T-cell help, the self-assembling unit of LuS, and two repeats of the CIS43 epitope with KQPADG (SEQ ID NO: 82) added at the N-terminus and with a ggsgg linker (SEQ ID NO: 31). Lumazine synthase 3-tandem (LuS-3T) comprised the same as LuS-2T with an additional tandem repeat. Expression and purification of these two constructs yielded a single major peak with both constructs eluting at ~12 minutes; SDS-PAGE analysis revealed each of the major peaks to comprise a single band (FIG. IK). Negative stain electron microscopy (EM) showed properly assembled nanoparticles (FIG. IL), and the antibody binding revealed strong recognition for PfCSP-directed antibodies including CIS43, D3, and P3-43 (targeting the CIS43 epitope), L9 (targeting the minor repeat region), and 317 (targeting the major repeat region) (see, FIG. IM). Strong recognition was also observed for CIS42, a non-neutralizing antibody.

[0375] Thus, two self-assembling nanoparticle immunogens based on the CIS43 epitope on PfCSP formed homogenous nanoparticles that bound known CSP-directed non-neutralizing and neutralizing antibodies directed at not only the CIS43 epitope, but also to minor and major repeats. Notably, despite developing a vaccine displaying only the unique junctional epitope, broad recognition was observed by antibodies that targeted both major and minor repeats as well as the junctional epitope.

[0376] Example 3

[0377] Tandem-Displayed CIS43 Epitopes on LuS Nanoparticles Induced Protective Anti-Malarial Responses

[0378] To test if tire tandem display of junctional epitope increased tire efficacy of tire designed immunogens, mice were immunized with a single and double repeat of the CIS43 epitope displayed4239-112675-02

[0379] on lumazine synthase. R21 and junctional epitope coupled to KLH (NPDP19-KLH) were used as controls. After two immunizations four weeks apart, mice were challenged with sporozoites (FIG.

[0380] 2A). Sera collected at weeks 2 and 6 were analyzed with biolayer interferometry (BLI) to probe elicited antibody responses against PfCSP, major epitope (NANP,), and junctional epitope (NPDP19) (FIGS. 2B-2D). At week 2, R21 -immunized group elicited significant responses against PfCSP, NANP9, and almost negligible responses against NPDP19, as expected. In contrast, only tire 2-tandem containing immunogen group showed measurable responses against NPDP19 at week 2. Interestingly, at week 6, the R21 elicited responses stay at similar' levels as week 2, however, all the other tested immunogens showed a drastic increase in elicited antibody levels. The NPDP19-KLH control and the 2-tandem LuS groups trended to have the highest responses when compared across all three probes. An interesting dose response was observed for the 1 -tandem LuS groups with the lower dose of 1 pg trending to higher antibody responses compared to the 10 pg dose. Moreover, the 2-tandem group seemed better than the 1 -tandem group.

[0381] Vaccine efficacy was assessed by calculating the liver burden at day 2 after sporozoite challenge. The R21 group at 1 pg showed 87.8% reduction in liver burden (FIG. 2E), whereas the 2-tandem LuS group at 10 pg showed 60.3% reduction, which was the best among the LuS-based immunogens. The inverse dose response that was observed with antibody titers (FIGS. 2B-2D) was recapitulated in the liver burden assay, with the lower dose group (1 pg) being more protective than the higher dose (10 pg) group (FIG. 2E). Thus, overall, a 1 pg dose provided a better protection than 10 pg dose for the LuS immunogens, and tandem display of junctional epitope appeared to increase efficacy.

[0382] Example 4

[0383] Low Dose of Tandem LuS-Displayed CIS43 Epitope

[0384] Provides Protection Equivalent to R21

[0385] As the 1 pg dose of the LuS-displayed CIS43 vaccine trended to better protection than tire 10 pg dose, the most promising immunogen (2-tandem LuS) was next assessed at three different doses of 0.2, 1, and 5 pg alongside the R21 control at 1 pg, following die same immunization schema as the previous experiment (Fig.3A). All three 2-tandem LuS groups showed significantly enhanced BLI responses at week 6 and more intriguingly, tire lowest dose of 0.2 pg had the highest antibody response against all three probes: PfCSP, NANPg, NPDP19 (FIGS.3B-3D). R21 by contrast showed responses to only PfCSP and NANPg, and the antibody responses elicited by R21 did not increase significantly after the second immunization (FIGS. 3B-3D).

[0386] To assess whether placement of the added T-cell epitope (PADRE) impacted immunogenicity, PADRE was added to the N-terminus instead of the C-terminus of the 2-tandem LuS immunogen. At 1 pg, the N-terminal positioned PADRE immunogen yielded significantly higher recognition than the C-terminal positioned PADRE immunogen. In addition, in the context of an N-4239-112675-02

[0387] terminal PADRE, 3 versus 2-tandem repeats were tested and no significant differences were observed (FIGS. 3B-3D).

[0388] Lastly, liver burden after sporozoite challenge was assessed at week 8. In general, the protection recapitulated the probe-assessed binding, with the N-tenninal PADRE-based immunogens containing either 2 or 3 NPDP19 repeats yielding protection equal to R21 at a dose of 1 pg (FIG.3E).

[0389] In view of results indicating that lower doses could provide increased protection, lower doses of PADRE-LuS immunogen containing 2 or 3 tandem repeats of NPDP19 was assessed, as well as potential synergy with R21. The immunization schema is shown in FIG.4A. As observed previously, the lower dose of PADRE-LuS-NPDP19 at 0.25 pg yielded increased BLI titers than the 1 pg dose for PfCSP, NANP9, and NPDP19 (FIGS. 4B-4D). By contrast, the R21 titers were lower at 0.25 pg than at 1 pg dose; moreover, the R21 titers were substantially lower against NPDP19 than NANP9 or PfCSP (FIGS. 4B-4D).

[0390] Notably, the PADRE-LuS-3T immunogen at 0.25 pg dose provided protection of 83.8 %, which was significantly better than the protection of 49.9 % from R21 at 0.25 pg dose. Also, synergy between PADRE-LuS-3T and R21 was observed, with the combination yielding protection of 89.1 % at 0.25 + 0.25 pg dose and a protection of 97.2 % at 1 + 1 pg dose. Thus, synergistic protection was induced by the combination of PADRE-LuS-3T and R21 , and at a lower dose of 0.25 pg, the NPDP19 tandem immunogen provided superior protection than R21 (observed with significance when repeated; see, FIGS.4E-4F).

[0391] Example 5

[0392] Assessment of PADRE-LuS-3T in SMNP Formulation

[0393] Mice (N=10) were immunized with PADRE-LuS-3T (0.25 pg) or PADRE-LuS-3T (1.0 pg), both in SMNP formulation, twice, four weeks apart. SMNP (saponin / MPLA nanoparticles) is a potent adjuvant that combines saponin and monophosphoryl lipid A (MPLA). Mice were challenged with sporozoites four weeks after the second immunization (.see, FIG. 5A). Naive and max burden controls were also included. Liver burden was assessed day 2 post Pb-PfCSP-Luc sporozoite challenge. The percent reduction was calculated based on geometric mean of each group see, FIG.

[0394] 5B). Vaccine efficacy was comparable between the SMNP formulation see, FIG. 5B) and the ALFQ-adjuvanted formulation {see, FIG. 4E). Specifically, PADRE-LuS-3T achieved 83.8% efficacy with ALFQ and 84.5% with SMNP.

[0395] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of this disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239412675-02Claims:

1. A nanoparticle, comprising a fusion protein comprising multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide, wherein:each PfCSP peptide in the fusion protein comprises 13 to 55 consecutive residues of PfCSP, wherein the 13 to 55 consecutive residues comprise SEQ ID NO: 32 (NPDPNANPNVDPN); and the multiple copies of the PfCSP peptide in the fusion protein are fused by peptide linkers.

2. The nanoparticle of claim 1, wherein each PfCSP peptide in the fusion protein comprises 19 to 25 consecutive residues of PfCSP, and the 19 to 25 consecutive residues comprise SEQ ID NO: 1 (KQPADGNPDPNANPNVDPN).

3. The nanoparticle of claim 1 or claim 2, wherein each PfCSP peptide in the fusion protein comprises 19 to 21 consecutive residues of PfCSP, and the 19 to 21 consecutive residues comprise SEQ ID NO: 1.

4. The nanoparticle of any one of the prior claims, wherein each PfCSP peptide in the fusion protein consists of SEQ ID NO: 1.

5. The nanoparticle of any one of the prior claims, wherein the nanoparticle is a protein nanoparticle, metallic nanoparticle, gold shell nanoparticle, carbon nanoparticle, or liposome.

6. The nanoparticle of any one of the prior claims, wherein the nanoparticle is a selfassembling protein nanoparticle and the fusion protein further comprises a subunit of the selfassembling protein nanoparticle.

7. The nanoparticle of claim 6, wherein:(i) the self-assembling protein nanoparticle is a lumazine synthase nanoparticle and the fusion protein comprises a lumazine synthase nanoparticle subunit;(ii) the self-assembling protein nanoparticle is a ferritin nanoparticle and the fusion protein comprises a ferritin nanoparticle subunit;(iii) the self-assembling protein nanoparticle is a BP26 nanoparticle and the fusion protein comprises a BP26 nanoparticle subunit;(iv) the self-assembling protein nanoparticle is an encapsulin nanoparticle and the fusion protein comprises an encapsulin nanoparticle subunit;(v) the self-assembling protein nanoparticle is a sulfur oxygenase reductase (SOR) nanoparticle and the fusion protein comprises a SOR nanoparticle subunit; or4239-112675-02(vi) the self-assembling protein nanoparticle is a pyruvate dehydrogenase nanoparticle and the fusion protein comprises a pyruvate dehydrogenase nanoparticle subunit.

8. The nanoparticle of claim 7, wherein:(i) the lumazine synthase nanoparticle subunit comprises any one of SEQ ID NOs: 3 or 36-46, or an amino acid sequence at least 90% identical thereto;(ii) the ferritin nanoparticle subunit comprises any one of SEQ ID NOs: 47-57 or 65, or an amino acid sequence at least 90% identical thereto; or(hi) the BP26 nanoparticle subunit comprises SEQ ID NO: 62, or an amino acid sequence at least 90% identical thereto.

9. The nanoparticle of claim 7 or claim 8, wherein the self-assembling protein nanoparticle is the lumazine synthase nanoparticle and the lumazine synthase nanoparticle subunit comprises or consists of SEQ ID NO: 3, or an amino acid sequence at least 90% identical thereto.

10. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises two to five copies of the PfCSP peptide.

11. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises three copies of the PfCSP peptide.

12. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises two copies of the PfCSP peptide.

13. The nanoparticle of any one of the prior claims, wherein the fusion protein further comprises a T cell epitope.

14. The nanoparticle of claim 13, wherein the T-cell epitope is a pan HLA-DR epitope (PADRE), a P2 epitope, or TpD epitope.

15. The nanoparticle of claim 13 or 14, wherein the T-cell epitope is PADRE, wherein PADRE comprises or consists of the amino acid sequence set forth as SEQ ID NO: 2, or an amino acid sequence at least 90% identical thereto.

16. The nanoparticle of any one of the prior claims, wherein the peptide linkers comprise or consist of glycine-serine peptide linkers.4239-112675-0217. The nanoparticle of claim 16, wherein tire peptide linkers comprise or consist of the amino acid sequence set forth as SEQ ID NO: 31.

18. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises from N-terminal to C-terminal: a T cell epitope, a protein nanoparticle subunit, and the multiple copies of the PfCSP peptide.

19. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises three copies of the PfCSP peptide, and the fusion protein comprises SEQ ID NO: 5 or 7.

20. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises two copies of the PfCSP peptide, and the fusion protein comprises SEQ ID NO: 4 or 6.

21. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises or consists of SEQ ID NO: 9.

22. The nanoparticle of any one of the prior claims, wherein the fusion protein comprises or consists of any one of SEQ ID NOs: 9, 8, or 13-26.

23. A fusion protein, comprising a protein nanoparticle subunit fused to multiple copies of a Plasmodium falciparum circumsporozoite protein (PfCSP) peptide,wherein the PfCSP peptide comprises 13 to 55 consecutive residues of PfCSP, the 13 to 55 consecutive residues comprise SEQ ID NO: 32 (NPDPNANPNVDPN), and the protein nanoparticle subunit and the multiple copies of the PfCSP peptide are fused by peptide linkers.

24. The fusion protein of claim 23, further comprising a T cell epitope.

25. The fusion protein of claim 23 or 24, wherein the protein nanoparticle subunit comprises a lumazine synthase, ferritin, encapsulin, sulfur oxygenase reductase (SOR), pyruvate dehydrogenase, or BP26 subunit.

26. The fusion protein of any one of claims 23-25, wherein the fusion protein comprises two to five copies of the PfCSP peptide.

27. The fusion protein of any one of claims 23-26, wherein the fusion protein comprises any one of SEQ ID NOs: 5, 7, 4, or 6.4239-112675-0228. The fusion protein of any one of claims 23-27, wherein the fusion protein comprises or consists of SEQ ID NO: 9 or 8.

29. A nanoparticle, comprising a multimer of the fusion protein of any one of claims 23-28.

30. A nucleic acid molecule encoding the fusion protein of any one of claims 1-28.

31. The nucleic acid molecule of claim 30, wherein the nucleic acid molecule comprises or consists of SEQ ID NO: 35 or 34.

32. A vector comprising the nucleic acid molecule of claim 30 or 31.

33. A method of producing nanoparticles, comprising:expressing the nucleic acid or vector of any one of claims 30-32 in a host cell; and purifying assembled nanoparticles from the host cell or culture thereof, thereby producing the nanoparticles.

34. Nanoparticles produced by the method of claim 33.

35. An immunogenic composition comprising the nanoparticles of any one of claims 1-22, 29, or 34 and a pharmaceutically acceptable carrier,wherein the immunogenic composition elicits an immune response in a subject to Plasmodium falciparum.

36. A method of eliciting an immune response to Plasmodium falciparum in a subject, comprising administering to the subject an effective amount of the immunogenic composition of claim 35.

37. The method of claim 36, wherein administering the immunogenic composition to the subject induces a protective immune response to Plasmodium falciparum in the subject.

38. The method of claim 36 or claim 37, wherein the immune response inhibits development of malaria in the subject.

39. Use of the immunogenic composition of claim 35 to elicit an immune response to Plasmodium falciparum in a subject.

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