Compositions and related methods for delivery of Plasmodium CSP antigens

Abstract

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

Description

[Technical Field]

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

[0002] The present disclosure provides techniques (e.g., compositions, methods, etc.) for delivering Plasmodium antigens (also referred to herein as "malaria antigens" or "malarial antigens"). In one aspect, provided herein is a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more Plasmodium CSP polypeptide regions or portions thereof. In some embodiments, the one or more Plasmodium CSP polypeptide regions or portions thereof each comprise 25 or more contiguous amino acids of an amino acid sequence according to SEQ ID NO: 1. In some embodiments, a "fragment" of a polypeptide is a "portion" of a polypeptide.

[0003] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), and the polypeptides do not comprise the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise five or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102).

[0004] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, the polypeptide comprising (i) a heterologous secretion signal and (ii) one or more Plasmodium CSP polypeptide regions or portions thereof.

[0005] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, the polypeptide comprising (i) one or more Plasmodium CSP polypeptide domains or portions thereof, and (ii) a heterologous transmembrane domain.

[0006] In some embodiments, the polypeptide encoded by the polyribonucleotide comprises one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the polypeptide comprises two or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the polypeptide comprises between two and twelve repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the polypeptide comprises exactly three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the polypeptide comprises between four and twelve repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the polypeptide comprises (i) exactly eight repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), or (ii) exactly nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) are all contiguous with one another. In some embodiments, the repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) are not all contiguous with one another.

[0007] In some embodiments, the polypeptide encoded by the polyribonucleotide comprises four portions of a Plasmodium CSP polypeptide, each portion comprising two consecutive repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102).

[0008] In some embodiments, the polypeptide encoded by the polyribonucleotide comprises one or more Plasmodium CSP C-terminal regions or portions thereof. In some embodiments, the polypeptide comprises exactly one Plasmodium CSP C-terminal region, wherein the Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide comprises two or more portions of the Plasmodium CSP C-terminal region. In some embodiments, the polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprises or consists of (i) the amino acid sequence of SEQ ID NO: 111, (ii) the amino acid sequence of SEQ ID NO: 114, (iii) the amino acid sequence of SEQ ID NO: 117, (iv) the amino acid sequence of SEQ ID NO: 120, or (v) a combination thereof. In some embodiments, the polypeptide comprises a portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of (i) the amino acid sequence according to SEQ ID NO: 111, (ii) the amino acid sequence according to SEQ ID NO: 114, (iii) the amino acid sequence according to SEQ ID NO: 117, (iv) the amino acid sequence according to SEQ ID NO: 120, or (v) a combination thereof. In some embodiments, the polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein the one or more portions collectively comprise or consist of (i) the amino acid sequence according to SEQ ID NO: 111, (ii) the amino acid sequence according to SEQ ID NO: 114, (iii) the amino acid sequence according to SEQ ID NO: 117, and (iv) the amino acid sequence according to SEQ ID NO: 120.

[0009] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise a serine immediately following the Plasmodium CSP C-terminal region, hi some embodiments, the polypeptides comprise a serine-valine sequence immediately following the Plasmodium CSP C-terminal region.

[0010] In some embodiments, polypeptides encoded by provided polyribonucleotides comprise one or more Plasmodium CSP junction regions or portions thereof. In some embodiments, the polypeptide comprises two or more Plasmodium CSP junction regions or portions thereof. In some embodiments, the two or more Plasmodium CSP junction regions consist of the amino acid sequence according to SEQ ID NO: 126. In some embodiments, the polypeptide comprises two or more Plasmodium CSP junction regions. In some embodiments, two or more portions of the Plasmodium CSP junction region comprise a deletion of one or more of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, two or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, and Q96, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, two or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO: 1.

[0011] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises exactly one Plasmodium CSP junction region. In some embodiments, the Plasmodium CSP junction region consists of the amino acid sequence according to SEQ ID NO: 126. In some embodiments, the polypeptide comprises one or more portions of a Plasmodium CSP junction region. In some embodiments, the one or more portions of the Plasmodium CSP junction region comprise a deletion of one or more of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the one or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, and Q96, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the one or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, each portion of the Plasmodium CSP junction region comprises or consists of the amino acid sequence of SEQ ID NO: 129. In some embodiments, each portion of the Plasmodium CSP junction region comprises or consists of the amino acid sequence of SEQ ID NO: 132. In some embodiments, each portion of the Plasmodium CSP junction region comprises or consists of the amino acid sequence of SEQ ID NO: 129.

[0012] In some embodiments, polypeptides encoded by the provided polyribonucleotides comprise one or more Plasmodium CSP junction region variants. In some embodiments, the Plasmodium CSP junction region variants comprise one or more substitution mutations. In some embodiments, the one or more substitution mutations comprise a K93A mutation, an L94A mutation, or both, wherein the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, each Plasmodium CSP junction region variant comprises the amino acid sequence of AAKQ (SEQ ID NO: 426).

[0013] In some embodiments, polypeptides encoded by the provided polyribonucleotides comprise one or more Plasmodium CSP N-terminal end regions or portions thereof. In some embodiments, the polypeptides comprise two or more Plasmodium CSP N-terminal end regions or portions thereof. In some embodiments, each Plasmodium CSP N-terminal end region consists of the amino acid sequence according to SEQ ID NO: 135.

[0014] In some embodiments, the polypeptides encoded by the provided polyribonucleotides do not include the Plasmodium CSP N-terminal end region or any portion thereof. In some embodiments, the polypeptides include one or more Plasmodium CSP N-terminal regions or portions thereof. In some embodiments, the polypeptides include two or more Plasmodium CSP N-terminal regions or portions thereof. In some embodiments, each Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 138.

[0015] In some embodiments, the polypeptides encoded by the provided polyribonucleotides do not include the Plasmodium CSP N-terminal region or any portion thereof.

[0016] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise one or more Plasmodium CSP major repeat regions or portions thereof. In some embodiments, the one or more Plasmodium CSP major repeat regions or portions thereof comprise the amino acid sequence NANPNA (SEQ ID NO: 153) or NPNANP (SEQ ID NO: 150). In some embodiments, the polypeptides comprise exactly one Plasmodium CSP major repeat region or portions thereof, wherein the Plasmodium CSP major repeat region or portions thereof comprises a total of at least two and at most 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147). In some embodiments, the Plasmodium CSP major repeat region or portions thereof comprises two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 147), wherein the two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 147) are flanked by the amino acid sequence of NVDP (SEQ ID NO: 144). In some embodiments, the major repeat region of Plasmodium CSP comprises, from N-terminus to C-terminus, 17 repeats of the amino acid sequence NANP (SEQ ID NO:147), the amino acid sequence of NVDP (SEQ ID NO:144), and 18 repeats of the amino acid sequence NANP (SEQ ID NO:147). In some embodiments, the portion of the major repeat region of Plasmodium CSP consists of up to 18 consecutive repeats of the amino acid sequence NANP (SEQ ID NO:147). In some embodiments, the portion of the major repeat region of Plasmodium CSP consists of two consecutive repeats of the amino acid sequence NANP (SEQ ID NO:147). In some embodiments, the major repeat region of Plasmodium CSP comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:156.

[0017] In some embodiments, the polypeptides encoded by the provided polyribonucleotides do not include the major repeat region of Plasmodium CSP or the portion of the major repeat region of Plasmodium CSP that includes the amino acid sequence NPNA (SEQ ID NO: 141).

[0018] In some embodiments, the one or more Plasmodium CSP polypeptide regions or portions thereof, when present in a polypeptide encoded by the provided polyribonucleotides, are, in order from N-terminus to C-terminus, (i) one or more Plasmodium CSP N-terminal regions or portions thereof, (ii) one or more Plasmodium CSP N-terminal end regions or portions thereof, (iii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof, (iv) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), (v) one or more Plasmodium CSP major repeat regions or portions thereof, and (vi) one or more Plasmodium CSP C-terminal regions or portions thereof.

[0019] In some embodiments, the one or more Plasmodium CSP polypeptide regions or portions thereof, when present in a polypeptide encoded by the provided polyribonucleotides, are, in order from N-terminus to C-terminus, (i) one Plasmodium CSP N-terminal region or portion thereof, (ii) one Plasmodium CSP N-terminal end region or portion thereof, (iii) one Plasmodium CSP junction region, portion thereof, or variant thereof, (iv) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), (v) one Plasmodium CSP major repeat region or portion thereof, and (vi) one Plasmodium CSP C-terminal region or portion thereof.

[0020] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises one or more helper antigens. In some embodiments, the one or more helper antigens comprise a Plasmodium antigen. In some embodiments, the one or more helper antigens are Plasmodium 2-phospho-D-glycerate hydrolylase antigen, Plasmodium liver stage antigen 1(a), (LSA-1(a)), Plasmodium liver stage antigen 1(b) (LSA-1(b)), Plasmodium thrombospondin-related anonymous protein (TRAP), Plasmodium liver stage associated protein 1 (LSAP1), Plasmodium liver stage associated protein 2 (LSAP2), Plasmodium UIS3, Plasmodium UIS4, Plasmodium ETRAMP10.3, Plasmodium liver specific protein 1 (LISP-1), Plasmodium liver specific protein 2 (LISP-2), Plasmodium liver stage antigen 3 (LSA-3), Plasmodium EXP1, Plasmodium E140, Plasmodium reticulocyte-binding protein homolog 5 (Rh5), Plasmodium glutamic acid-rich protein (GARP), Plasmodium parasite-infected erythrocyte surface protein 2 (PIESP2), Plasmodium cysteine-rich protective antigen (CyRPA), Plasmodium Ripr, Plasmodium P113, or a combination thereof. In some embodiments, the one or more helper antigens comprise or consist of a P. falciparum 2-phospho-D-glycerate hydrolylase antigen. In some embodiments, the P. falciparum 2-phospho-D-glycerate hydrolylase antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 240. In some embodiments, the one or more helper antigens comprise or consist of a P. falciparum liver stage antigen 3. In some embodiments, the P. falciparum liver stage antigen 3 comprises or consists of an amino acid sequence according to SEQ ID NO: 243. In some embodiments, the one or more helper antigens comprise an Anopheles antigen.In some embodiments, the helper antigen comprises or consists of Anopheles gambiae TRIO. In some embodiments, Anopheles gambiae TRIO comprises or consists of an amino acid sequence according to SEQ ID NO:246.

[0021] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises a secretion signal and the helper antigen immediately follows the secretion signal.

[0022] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises a helper antigen at the C-terminus of the polypeptide.

[0023] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises a multimerization region. In some embodiments, the multimerization region comprises or consists of a trimerization region. In some embodiments, the trimerization region comprises or consists of a fibritin region. In some embodiments, the fibritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 255. In some embodiments, the polypeptide comprises a multimerization region at the N-terminus of the polypeptide.

[0024] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises a secretion signal. In some embodiments, the secretion signal comprises or consists of a Plasmodium secretion signal. In some embodiments, the Plasmodium secretion signal comprises or consists of a Plasmodium CSP secretion signal. In some embodiments, the Plasmodium CSP secretion signal comprises or consists of an amino acid sequence according to SEQ ID NO: 174. In some embodiments, the secretion signal comprises or consists of a heterologous secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a non-human secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a viral secretion signal. In some embodiments, the viral secretion signal comprises or consists of an HSV secretion signal. In some embodiments, the HSV secretion signal comprises or consists of an HSV-1 or HSV-2 secretion signal. In some embodiments, the HSV secretion signal comprises or consists of an HSV glycoprotein D (gD) secretion signal. In some embodiments, the HSV gD secretion signal comprises or consists of the amino acid sequence set forth in SEQ ID NO: 159. In some embodiments, the HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 165. In some embodiments, the secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, the Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal. In some embodiments, the Ebola virus SGP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 177.

[0025] In some embodiments, the secretion signal present in the polypeptide encoded by the provided polyribonucleotide is located at the N-terminus of the polypeptide.

[0026] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises a transmembrane region. In some embodiments, the transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, the Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, the Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 231.

[0027] In some embodiments, the transmembrane region present in the polypeptide encoded by the provided polyribonucleotide comprises or consists of a heterologous transmembrane region. In some embodiments, the heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, the HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 234.

[0028] In some embodiments, the transmembrane region present in the polypeptide encoded by the provided polyribonucleotide comprises or consists of a human transmembrane region. In some embodiments, the human transmembrane region comprises or consists of a human decay-accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, the hDAF-GPI anchor region comprises or consists of the amino acid sequence of SEQ ID NO: 237.

[0029] In some embodiments, the polypeptides encoded by the provided polyribonucleotides do not include a secretory signal.

[0030] In some embodiments, the polypeptide encoded by the provided polyribonucleotide does not include a transmembrane region.

[0031] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise one or more linkers. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 258. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 279. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 270. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 282.

[0032] In some embodiments where a transmembrane is present, the polypeptide encoded by the provided polyribonucleotide comprises a linker between the C-terminal region or portion thereof and the transmembrane region.

[0033] In some embodiments, in which the polypeptide encoded by the provided polyribonucleotide comprises the amino acid sequence of NANPNVDP (SEQ ID NO: 102), the polypeptide comprises a linker after the amino acid sequence of NANPNVDP (SEQ ID NO: 102).

[0034] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof (e.g., according to certain embodiments described herein), (ii) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102 (e.g., according to certain embodiments described herein), (iii) one or more Plasmodium CSP C-terminal regions or portions thereof (e.g., according to certain embodiments described herein), (iv) a secretion signal (e.g., according to certain embodiments described herein), and (v) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise (a) the amino acid sequence of NPNA (SEQ ID NO: 141), and (b) a Plasmodium CSP N-terminal region or portions thereof. In some embodiments, the polypeptide does not comprise a Plasmodium CSP N-terminal end region. In some embodiments, the polypeptide comprises one or more Plasmodium CSP N-terminal end regions or portions thereof (e.g., according to certain embodiments described herein). In some embodiments, the polypeptide comprises one or more helper antigens (e.g., according to certain embodiments described herein).

[0035] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (vi) five antigenic repeat regions, each antigenic repeat region comprising (A) a linker (e.g., according to certain embodiments described herein), and (B) a helper antigen (e.g., according to certain embodiments described herein), and the polypeptide does not comprise any of (a) the amino acid sequence of NPNA (SEQ ID NO: 141), (b) the Plasmodium CSP N-terminal region or a portion thereof, and (c) a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:36.

[0036] In some embodiments, the polypeptides encoded by the provided polyribonucleotides contain (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a helper antigen (e.g., according to certain embodiments described herein), (iii) a linker (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (vi) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (viii) a Plasmodium CSP (ix) a linker (e.g., according to certain embodiments described herein), and (x) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not contain (a) the amino acid sequence of NPNA (SEQ ID NO: 141) and (b) the Plasmodium CSP N-terminal region or portion thereof. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 39.

[0037] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a portion of the Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) the Plasmodium CSP N-terminal region or a portion thereof; (b) the Plasmodium CSP N-terminal end region or a portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:57.

[0038] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a portion of the Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) the Plasmodium CSP N-terminal region or a portion thereof; (b) the Plasmodium CSP N-terminal end region or a portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:60.

[0039] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) a Plasmodium CSP N-terminal end region or portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:63.

[0040] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) a Plasmodium CSP N-terminal end region or portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:66.

[0041] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP junction region variant (e.g., according to certain embodiments described herein), (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) a Plasmodium CSP N-terminal end region or portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:69.

[0042] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP junction region variant (e.g., according to certain embodiments described herein), (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) a Plasmodium CSP N-terminal end region or portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:72.

[0043] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a portion of the Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) the Plasmodium CSP N-terminal region or a portion thereof; (b) the Plasmodium CSP N-terminal end region or a portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:75.

[0044] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a portion of the Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of: (a) the Plasmodium CSP N-terminal region or a portion thereof; (b) the Plasmodium CSP N-terminal end region or a portion thereof; and (c) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:78.

[0045] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vii) a linker (e.g., according to certain embodiments described herein), and (viii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of (a) the Plasmodium CSP N-terminal region or a portion thereof, and (b) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:81.

[0046] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP junction region variant (e.g., according to certain embodiments described herein), (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vii) a linker (e.g., according to certain embodiments described herein), and (viii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of (a) the Plasmodium CSP N-terminal region or a portion thereof, and (b) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:84.

[0047] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (vi) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of (a) the Plasmodium CSP N-terminal region or a portion thereof, and (b) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:96.

[0048] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (vi) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of (a) the Plasmodium CSP N-terminal region or a portion thereof, and (b) the amino acid sequence of NPNA (SEQ ID NO: 141). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:99.

[0049] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) two or more Plasmodium CSP neutralization region repeats, each Plasmodium CSP neutralization region repeat comprising or consisting of: (a) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (b) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (c) two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), and (d) a linker (e.g., according to certain embodiments described herein), (iii) a portion of the major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (v) a Plasmodium CSP (vi) a serine-valine sequence immediately following the C-terminal region (e.g., according to certain embodiments described herein), (vi) a linker (e.g., according to certain embodiments described herein), and (vii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not include the Plasmodium CSP N-terminal region or a portion thereof. In some embodiments, the polypeptide includes exactly four Plasmodium CSP neutralizing region repeats. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 87.

[0050] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) one Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP major repeat region (e.g., according to certain embodiments described herein), (v) one Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (vii) a linker (e.g., according to certain embodiments described herein), and (viii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise any of (a) the Plasmodium CSP N-terminal region or portion thereof, and (b) the Plasmodium CSP N-terminal end region or portion thereof. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:30.

[0051] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a portion of the Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (viii) a serine immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:27.

[0052] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (viii) a serine immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:6.

[0053] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (viii) a serine immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:24.

[0054] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:93.

[0055] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a Plasmodium CSP major repeat region (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (viii) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 33.

[0056] In some embodiments, the polypeptides encoded by the provided polyribonucleotides comprise (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a Plasmodium CSP major repeat region (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (ix) a linker (e.g., according to certain embodiments described herein), (x) a multimerization region (e.g., according to certain embodiments described herein), and the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:42.

[0057] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a Plasmodium CSP major repeat region (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (viii) a serine immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (ix) a linker (e.g., according to certain embodiments described herein), and (x) a transmembrane region (e.g., according to certain embodiments described herein), wherein the polypeptide does not comprise a transmembrane region. In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:48.

[0058] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a Plasmodium CSP major repeat region (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (ix) a linker (e.g., according to certain embodiments described herein), (x) a transmembrane region (e.g., according to certain embodiments described herein). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 90.

[0059] In some embodiments, the polypeptide encoded by the provided polyribonucleotide comprises (i) a secretion signal (e.g., according to certain embodiments described herein), (ii) a Plasmodium CSP N-terminal region (e.g., according to certain embodiments described herein), (iii) a Plasmodium CSP N-terminal end region (e.g., according to certain embodiments described herein), (iv) a Plasmodium CSP junction region (e.g., according to certain embodiments described herein), (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) (e.g., according to certain embodiments described herein), (vi) a major repeat region of Plasmodium CSP (e.g., according to certain embodiments described herein), (vii) a Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), (viii) a serine immediately following the Plasmodium CSP C-terminal region (e.g., according to certain embodiments described herein), and (ix) a transmembrane region (e.g., according to certain embodiments described herein). In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO:21.

[0060] In some embodiments, the polypeptide has features (i)-(x) (as described above), if present, in numerical order from C-terminus to N-terminus.

[0061] In some embodiments, the Plasmodium is Plasmodium falciparum. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof present in the polypeptides of the provided polyribonucleotides are one or more P. falciparum CSP polypeptide regions or portions thereof. In some embodiments, the Plasmodium falciparum is Plasmodium falciparum isolate 3D7.

[0062] In some embodiments, the provided polyribonucleotide is an isolated polyribonucleotide. In some embodiments, the provided polyribonucleotide is an engineered polyribonucleotide. In some embodiments, the provided polyribonucleotide is a codon-optimized polyribonucleotide.

[0063] In one aspect, provided herein is an RNA construct comprising a polyribonucleotide described herein. In some embodiments, the RNA construct comprises, in 5' to 3' order, (i) a 5'UTR comprising or consisting of a modified human alpha-globin 5'-UTR, (ii) a polyribonucleotide as described herein, (iii) a 3'UTR comprising a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA, and (iv) a polyA tail sequence. In some embodiments, the 5'UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 415. In some embodiments, the 3'UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 416. In some embodiments, the polyA tail sequence is a split polyA tail sequence. In some embodiments, the split polyA tail sequence comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 417.

[0064] In some embodiments, the provided RNA constructs further comprise a 5' cap. In some embodiments, the provided RNA constructs further comprise a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide. In some embodiments, the 5' cap comprises or consists of a Cap1 structure comprising m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is at position +1 of the polyribonucleotide and G2 is at position +2 of the polyribonucleotide. In some embodiments, the cap-proximal sequence comprises a sequence comprising A3A4U5 (SEQ ID NO: 424) at positions A1 and G2 of the Cap1 structure and positions +3, +4, and +5 of the polyribonucleotide, respectively.

[0065] In some embodiments, provided polyribonucleotides include modified uridines in place of every uridine. In some embodiments, each modified uridine is N1-methyl-pseudouridine.

[0066] Compositions comprising the provided polynucleotides or the provided RNA constructs are also within the scope of the present disclosure. In some embodiments, such compositions further comprise lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes. In some embodiments, one or more polyribonucleotides or one or more RNA constructs are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes. In some embodiments, the composition further comprises lipid nanoparticles, and one or more polyribonucleotides or one or more RNA constructs are fully or partially encapsulated within the lipid nanoparticles. In some embodiments, the lipid nanoparticles target hepatocytes. In some embodiments, the lipid nanoparticles target secondary lymphoid organ cells. In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles. In some embodiments, the lipid nanoparticles each comprise (a) a polymer-conjugated lipid, (b) a cationically ionizable lipid, and (c) one or more neutral lipids. In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, the polymer-bound lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the one or more neutral lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC). In some embodiments, the one or more neutral lipids comprise cholesterol. In some embodiments, the cationically ionizable lipid comprises [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate). In some embodiments, the lipid nanoparticles have an average diameter of about 50-150 nm.

[0067] Another aspect provided herein relates to a pharmaceutical composition comprising a composition as described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a cryoprotectant, optionally the cryoprotectant is sucrose. In some embodiments, the pharmaceutical composition comprises an aqueous buffer solution, optionally the aqueous buffer solution comprises one or more of Tris base, Tris-HCl, NaCl, KCl, NaHPO, and KHPO.

[0068]

[0010] A further aspect provided herein relates to a combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide encodes a first polypeptide, wherein the first polypeptide comprises one or more Plasmodium CSP polypeptide regions or portions thereof; and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, wherein the second polypeptide comprises one or more Plasmodium T cell antigens. In some embodiments, the first polyribonucleotide is a polyribonucleotide according to certain embodiments described herein or an RNA construct according to certain embodiments described herein.

[0069] Also within the scope of the present disclosure is a method for administering to a subject a provided polyribonucleotide, a provided RNA construct, a provided composition, or a provided pharmaceutical composition.In some embodiments, the method comprises administering to a subject one or more doses of the pharmaceutical composition described herein.

[0070] In one aspect, a pharmaceutical composition as described herein for use in the treatment of a malaria infection, comprising administering to a subject one or more doses of the pharmaceutical composition. In another aspect, a pharmaceutical composition as described herein for use in the prevention of a malaria infection, comprising administering to a subject one or more doses of the pharmaceutical composition.

[0071] In some embodiments, two or more doses of pharmaceutical composition as described herein are administered to the subject.In some embodiments, three or more doses of pharmaceutical composition as described herein are administered to the subject.In some embodiments, the second dose of the three or more doses is administered to the subject at least four weeks after the first dose of the three or more doses is administered to the subject.In some embodiments, the third dose of the three or more doses is administered to the subject at least four weeks after the second dose of the three or more doses is administered to the subject.

[0072] In some embodiments, a fourth dose of the pharmaceutical composition described herein is administered to the subject. In some embodiments, the fourth dose is administered to the subject at least one year after the third dose of the three or more doses is administered to the subject.

[0073] Also provided herein are methods comprising administering to a subject a combination according to certain embodiments described herein. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on different days. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered to a subject at different locations on the subject's body.

[0074] In some embodiments, the technology described herein may be useful for treating a malaria infection. In some embodiments, the technology described herein may be useful for preventing a malaria infection. In some embodiments, a subject amenable to the technology described herein has or is at risk of developing a malaria infection. In some embodiments, a subject amenable to the technology described herein is a human.

[0075] In some embodiments, the administration induces an anti-malarial immune response in the subject. In some embodiments, the anti-malarial immune response in the subject comprises an adaptive immune response. In some embodiments, the anti-malarial immune response in the subject comprises a T cell response. In some embodiments, the T cell response is or comprises a CD4+ T cell response. In some embodiments, the T cell response is or comprises a CD8+ T cell response. In some embodiments, the anti-malarial immune system response comprises a B cell response. In some embodiments, the anti-malarial immune response comprises the production of antibodies directed against one or more Plasmodium antigens.

[0076] Also included within the scope of the present disclosure is the use of a pharmaceutical composition as described herein in the treatment of a malaria infection, the use of a pharmaceutical composition as described herein in the prevention of a malaria infection, and the use of a pharmaceutical composition as described herein in inducing an anti-malaria immune response in a subject.

[0077] Also within the scope of the present disclosure are polypeptides encoded by the polyribonucleotides according to the various embodiments described herein, polypeptides encoded by the RNA constructs according to the various embodiments described herein, host cells comprising the polyribonucleotides described herein, host cells comprising the RNA constructs described herein, and host cells comprising the polypeptides described herein. [Brief explanation of the drawings]

[0078] [Figure 1]Figure 1 shows in vitro expression of unformulated RNA constructs encoding different Plasmodium polypeptide constructs in HEK293T cells. A shows transfection rates as measured by the percentage of the total HEK293T population positive for the presence of expressed protein. B shows total expression as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), while non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Each sample was stained in triplicate, and bars represent the mean and SD; NT is non-transfected. [Figure 2A] Figure 1 shows in vitro expression of formulated RNA constructs in HEK293T cells. Transfection rates, as measured by the percentage of the total HEK293T population positive for the presence of expressed protein, are shown. [Figure 2B] Figure 1 shows in vitro expression of formulated RNA constructs in HEK293T cells. Total expression is shown as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), while non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Each sample was stained in triplicate, and bars represent the mean and SD. [Figure 2C] Figure 1 shows in vitro expression of formulated RNA constructs in HEK293T cells. The amount of protein detected in the culture supernatant is shown; each data point represents triplicate, NT = non-transfected. [Figure 3]Figure 1 shows in vitro expression of unformulated RNA constructs 59 and 60 encoding different Plasmodium polypeptides in HEK293T cells. A shows the transfection rate as measured by the percentage of the total HEK293T population positive for the presence of expressed protein. B shows total expression as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), while non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Protein was detected using anti-PfCSP 2A10 antibody. Each sample was stained in triplicate, and bars represent the mean and SD; NT is non-transfected. [Figure 4] Figure 1 shows in vitro expression of unformulated RNA constructs 91, 100, and 104 encoding different Plasmodium polypeptides in HEK293T cells. A shows the transfection rate as measured by the percentage of the total HEK293T population positive for the presence of expressed protein. B shows total expression as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), while non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Protein was detected using anti-PfCSP L9 antibody. Each sample was stained in triplicate, and bars represent the mean and SD; NT is non-transfected. [Figure 5]Figure 1 shows in vitro expression of unformulated RNA constructs 87 and 88 encoding different Plasmodium polypeptides in HEK293T cells. A shows transfection efficiency as measured by the percentage of the total HEK293T population positive for the presence of expressed protein. B shows total expression as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), while non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Protein was detected using anti-PfCSP L9 antibody. Each sample was stained in triplicate, and bars represent the mean and SD; NT is non-transfected. [Figure 6] Figure 1 shows in vitro expression of formulated RNA constructs 87, 88, 91, 100, and 104 encoding different Plasmodium polypeptides in HEK293T cells. A shows transfection rates as measured by the percentage of the total HEK293T population positive for the presence of expressed protein. B shows total expression as measured by the mean fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bars, intracellular staining), whereas non-permeabilized cells show only surface-expressed protein (gray bars, surface staining). Protein was detected using anti-PfCSP L9 antibody. Each sample was stained in triplicate; bars represent the mean and SD; NT is non-transfected. [Figure 7] Figure 1 shows immunogenicity induced in mice by formulated RNA constructs. A shows antibodies against Plasmodium falciparum (Pf) CSP full-length protein ("PfCSP-FL"). B shows antibodies against the PfCSP C-terminal domain ("PfCSP-C"). Each data point represents one mouse, and bars show the mean and SEM. LDL is the lower limit of detection. C shows antibodies against a region spanning the end of the N-terminal domain to the end of the minority repeat ("PfCsp-76-140"). [Figure 8]Figure 1 shows the immunogenicity induced in mice by formulated RNA constructs 87, 88, 91, 100, and 104. A shows antibodies against Plasmodium falciparum (Pf) CSP full-length protein ("PfCSP-FL"). B shows antibodies against the PfCSP C-terminal domain ("PfCSP-C-term(3D7)"). Each data point represents one mouse, and bars indicate the mean and SEM. LDL is the lower limit of detection. [Figure 9A] 9B-9K show binding to various epitopes of antibodies generated from mice immunized with different formulated RNA constructs. A visual summary of the data in Figures 9B-9K is shown in the form of a heat map. [Figure 9B] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9C] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9D] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9E] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9F] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9G]Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9H] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9I] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9J] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 9K] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes, each showing a bar representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 10] Binding of antibodies to various epitopes generated from mice immunized with different formulated RNA constructs 87, 88, 91, 104, and 100 during a challenge study is shown in heat map format. [Figure 11A] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11B] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11C]Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11D] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11E] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11F] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11G] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11H] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11I] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 11J] Figure 1 shows the binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes. Bars are shown representing the area under the curve (AUC) generated when plotting the dilution steps against the ECL signal. [Figure 12]Figure 1 shows the binding specificity of antibodies generated from mice immunized with different formulated RNA constructs to the CSP protein in Plasmodium falciparum sporozoite lysate. (A) shows the binding between antibodies (serum dilution 1:1250) and the CSP protein in sporozoite (spz) lysate as assessed by luminescence (cps, counts per second). (B) shows the binding between mouse anti-Pfs25 mAb 32F81, used as a negative control, and mouse anti-CSP mAb 3SP2, used as a positive control. [Figure 13A] Figure 1 shows the evaluation of antibodies generated from mice immunized with different formulated RNA constructs for their ability to inhibit P. falciparum sporozoite traversal. Results are shown as the percentage of inhibition of traversal activity (mean and SEM) compared to the vehicle control, which was set as 0% inhibition. [Figure 13B] 13B shows an evaluation of antibodies generated from mice immunized with different formulated RNA constructs for their ability to inhibit P. falciparum sporozoite traversal. Results for a negative control (serum from vehicle mice, FIG. 13B) and a positive control (mAb317, an antibody known to bind to the NANP (SEQ ID NO: 147) repeat in the major repeat region and inhibit traversal, FIG. 13C) are shown, with 002, 003, 005, 012, 014, and 018 representing different experimental runs. [Figure 13C] 13B shows an evaluation of antibodies generated from mice immunized with different formulated RNA constructs for their ability to inhibit P. falciparum sporozoite traversal. Results for a negative control (serum from vehicle mice, FIG. 13B) and a positive control (mAb317, an antibody known to bind to the NANP (SEQ ID NO: 147) repeat in the major repeat region and inhibit traversal, FIG. 13C) are shown, with 002, 003, 005, 012, 014, and 018 representing different experimental runs. [Figure 14]Figure 1 shows the evaluation of antibodies generated from mice immunized with different formulated RNA constructs for their ability to inhibit P. falciparum sporozoite infection of primary human hepatocytes. A-D show results as percentage of inhibition of infection activity (mean and SEM) compared to the vehicle control, which was set as 0% inhibition. E-F show results from a negative control (serum from vehicle mice, E) and a positive control (mAb317, an antibody known to inhibit hepatocyte infection, F). [Figure 15] Figure 1 shows the evaluation of antibodies generated from mice immunized with different formulated RNA constructs for their ability to inhibit P. falciparum sporozoite infection of primary human hepatocytes. A-C show results as percentage of inhibition of infection activity (mean and SEM) compared to the vehicle control, which was set as 0% inhibition. D-E show results from a negative control (serum from vehicle mice, D) and a positive control (mAb317, an antibody known to inhibit hepatocyte infection, E). [Figure 16] Figure 1 shows the ability of antibodies generated from mice immunized with different formulated RNA constructs to bind human complement and induce PfCSP sporozoite lysis. Results are presented as viable (unlysed) sporozoites as a percentage of total events recorded. A-C represent the same data at increasing dilutions (1:10, 1:100, and 1:1000, respectively). D represents the vehicle serum control at the same dilution. E shows results using the positive control mAb317 antibody, which binds to PfCSP and induces complete sporozoite lysis, and the negative control mAb1245 antibody, which binds to Pf proteins not expressed within sporozoites and therefore does not induce sporozoite lysis. Each data point represents a technical replicate of a pooled serum sample, and the bars show the mean and SEM. [Figure 17]Figure 1 shows the binding and dissociation of antibodies generated from mice immunized with different formulated RNA constructs to full-length PfCSP and two peptides (junction + minor repeat and major repeat). Serum samples from all animals immunized with the same construct were pooled before analysis. A shows the binding level of antibodies to each binding partner in RU. B represents the percentage of residual response, calculated from the initial binding, which means the percentage of antibody:antigen complexes still measurable 15 minutes after dissociation. RU is a relative unit. [Figure 18A] Figure 18 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (PfCSP_FL_pep, Figure 18A), MHC-I (Figure 18B), MHC-II (Figure 18C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 18D) at 4 μg / mL, positive control: concanavalin A at 2 μg / mL (Figure 18E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 18B] Figure 18 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (PfCSP_FL_pep, Figure 18A), MHC-I (Figure 18B), MHC-II (Figure 18C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 18D) at 4 μg / mL, positive control: concanavalin A at 2 μg / mL (Figure 18E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 18C]Figure 18 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (PfCSP_FL_pep, Figure 18A), MHC-I (Figure 18B), MHC-II (Figure 18C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 18D) at 4 μg / mL, positive control: concanavalin A at 2 μg / mL (Figure 18E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 18D] Figure 18 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (PfCSP_FL_pep, Figure 18A), MHC-I (Figure 18B), MHC-II (Figure 18C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 18D) at 4 μg / mL, positive control: concanavalin A at 2 μg / mL (Figure 18E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 18E]Figure 18 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (PfCSP_FL_pep, Figure 18A), MHC-I (Figure 18B), MHC-II (Figure 18C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 18D) at 4 μg / mL, positive control: concanavalin A at 2 μg / mL (Figure 18E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 19A] Figure 19 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 19A), MHC-I (Figure 19B), and MHC-II (Figure 19C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 19D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 19E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 19B]Figure 19 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 19A), MHC-I (Figure 19B), and MHC-II (Figure 19C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 19D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 19E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 19C] Figure 19 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 19A), MHC-I (Figure 19B), and MHC-II (Figure 19C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 19D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 19E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 19D]Figure 19 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 19A), MHC-I (Figure 19B), and MHC-II (Figure 19C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 19D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 19E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 19E] Figure 19 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 19A), MHC-I (Figure 19B), and MHC-II (Figure 19C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 19D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 19E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 20A]Figure 20 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 20A), MHC-I (Figure 20B), and MHC-II (Figure 20C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 20D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 20E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 20B] Figure 20 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 20A), MHC-I (Figure 20B), and MHC-II (Figure 20C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 20D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 20E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 20C]Figure 20 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 20A), MHC-I (Figure 20B), and MHC-II (Figure 20C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 20D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 20E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 20D] Figure 20 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 20A), MHC-I (Figure 20B), and MHC-II (Figure 20C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 20D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 20E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 20E]Figure 20 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 20A), MHC-I (Figure 20B), and MHC-II (Figure 20C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 20D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 20E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 21A] Figure 21 shows T cell activation, as assessed by IL-2 and IFN-γ secretion. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 21A), MHC-I (Figure 21B), and MHC-II (Figure 21C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 21D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 21E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 21B]Figure 21 shows T cell activation, as assessed by IL-2 and IFN-γ secretion. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 21A), MHC-I (Figure 21B), and MHC-II (Figure 21C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 21D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 21E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 21C] Figure 21 shows T cell activation, as assessed by IL-2 and IFN-γ secretion. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 21A), MHC-I (Figure 21B), and MHC-II (Figure 21C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 21D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 21E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 21D]Figure 21 shows T cell activation, as assessed by IL-2 and IFN-γ secretion. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 21A), MHC-I (Figure 21B), and MHC-II (Figure 21C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 21D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 21E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 21E] Figure 21 shows T cell activation, as assessed by IL-2 and IFN-γ secretion. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 21A), MHC-I (Figure 21B), and MHC-II (Figure 21C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 21D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 21E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 22A]Figure 22 shows T cell activation, as assessed by TNF-α and IFN-γ secretion. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 22A), MHC-I (Figure 22B), and MHC-II (Figure 22C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 22D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 22E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 22B] Figure 22 shows T cell activation, as assessed by TNF-α and IFN-γ secretion. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 22A), MHC-I (Figure 22B), and MHC-II (Figure 22C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 22D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 22E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 22C]Figure 22 shows T cell activation, as assessed by TNF-α and IFN-γ secretion. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 22A), MHC-I (Figure 22B), and MHC-II (Figure 22C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 22D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 22E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 22D] Figure 22 shows T cell activation, as assessed by TNF-α and IFN-γ secretion. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 22A), MHC-I (Figure 22B), and MHC-II (Figure 22C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 22D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 22E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 22E]Figure 22 shows T cell activation, as assessed by TNF-α and IFN-γ secretion. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 22A), MHC-I (Figure 22B), and MHC-II (Figure 22C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 22D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 22E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 23A] Figure 23 shows T cell activation, as assessed by TNF-α and IL-2 secretion. TNF-α and IL-2 secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 23A), MHC-I (Figure 23B), and MHC-II (Figure 23C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 23D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 23E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 23B]Figure 23 shows T cell activation, as assessed by TNF-α and IL-2 secretion. TNF-α and IL-2 secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 23A), MHC-I (Figure 23B), and MHC-II (Figure 23C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 23D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 23E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 23C] Figure 23 shows T cell activation, as assessed by TNF-α and IL-2 secretion. TNF-α and IL-2 secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 23A), MHC-I (Figure 23B), and MHC-II (Figure 23C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 23D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 23E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 23D]Figure 23 shows T cell activation, as assessed by TNF-α and IL-2 secretion. TNF-α and IL-2 secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 23A), MHC-I (Figure 23B), and MHC-II (Figure 23C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 23D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 23E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 23E] Figure 23 shows T cell activation, as assessed by TNF-α and IL-2 secretion. TNF-α and IL-2 secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 23A), MHC-I (Figure 23B), and MHC-II (Figure 23C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 23D) at 4 μg / mL; positive control: concanavalin A at 2 μg / mL (Figure 23E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes; ve is vehicle. [Figure 24A]Figure 24 shows T cell activation, as assessed by secretion of TNF-α, IL-2, and IFN-γ. TNF-α, IL-2, and IFN-γ secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 24A), MHC-I (Figure 24B), MHC-II (Figure 24C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 24D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 24E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, bars represent group mean spot-forming units (SFU) per 5×10 5 splenocytes ±SD, ve is vehicle. [Figure 24B] Figure 24 shows T cell activation, as assessed by secretion of TNF-α, IL-2, and IFN-γ. TNF-α, IL-2, and IFN-γ secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 24A), MHC-I (Figure 24B), MHC-II (Figure 24C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 24D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 24E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, bars represent group mean spot-forming units (SFU) per 5×10 5 splenocytes ±SD, ve is vehicle. [Figure 24C]Figure 24 shows T cell activation, as assessed by secretion of TNF-α, IL-2, and IFN-γ. TNF-α, IL-2, and IFN-γ secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 24A), MHC-I (Figure 24B), MHC-II (Figure 24C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 24D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 24E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, bars represent group mean spot-forming units (SFU) per 5×10 5 splenocytes ±SD, ve is vehicle. [Figure 24D] Figure 24 shows T cell activation, as assessed by secretion of TNF-α, IL-2, and IFN-γ. TNF-α, IL-2, and IFN-γ secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 24A), MHC-I (Figure 24B), MHC-II (Figure 24C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 24D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 24E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, bars represent group mean spot-forming units (SFU) per 5×10 5 splenocytes ±SD, ve is vehicle. [Figure 24E]Figure 24 shows T cell activation, as assessed by secretion of TNF-α, IL-2, and IFN-γ. TNF-α, IL-2, and IFN-γ secretion were assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering epitopes predicted to be present on the full-length CSP protein (PfCSP_FL_pep, Figure 24A), MHC-I (Figure 24B), MHC-II (Figure 24C), or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 425], Figure 24D), 4 μg / mL; positive control: concanavalin A, 2 μg / mL (Figure 24E)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, bars represent group mean spot-forming units (SFU) per 5×10 5 splenocytes ±SD, ve is vehicle. [Figure 25] Figure 1 shows T cell activation, as assessed by IFN-γ secretion. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice, and ve is vehicle. [Figure 26]Figure 1 shows T cell activation, as assessed by IL-2 secretion. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice, and ve is vehicle. [Figure 27] Figure 1 shows T cell activation, as assessed by TNF-α secretion. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice, and ve is vehicle. [Figure 28] Figure 1 shows T cell activation, as assessed by both IFN-γ and IL-2 secretion. IFN-γ + IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice, and ve is vehicle. [Figure 29] Figure 1 shows T cell activation, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ + TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the media and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice, and ve is vehicle. [Figure 30] Figure 1 shows T cell activation, as assessed by the secretion of both IL-2 and TNF-α. IL-2 + TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice. ve is vehicle. [Figure 31]Figure 1 shows T cell activation, as assessed by secretion of IFN-γ, IL-2, and TNF-α. IFN-γ, IL-2, and TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C)). Samples were measured in triplicate, and negative controls were measured in duplicate. Each data point represents a single mouse, and bars represent group mean spot-forming units (SFU) ± SD per 5 × 10 splenocytes. Each data point in the medium and concanavalin A controls represents the mean of triplicates of a pool of splenocytes from all mice. ve is vehicle. [Figure 32] Only CD4 T cell activation, as assessed by IFN-γ secretion, is shown. IFN-γ secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4 T cells, and ve is vehicle. [Figure 33]Activation of CD4+ T cells only, as assessed by IL-2 secretion, is shown. IL-2 secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4+ T cells, and ve is vehicle. [Figure 34] Only CD4 T cell activation, as assessed by TNF-α secretion, is shown. TNF-α secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4 T cells; ve is vehicle. [Figure 35]Activation of CD4+ T cells alone, as assessed by secretion of both IFN-γ and IL-2, is shown. IFN-γ + IL-2 secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4+ T cells, and ve is vehicle. [Figure 36] Activation of CD4+ T cells alone, as assessed by secretion of both IFN-γ and TNF-α, is shown. IFN-γ + TNF-α secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) per 1 × 105 CD4+ T cells ± SD; ve is vehicle. [Figure 37]Activation of CD4+ T cells alone, as assessed by secretion of both IL-2 and TNF-α, is shown. IL-2 + TNF-α secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4+ T cells; ve is vehicle. [Figure 38] Activation of CD4+ T cells alone, as assessed by secretion of IFN-γ, IL-2, and TNF-α, is shown. IFN-γ, IL-2, and TNF-α secretion was assessed by fluorospot assay after isolating CD4+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD4+ T cells; ve is vehicle. [Figure 39]Activation of CD8+ T cells only, as assessed by IFN-γ secretion, is shown. IFN-γ secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells, and ve is vehicle. [Figure 40] Activation of CD8+ T cells only, as assessed by IL-2 secretion, is shown. IL-2 secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering the full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells, and ve is vehicle. [Figure 41]Figure 1 shows activation of CD8+ T cells alone, as assessed by TNF-α secretion. TNF-α secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells; ve is vehicle. [Figure 42] Only CD8 T cell activation, as assessed by both IFN-γ and IL-2 secretion, is shown. IFN-γ + IL-2 secretion was assessed by fluorospot assay after isolating CD8 T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 10 CD8 T cells; ve is vehicle. [Figure 43]Activation of CD8+ T cells alone, as assessed by secretion of both IFN-γ and TNF-α, is shown. IFN-γ + TNF-α secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells, and ve is vehicle. [Figure 44] Activation of CD8+ T cells alone, as assessed by secretion of both IL-2 and TNF-α, is shown. IL-2 + TNF-α secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells; ve is vehicle. [Figure 45]Activation of CD8+ T cells alone, as assessed by secretion of IFN-γ, IL-2, and TNF-α, is shown. IFN-γ, IL-2, and TNF-α secretion was assessed by fluorospot assay after isolating CD8+ T cells (from pools of splenocytes from mice immunized with different formulated RNA constructs) using MACS separation. Cells were then incubated with overlapping peptide pools covering full-length CSP protein (A) or controls (e.g., negative control: Trp1, 2 μg / mL (B); positive control: concanavalin A, 2 μg / mL (C); medium control (D)). Pooled samples were measured in triplicate, and negative controls were measured in duplicate. Data points and bars represent group mean spot-forming units (SFU) ± SD per 1 × 105 CD8+ T cells; ve is vehicle. [Figure 46A] Protection of mice immunized with formulated RNA constructs against challenge with PfCSP-expressing P. berghei sporozoites and the immunogenicity induced by this immunization are shown. The percentage of protected mice is shown for mice immunized with formulated RNA constructs, vehicle, or a positive control up to day 11 after challenge with PfCSP-expressing P. berghei sporozoites. Mice that received 100 μg of 2A10 monoclonal antibody 24 hours before challenge were used as a positive control. [Figure 46B] Protection of mice immunized with the formulated RNA construct against challenge with PfCSP-expressing P. berghei sporozoites and the immunogenicity induced by this immunization are shown. Endpoint titers against full-length PfCSP are shown for mice immunized with the formulated RNA construct and mice injected with vehicle alone, 2 weeks after the boost (day 35, Figure 46B) and 1 day before challenge (day 49, Figure 46C). Mean ± SEM and individual animal values ​​are shown. [Figure 46C]Protection of mice immunized with the formulated RNA construct against challenge with PfCSP-expressing P. berghei sporozoites and the immunogenicity induced by this immunization are shown. Endpoint titers against full-length PfCSP are shown for mice immunized with the formulated RNA construct and mice injected with vehicle alone, 2 weeks after the boost (day 35, Figure 46B) and 1 day before challenge (day 49, Figure 46C). Mean ± SEM and individual animal values ​​are shown. [Figure 46D] Figure 1 shows protection of mice immunized with formulated RNA constructs against challenge with PfCSP-expressing P. berghei sporozoites and the immunogenicity induced by this immunization. Figure 2 shows a visual summary of antibody binding to specific PfCSP epitopes (generated by immunization with RNA constructs 2, 23, and 39 during the challenge study). [Figure 47] Figure 1 shows the evaluation of antibodies generated from mice immunized with formulated RNA constructs for their ability to recognize native PfCSP on sporozoites and inhibit sporozoite viability and motility. (A) Log endpoint titers using fixed PfCSP-expressing P. berghei sporozoites. Symbols represent the mean ± SEM using sera from individual mice. (B) Estimated length of circumsporozoite precipitation reaction (CSPR) elicited by serum samples from immunized mice, as measured by flow cytometry (forward scatter width (FSC-W)). Symbols represent the mean ± SEM using sera from individual mice. (C) Cytotoxicity of serum samples from immunized mice against sporozoites in suspension (PBS). Symbols represent the mean ± SEM using sera from individual mice. (D) Cytotoxicity in 3D (Matrigel). Symbols represent the mean ± SEM using sera from individual mice. (E) Inhibition of sporozoite gliding velocity. Circles represent the mean ± SEM of duplicate pooled serum samples from each group. [Figure 48A]Protection of mice immunized with formulated RNA constructs against challenge with PfCSP-expressing P. berghei sporozoites and immunogenicity induced by immunization from three separate experiments is shown. The percentage of protected mice is shown for mice immunized with formulated RNA constructs, vehicle (saline), or a positive control up to day 11 after challenge with PfCSP-expressing P. berghei sporozoites. Mice receiving 100 μg of 2A10 monoclonal antibody or Mosquirix® 24 hours before challenge were used as positive controls. [Figure 48B] Protection of mice immunized with formulated RNA constructs against challenge with PfCSP-expressing P. berghei sporozoites and immunogenicity induced by immunization from three separate experiments are shown. Endpoint titers against full-length PfCSP 2 weeks after boost (day 35) and 1 day before challenge (day 49) are shown for mice immunized with formulated RNA constructs and mice injected with vehicle alone. Mean ± SEM and individual animal values ​​are shown. Mice receiving 100 μg of 2A10 monoclonal antibody 24 hours prior to challenge were used as a positive control in experiment 1. Serum samples from this group were collected 20 hours after passive immunization with 2A10. Mice immunized twice IM with 5 μg of Mosquirix® were used as a positive control in experiments 2 and 3. Mice injected with vehicle were used as a negative control in all experiments. [Figure 49A] Figure 1 shows the evaluation of antibodies generated from mice immunized with the formulated RNA construct. Figure 1 shows the evaluation of antibodies generated from mice immunized with the formulated RNA construct for their ability to recognize native PfCSP sporozoites. The graph shown corresponds to experiment 1 of the challenge study and shows the logarithm of the anti-sporozoite endpoint titer using fixed PfCSP-expressing P. berghei sporozoites. Symbols represent the mean ± SEM using sera from individual mice. 2A10 is a positive antibody control; IFA is an immunofluorescence assay. [Figure 49B]Figure 1 shows an evaluation of antibodies generated from mice immunized with formulated RNA constructs. Figure 1 shows an evaluation of antibodies generated from mice immunized with formulated RNA constructs for their ability to inhibit sporozoite gliding motility. The graph shown corresponds to experiment 1 of the challenge study and shows sporozoite gliding velocity (μm / s). Symbols represent the mean ± SEM of duplicate pooled serum samples from each group. 2A10 is a positive antibody control, and Veh is vehicle. [Figure 49C] Figure 1 shows the evaluation of antibodies generated from mice immunized with formulated RNA constructs. Figure 1 shows the evaluation of antibodies generated from mice immunized with formulated RNA constructs for their ability to bind and crosslink native PfCSP on the sporozoite surface. The graph shown corresponds to experiment 1 of the challenge study and represents the estimated length of the circumsporozoite precipitation reaction (CSPR) elicited by 17% of immune sera as measured by flow cytometry (forward scatter width (FSC-W)). Symbols represent the mean ± SEM using sera from individual mice. 2A10 is a positive antibody control, and Veh is vehicle. [Figure 49D] Figure 49A shows an evaluation of antibodies generated from mice immunized with the formulated RNA constructs. The cytotoxicity of antibodies present in serum samples from immunized mice against sporozoites in suspension (PBS) above and in 3D (Matrigel) below is shown. Figure 49B shows the cytotoxicity of 17% immune serum measured against PfCSP-expressing P. berghei sporozoites in suspension, presented as the percentage of viable sporozoites. [Figure 49E]Figure 49A shows the evaluation of antibodies generated from mice immunized with formulated RNA constructs. Figure 49B shows the cytotoxicity of antibodies present in serum samples from immunized mice against sporozoites in suspension (PBS) as above and in 3D (Matrigel) as below. Figure 49C shows the cytotoxicity of 17% immune serum measured against PfCSP-expressing P. berghei sporozoites in suspension, presented as the percentage of viable sporozoites. Figure 49D shows the cytotoxicity of 17% immune serum measured against PfCSP-expressing P. berghei sporozoites in 3D Matrigel and normalized to the saline group (viability = 100%). Symbols represent the mean ± SEM using serum from individual mice. 2A10 is a positive antibody control, Veh is vehicle, and PBS is phosphate-buffered saline. [Figure 50] Figure 1 shows the evaluation of antibodies generated from mice immunized with formulated RNA of five prioritized constructs for their ability to recognize native PfCSP sporozoites. Each graph represents a different experiment and shows the logarithm of the anti-sporozoite endpoint titer using fixed PfCSP-expressing P. berghei sporozoites. Bars represent the mean ± SEM using sera from individual mice. 2A10 is a positive antibody control, Mos is a Mosquirix® positive control, and IFA is an immunofluorescence assay. [Figure 51] Figure 1 shows an evaluation of antibodies generated from mice immunized with formulated RNA of five prioritized constructs for their ability to inhibit sporozoite gliding motility. Each graph represents a different experiment and shows sporozoite gliding velocity (μm / s). Bars represent the mean ± SEM of duplicate (in experiment 1) or single replicates (experiments 2 and 3) of pooled serum samples from each group. 2A10 is a positive antibody control, Mos is the Mosquirix® positive control, and Veh is vehicle. [Figure 52]Figure 1 shows the evaluation of antibodies generated from mice immunized with formulated RNA of five prioritized constructs for their ability to bind and crosslink native PfCSP on the sporozoite surface. Each graph represents a different experiment and shows the estimated length of the circumsporozoite precipitation reaction (CSPR) elicited by 17% of immune sera, as measured by flow cytometry (forward scatter width (FSC-W)). Symbols represent the mean ± SEM using sera from individual mice. 2A10 is a positive antibody control, Mos is the Mosquirix® positive control, and Veh is vehicle. [Figure 53] Figure 1 shows the cytotoxicity of antibodies present in serum samples immunized with five prioritized formulated RNA constructs against sporozoites in suspension (PBS) as above and in 3D (Matrigel) as below. (A) shows the cytotoxicity of 17% immune serum measured against PfCSP-expressing P. berghei sporozoites in suspension, presented as the percentage of viable sporozoites. (B) shows the cytotoxicity of 17% immune serum measured against PfCSP-expressing P. berghei sporozoites in 3D Matrigel and normalized to the saline group (viability = 100%). Each graph represents an independent challenge experiment (designated Experiment 1, Experiment 2, and Experiment 3). Bars represent the mean ± SEM using serum from individual mice. 2A10 is a positive antibody control; Mosquirix® is a positive control; Veh is vehicle; and PBS is phosphate-buffered saline. [Figure 54] Schematics of exemplary Plasmodium polypeptide constructs are included. The circle in RNA construct 91 indicates the T337N mutation (numbered according to SEQ ID NO: 1) to remove the O-fucose site.

[0079] definition The compounds of the present disclosure include those outlined above and are further represented by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Edition: Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of each of which are incorporated herein by reference.

[0080] Unless otherwise specified, structures depicted herein are intended to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, R and S configurations of each stereocenter are contemplated as part of the present disclosure. Accordingly, single stereochemical isomers of the provided compounds, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures, are within the scope of the present disclosure. For example, in some cases, the provided compounds represent one or more stereoisomers of the compound, and unless otherwise specified, each stereoisomer is represented alone and / or as a mixture. Unless otherwise specified, all tautomeric forms of the provided compounds are within the scope of the present disclosure.

[0081] Unless otherwise stated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of a hydrogen by deuterium or tritium, or the replacement of a carbon by a C- or C-enriched carbon are within the scope of this disclosure.

[0082] About: The term "about," when used herein with respect to a value, refers to a value that is contextually similar to the referenced value. Generally, the appropriate degree of variation encompassed by "about" in that context will be apparent to one of ordinary skill in the art familiar with the context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0083] Drug: As used herein, the term "drug" may refer to a physical entity. In some embodiments, a drug may be characterized by particular characteristics and / or effects. For example, as used herein, the term "therapeutic agent" refers to a physical entity that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect. In some embodiments, a drug may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, a polypeptide, a nucleic acid, a monosaccharide, a lipid, a metal, or a combination or complex thereof.

[0084] Amino acid: In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including the carboxy- and / or amino-terminal amino acids in a polypeptide, may contain structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing an otherwise identical, unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid compared to one otherwise identical that comprises the unmodified amino acid. As will be clear from the context, the term "amino acid" may in some embodiments be used to refer to a free amino acid, and in some embodiments, to an amino acid residue of a polypeptide.

[0085] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response and / or that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody.

[0086] Anti-malarial immune response: As used herein, the term "anti-malarial immune response" refers to an immune response directed against one or more antigens derived from Plasmodium.

[0087] Associated: As used herein, two events or entities are "associated" with one another when the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence, susceptibility, severity, stage, etc. of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they directly or indirectly interact to bring them into and / or maintain close physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0088] C-terminal domain: As used herein, the term "C-terminal domain" refers to the region of the CSP polypeptide corresponding to amino acids 273 to 397 of the wild-type CSP sequence of Plasmodium falciparum (isolate 3D7) (SEQ ID NO: 1).

[0089] C-terminal region: As used herein, the term "C-terminal region" refers to the region of a CSP polypeptide corresponding to amino acids 273-375 of the wild-type CSP sequence (SEQ ID NO: 1). In some embodiments, a serine immediately follows the C-terminal region. In some embodiments, a serine and a valine immediately follow the C-terminal region.

[0090] Central domain: The term "central domain," as used herein, refers to the region of a CSP polypeptide corresponding to amino acids 105 to 272 of the wild-type CSP sequence (SEQ ID NO: 1).

[0091] Characteristic portion: As used herein, the term "characteristic portion" most broadly refers to a portion of a polypeptide or region thereof whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the polypeptide or region thereof. In some embodiments, a characteristic portion of a polypeptide or region thereof is a portion found in the polypeptide or region thereof and related polypeptides or regions thereof that share a particular characteristic, attribute, or activity, but not a portion that does not share the particular characteristic, attribute, or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact polypeptide or region thereof. For example, in some embodiments, a "characteristic portion" of a polypeptide or region thereof is one that includes a contiguous stretch of amino acids, or a collection of contiguous stretches of amino acids, that together are characteristic of the polypeptide or region thereof. In some embodiments, each such contiguous stretch generally contains 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a polypeptide or region thereof (e.g., CSP, its N-terminal domain, its major repeat region, etc.) is a portion that, in addition to the sequence and / or structural identity specified above, shares at least one functional characteristic with the related intact polypeptide or region thereof. In some embodiments, a characteristic portion is biologically active. In some embodiments, a fragment as described herein may be a portion. Thus, in some embodiments, a characteristic fragment may be a "characteristic portion."

[0092] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to the administration of any dose of a second regimen); and in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administering one or more agent(s) or modality(s) to a subject receiving other agent(s) or modality(s) being combined. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents or active portions thereof may be administered together in a combined composition.

[0093] The term "equivalent," as used herein, refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them where one of skill in the art would understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few different characteristics. A skilled artisan will understand the degree of identity required for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable in any given context. For example, a skilled artisan will understand that sets of circumstances, individuals, or populations are comparable to one another if they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by, or indicate, variations in those characteristics.

[0094] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position / identity of a structural element in one compound or composition relative to another compound or composition (e.g., relative to an appropriate reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as "corresponding" to a residue in an appropriate reference polymer. For example, those skilled in the art often designate residues in a polypeptide using a standard numbering system with reference to a reference related polypeptide for simplicity, and as a result, an amino acid "corresponding to," for example, residue 190 in a particular amino acid chain, does not necessarily correspond to the actual residue at position 190. 番目It will be understood that the amino acid sequence of the reference polypeptide need not be the amino acid sequence of the reference polypeptide, but rather is intended to correspond to the residue found at position 190 in the reference polypeptide, and one of skill in the art will readily understand how to identify a "corresponding" amino acid. For example, one of skill in the art will recognize various alignment strategies, e.g., software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which may be utilized to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. One of skill in the art will also recognize that the term "corresponding to" may, in some cases, be used to describe an entity or entity that bears a meaningful similarity to another entity or entity (e.g., an appropriate reference entity or entity). As an example, a gene or protein in one organism can be described as "corresponding to" a gene or protein from another organism to indicate that, in some embodiments, it plays a similar role or performs a similar function, and / or that it exhibits a particular degree of sequence identity or homology or shares certain characteristic sequence elements.

[0095] Dosing regimen: Those skilled in the art will appreciate that the term "dosing regimen" (or "therapeutic regimen") can be used to refer to a set of unit doses (typically two or more) administered individually to a subject, typically spaced apart over a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more doses.

[0096] Encode: As used herein, the terms "encode" or "encoding" refer to the sequence information of a first molecule that directs the production of a second molecule having a defined sequence of nucleotides (e.g., polyribonucleotides) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process involving a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, cDNA, or RNA molecule encodes a polypeptide if the polypeptide is produced in a cell or other biological system by transcription and translation of the RNA corresponding to that gene. In some embodiments, the coding region of a polyribonucleotide encoding a target antigen refers to the coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such target antigen. In some embodiments, the coding region of a polyribonucleotide encoding a target antigen refers to the non-coding strand of such target antigen, the non-coding strand being one that can be used as a template for transcription of the gene or cDNA.

[0097] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product, e.g., a polyribonucleotide as provided herein. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0098] Helper antigen: As used herein, the term "helper antigen" refers to an antigen contained in a polypeptide that includes one or more CSP polypeptide regions or portions thereof, where the antigen is not derived from a CSP polypeptide.

[0099] Heterologous: As used herein, the term "heterologous" in reference to a secretory signal or transmembrane region refers to a secretory signal or transmembrane region from a virus or organism other than Plasmodium.

[0100] Homology: As used herein, the terms "homology" or "homologues" refer to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be homologous to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be homologous to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). For example, as is well known to those of skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains. Substitution of one amino acid for another amino acid of the same type can often be considered a "homologous" substitution.

[0101] Identity: As used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "substantially identical" to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity of two nucleotide sequences can be determined using the algorithm of Meyers and Miller (1989) incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0102] Increased, induced, or reduced: As used herein, these terms, or grammatically equivalent comparative terms, refer to a value relative to a comparable reference measurement. For example, in some embodiments, an assessment value obtained using a provided composition (e.g., a pharmaceutical composition) may be "increased" compared to an assessment value obtained using a comparable reference composition. Alternatively or additionally, in some embodiments, an assessment value obtained in a subject may be "increased" compared to an assessment value obtained in the same subject under different conditions (e.g., before or after an event, or in the presence or absence of an event, such as administration of a composition (e.g., a pharmaceutical composition) described herein) or in a different comparable subject (e.g., in a comparable subject that differs from the subject of interest in that it has been exposed to a prior condition, such as the absence of administration of a composition (e.g., a pharmaceutical composition) described herein). In some embodiments, comparative terms refer to a statistically significant difference (e.g., of sufficient predominance and / or magnitude to achieve statistical significance). Determining the degree and / or extent of the difference necessary or sufficient to achieve such statistical significance in a given situation is recognized or readily possible by those skilled in the art. In some embodiments, the term "reduce" or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or more relative to a comparable reference. In some embodiments, the term "reduce" or equivalent terms refers to complete or substantially complete inhibition, i.e., a reduction to zero or substantially zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or more relative to an equivalent reference.

[0103] In order: As used herein with respect to a polynucleotide or polyribonucleotide, "in order" refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with respect to a polypeptide, "in order" refers to the order of features along the polypeptide, moving from the most N-terminal feature to the most C-terminal feature. "In order" does not mean that there cannot be additional features between the listed features. For example, if features A, B, and C of a polynucleotide are described herein as being "in order, feature A, feature B, and feature C," this description does not exclude, for example, feature D from being located between feature A and feature B.

[0104] Isolated: The term "isolated" means modified or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0105] Junction: As used herein, the term "junction" refers to the region of a CSP polypeptide corresponding to amino acids 98-104 of the wild-type CSP sequence (SEQ ID NO: 1).

[0106] Junction region: As used herein, the term "junction region" refers to the region of a CSP polypeptide corresponding to amino acids 93-104 of the wild-type CSP sequence (SEQ ID NO: 1).

[0107] Junction region variant: As used herein, the term "junction region variant" refers to a junction region that contains one or more substitution mutations compared to amino acids 93-104 of the wild-type CSP sequence (SEQ ID NO: 1).

[0108] Linker: As used herein, the term "linker" refers to a portion of a polypeptide that connects different regions, moieties, or antigens to one another.

[0109] Lipid: As used herein, the terms "lipid" and "lipid-like substance" are broadly defined as molecules that contain one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules that contain hydrophobic and hydrophilic moieties are also typically referred to as amphiphiles.

[0110] Major repeat region: As used herein, the term "major repeat region" refers to a region of a CSP polypeptide corresponding to amino acids 129-272 of the wild-type CSP sequence (SEQ ID NO: 1) and containing 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147). The 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147) are separated into two contiguous stretches, the first containing 17 repeats of the amino acid sequence NANP (SEQ ID NO: 147) and the second containing 18 repeats of the amino acid sequence NANP (SEQ ID NO: 147) adjacent to the amino acid sequence of NVDP (SEQ ID NO: 144). A portion of the major repeat region contains at least the amino acid sequence NPNA (SEQ ID NO: 141). Preferably, a portion of the major repeat region contains at least the amino acid sequences NANPNA (SEQ ID NO: 153) and NPNANP (SEQ ID NO: 150). As used herein, "repeat" with respect to sequence A refers to the presence of sequence A once, and "one or more repeats" of sequence A refers to the presence of sequence A more than once.

[0111] Merozoite stage-specific Plasmodium antigen: As used herein, the term "merozoite stage-specific Plasmodium antigen" refers to an antigen that is expressed during the merozoite stage of the Plasmodium life cycle.

[0112] Minority repeat region: As used herein, the term "minority repeat region" refers to a region of a CSP polypeptide corresponding to amino acids 105-128 of the wild-type CSP sequence (SEQ ID NO: 1) and containing three repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 102). The minority repeat region does not contain the amino acid sequence NPNA (SEQ ID NO: 141) and does not contain the amino acid sequences NANPNA (SEQ ID NO: 153) or NPNANP (SEQ ID NO: 150). As used herein, "repeat" with respect to sequence A refers to the presence of sequence A once, and "three repeats" of sequence A refers to the presence of sequence A three or more times.

[0113] Multimerization domain: As used herein, the term "multimerization domain" refers to a domain that directs the assembly of multimers into a complex, each multimer comprising a polypeptide associated with the multimerization domain.

[0114] N-terminal domain: As used herein, the term "N-terminal domain" refers to the region of a CSP polypeptide corresponding to amino acids 19-92 of the wild-type CSP sequence (SEQ ID NO: 1).

[0115] N-terminal end region: As used herein, the term "N-terminal end region" refers to the region of a CSP polypeptide corresponding to amino acids 81-92 of the wild-type CSP sequence (SEQ ID NO: 1).

[0116] N-terminal region: As used herein, the term "N-terminal region" refers to the region of a CSP polypeptide corresponding to amino acids 19-80 of the wild-type CSP sequence (SEQ ID NO: 1).

[0117] RNA lipid nanoparticles: As used herein, the term "RNA lipid nanoparticles" refers to nanoparticles comprising at least one lipid and RNA molecule(s), such as one or more polyribonucleotides provided herein. In some embodiments, the RNA lipid nanoparticles comprise at least one cationic amino lipid. In some embodiments, the RNA lipid nanoparticles comprise at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, the RNA lipid nanoparticles described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, the RNA-lipid nanoparticles may have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average particle size of the lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, the RNA-lipid nanoparticles may be prepared by mixing lipids with the RNA molecules described herein.

[0118] Neutralization: As used herein, the term "neutralization" refers to an event in which a binding agent, such as an antibody, binds to a biologically active site on a parasite, such as a receptor-binding protein, thereby inhibiting parasite infection of a cell. In some embodiments, the term "neutralization" refers to an event in which the ability of the binding agent to infect a cell is eliminated or significantly reduced.

[0119] Nucleic Acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone having both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid can comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof).In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 275, 3000, 3250, 3500, 375, 4000, 4250, 4500, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, and 500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0120] Pharmaceutically effective amount: The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that alone, or together with further doses, produces a desired response or a desired effect. In the case of treating a particular disease (e.g., malaria), the desired response in some embodiments relates to inhibiting the progression of the disease (e.g., malaria). In some embodiments, such inhibition may include slowing the progression of the disease (e.g., malaria) and / or halting or reversing the progression of the disease (e.g., malaria). In some embodiments, the desired response in treating a disease (e.g., malaria) may be or may include delaying or preventing the onset of the disease (e.g., malaria) or condition (e.g., a malaria-related condition). Effective amounts of the compositions (e.g., pharmaceutical compositions) described herein will depend on individual patient parameters, including, for example, the disease (e.g., malaria) or condition (e.g., a malaria-related condition) to be treated, the severity of such disease (e.g., malaria) or condition (e.g., a malaria-related condition), e.g., age, physiological state, size and weight, duration of treatment, type of concomitant therapy (if any), the particular route of administration, and similar factors. Thus, the dose of the compositions (e.g., pharmaceutical compositions) described herein may depend on various such parameters. If the patient does not respond to the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0121] Polypeptide: As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or produced by human activity. In some embodiments, a polypeptide can include or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can include only natural amino acids, only unnatural amino acids, or only natural amino acids, or only unnatural amino acids. In some embodiments, a polypeptide can include D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can include only D-amino acids. In some embodiments, a polypeptide can include only L-amino acids. In some embodiments, a polypeptide can include one or more pendant groups or other modifications, e.g., one or more amino acid side chains modified or attached to one or more amino acid side chains at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications include acetylation, amidation, lipidation, methylation, pegylation, and the like (including combinations thereof). In some embodiments, a polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be applied to the name of a reference polypeptide, activity, or structure, and in such cases, it is used to refer to polypeptides that share a related activity or structure and therefore may be considered members of the same class or family of polypeptides.For each such class, the present specification provides, and / or one of skill in the art would recognize, exemplary polypeptides within the class whose amino acid sequence and / or function are known. In some embodiments, such exemplary polypeptides are reference polypeptides for a class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments, with all polypeptides within the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, a similar level or activity within a specified range) with the reference polypeptide of the class (and in some embodiments, with all polypeptides within the class). For example, in some embodiments, a member polypeptide exhibits a degree of overall sequence homology or identity with a reference polypeptide of at least about 30-40%, often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or comprises at least one region (e.g., a conserved region, which in some embodiments may be or may include a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least three to four, and often up to 35 or more, amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more consecutive amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, the polypeptide is a Plasmodium polypeptide construct described herein. A Plasmodium polypeptide construct is a polypeptide comprising one or more Plasmodium proteins, or one or more portions thereof.In some embodiments, the Plasmodium polypeptide constructs described herein comprise at least one region or portion of a Plasmodium CSP, hi some embodiments, the Plasmodium polypeptide constructs further comprise one or more additional amino acid sequences, such as a secretion signal (e.g., a heterologous secretion signal), a transmembrane region (e.g., a heterologous transmembrane region), a helper antigen, a multimerization region, and / or a linker, as described herein.

[0122] As used herein, the terms "prevent" or "prevention," when used in reference to the occurrence of a disease, disorder, and / or condition, refer to a reduction in the risk of developing a disease, disorder, and / or condition and / or a delay in the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predefined period of time. In some embodiments, prevention refers to a reduction in the risk of developing clinical malaria.

[0123] R1: As used herein, the term "R1" refers to the region of the CSP polypeptide corresponding to amino acids 93-97 of the wild-type CSP sequence (SEQ ID NO: 1).

[0124] Reference: As used herein, the term "reference" refers to a standard or control for comparison. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or measured substantially simultaneously with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, a reference or control is measured or characterized under conditions or circumstances comparable to those being evaluated, as will be apparent to one of skill in the art. One of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control considered.

[0125] Ribonucleic acid (RNA) or polyribonucleotide: As used herein, the terms "ribonucleic acid," "RNA," or "polyribonucleotide" refer to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA contains both single-stranded and double-stranded portions. In some embodiments, the RNA may contain a backbone structure described in the definition of "nucleic acid / polynucleotide" above. The RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, the RNA is an mRNA. In some embodiments where the RNA is an mRNA, the RNA typically contains a poly(A) region at its 3' end. In some embodiments, the RNA is an mRNA, and the RNA typically contains an art-recognized cap structure at its 5' end for, for example, recognition and attachment of the mRNA to a ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic and / or chemical synthesis). In some embodiments, the polyribonucleotide encodes a polypeptide, which is preferably a Plasmodium polypeptide construct.

[0126] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can include one or more modifications, including, but not limited to, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (e.g., conjugation, inverted linkage, etc.), (b) base modifications, such as replacement with a modified base, a stabilized base, a destabilized base, or a base that base pairs with an expanded repertoire of partners, or a conjugated base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) internucleoside linkage modifications, including modification or replacement of a phosphodiester bond. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0127] Secretory signal: As used herein, the term "secretory signal" refers to an amino acid sequence motif that targets an associated polypeptide for entry into the secretory pathway.

[0128] Subject: As used herein, the term "subject" refers to an organism to which a composition described herein is administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mice, rats, rabbits, non-human primates, household pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition (e.g., malaria and / or a malaria-related condition). In some embodiments, the subject is susceptible to a disease, disorder, or condition (e.g., malaria and / or a malaria-related condition). In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and / or a malaria-related condition). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder, or condition (e.g., malaria and / or a malaria-related condition). In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and / or a malaria-related condition). In some embodiments, a subject is one who has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual who is being and / or has been administered a diagnosis and / or therapy.

[0129] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition (e.g., malaria and / or a malaria-related condition) has and / or displays one or more symptoms of the disease, disorder, and / or condition.

[0130] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) is an individual who has a higher risk of developing the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) than members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) may not have been diagnosed with the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) may exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) develops the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions) does not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions).

[0131] Therapy: The term "therapy" refers to the administration or delivery of an agent or intervention that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect (e.g., that has been demonstrated to be statistically likely to have such an effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, a therapeutic agent or therapy is a medical intervention that can be performed to alleviate, alleviate, inhibit, present, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0132] Transmembrane domain: As used herein, the term "transmembrane domain" refers to the region of a polypeptide that spans a biological membrane, such as the plasma membrane of a cell.

[0133] Treat: As used herein, "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). Treatment may be administered to a subject who does not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions), e.g., to reduce the risk of developing conditions associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later stage of the disease, disorder, and / or condition (e.g., malaria and / or malaria-related conditions).

[0134] Variant: As used herein, the term "variant" refers to a molecule that exhibits significant structural identity (e.g., primary or secondary) with a reference molecule, but that differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., to which the polypeptide or nucleic acid backbone is attached).

[0135] Detailed Description of Specific Embodiments I. Malaria Malaria is a mosquito-borne infectious disease caused by the unicellular eukaryotic Plasmodium parasite, which is transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et al. Malaria. Nat Rev Dis Primers 3, 17050, 2017, incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must be infected through a previous blood meal collected from an infected subject (e.g., a human). When a mosquito bites an infected subject, it collects a small amount of blood containing the Plasmodium parasite. The infected mosquito can then subsequently bite an uninfected subject and infect the subject.

[0136] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000 to 600,000 deaths annually. Although significant efforts have been made to develop therapeutic treatments for malaria, many Plasmodium parasites have developed resistance to available treatments. According to the Malaria Eradication Research Agenda Initiative, malaria eradication can only be achieved through effective vaccination.

[0137] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as "RTS,S," a milestone in malaria vaccine development. In 2019, the World Health Organization launched a pilot program to provide RTS,S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS,S / AS01 is an adjuvanted protein subunit vaccine consisting of the major repeat region and C-terminal portion of the CSP from Plasmodium falciparum fused to hepatitis B surface antigen (HBsAg). The vaccine is a mix of this PfCSP-HBsAg compound and HBsAg, which forms virus-like particles (RTS,S / AS01, Mosquirix®). RTS,S is administered according to a regimen requiring four doses: an initial three-dose schedule given at least one month apart, and a fourth dose 15-18 months after dose three (see, e.g., Vandoolaeghe & Schuerman Expert Rev Vaccines. 15:1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, which is incorporated herein by reference in its entirety). RTS,S has reportedly been shown to protect approximately 30%-50% of children from clinical disease over an 18-month period. RTS,S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T-cell responses (see, e.g., Morris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). A phase III trial of RTS,S delivered as a three-dose series with boosters to children aged 5-17 months showed moderate vaccine efficacy in children aged 5-17 months after 1 year, preventing 36% of clinical malaria cases over the entire study period, with a median follow-up of 4 years and transmission settings ranging from a high of 20% to a low of 66%.Furthermore, published literature suggests that protection wanes over time, including reports of potential negative efficacy after 5 years in children with high malaria exposure (see Olotu et al. 2016, N. Engl. J. Med. 374:2519-29, which is incorporated herein by reference in its entirety). Thus, an effective malaria vaccine remains a critical unmet medical need for global health.

[0138] A. Life Cycle During the blood meal, infected mosquitoes inject sporozoites, known as the hepatic stage of Plasmodium spp., along with their anticoagulant saliva. The sporozoites migrate through the skin into lymphatic vessels and then to hepatocytes in the liver. This migration occurs very rapidly and can be completed in just a few minutes (see Sinnis et al., Parasitol Int. 2007 Sep;56(3):171-8, incorporated herein by reference in its entirety). This is known to be the bottleneck time in malaria infection, making it most favorable for therapeutic intervention, as only a small number of sporozoites (thought to be up to several hundred) are injected by the mosquito, and only a small fraction of that number establish an infection in the liver and develop into mature hepatic stage parasites (Flores-Garcia et al., mBio. 2018 Nov20;9(6):e02194-18, incorporated herein by reference in its entirety). Thus, subjects whose immune systems are primed to clear sporozoites before they enter hepatocytes are able to efficiently clear the infection.

[0139] One particular challenge associated with clearing malaria infection during this bottleneck is that the most abundant and immunogenic protein on the sporozoite surface, the circumsporozoite protein (CSP), is exposed to the immune system in small amounts and for only a short time due to the variably low inoculum from the mosquito and the kinetics of hepatocyte infection after inoculation. After liver infection is established, parasites no longer express CSP and instead differentiate into stages with a different mosaic of surface antigens. Furthermore, due to the bivalency of antibodies and the density and proximity of adjacent CSPs on the surface of the bound parasite, antibody binding to CSPs can result in a phenomenon called CSP precipitation, whereby the antibody can crosslink adjacent CSPs, precipitating them and causing them to fall off the parasite surface, leaving a trail of precipitated, antibody-bound CSPs that the parasite can replace during its normal CSP translocation process (described in Livingstone et al., Sci Rep 11, 5318 (2021); Steward et al., J Protozool. 1991 Jul-Aug;38(4):411-21, each of which is incorporated herein by reference in its entirety).

[0140] As they migrate from the skin inoculation site to the liver, sporozoites traverse host cells (Mota et al., Science 2001 Jan 5;291(5501):141-4). To gain access to hepatocytes, sporozoites traverse different types of host cells in the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe. 2008 Feb 14;3(2):88-96, which is incorporated herein by reference in its entirety), and the liver sinusoidal barrier, which contains liver endothelial cells and Kupffer cells (Frevert et al., PLoS Biol 3(6):e192.2005, which is incorporated herein by reference in its entirety), and sinusoidal endothelial cells (Tavares et al., J Exp Med 2013 May 6;210(5):905-15, which is incorporated herein by reference in its entirety). Sporozoites preferentially traverse cells with low-sulfated heparan sulfate proteoglycans (HSPGs), but preferentially invade cells with high-sulfated HSPGs (Coppi et al., Cell Host & Microbe 2, 316-327, November 2007, which is incorporated herein by reference in its entirety).

[0141] Cell traversal was first observed as the non-phagocytic entry of P. berghei sporozoites into macrophages, followed by "escape" from these cells (Vanderberg et al., J. Euk. Microbiol. 37:528-536, 1990, which is incorporated herein by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being studied. However, electron microscopy suggests that destruction of the host cell occurs during entry and exit from the host cell (Mota et al., 2001; Tavares et al., 2013, each of which is incorporated herein by reference in its entirety). It has also been shown that P. yoelii sporozoites can enter hepatocytes via transient vacuoles, and that disruption of the host membrane occurs upon exit from the cell rather than upon entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov11;18(5):593-603, which is incorporated herein by reference in its entirety).

[0142] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is incorporated herein by reference in its entirety). Several possibilities have emerged as to why this occurs. The first hypothesis suggested that translocation through hepatocytes primes the parasite for invasion by activating apical exocytosis (Mota et al., Nat Med 2002 Nov;8(11):1318-22, which is incorporated herein by reference in its entirety. The second theory suggested that traversal releases hepatocyte growth factor (HGF), making adjacent hepatocytes more susceptible to infection (Carrolo et al., Nat Med. 2003 Nov;9(11):1363-9, which is incorporated herein by reference in its entirety). Finally, other studies suggest that it takes time for sporozoites to turn off the machinery for traversal and activate the invasion machinery (Amino et al., 2008; Coppi et al., 2007, each of which is incorporated herein by reference in its entirety), and that traversal functions primarily to penetrate the cellular barrier and evade phagocytosis on the way to the liver (Amino et al., 2008). al., 2008, Coppi et al., 2007, Tavares et al., 2013, each of which is incorporated herein by reference in its entirety).

[0143] Although sporozoites have been shown to traverse human cells (Behet et al., Malar J 2014 Apr 5;13:136; Cha et al., J Exp Med 2015 Aug 24;212(9):1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6), e0129623; van Schaijk et al., PLoS ONE,3(10)e3549 2008, each of which is incorporated by reference in its entirety), the molecular basis for the traversal process has remained largely unexplored. Antibodies against circumsporozoite proteins (CSPs) impair traversal (Dumoulin et al., 2015, incorporated herein by reference in their entirety), but this is likely due to inhibition of motility rather than a direct effect (Cha et al., J Exp Med 2016 Sep 19;213(10):2099-112, incorporated herein by reference in their entirety). Furthermore, antibodies induced by chloroquine prophylaxis with sporozoites interfere with cell traversal, and these may also target CSPs (Behet et al., 2014, incorporated herein by reference in their entirety). Recently, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the parasite surface has been shown to interact with CD68 on Kupffer cells during traversal (Cha et al., 2015, Cha et al., 2016, each of which is incorporated herein by reference in its entirety).

[0144] In rodent Plasmodium parasites such as P. berghei, two sporozoite microneme proteins that appear to be essential for cell traversal have been identified (SPECT1; Ishino et al., PLoS Biol., 2 (2004), pp. 77-84, which is incorporated herein by reference in its entirety) and SPECT2 (Ishino et al., Cell. Microbiol., 7 (2005), pp. 199-208, which is incorporated herein by reference in its entirety; perforin-like protein 1, also known as protein 1 [PLP1] [Kaiser et al., Mol. Biochem. Parasitol., 133 (2004), pp. 15-26, which is incorporated herein by reference in its entirety). Genetic disruption of SPECT1 or SPECT2 rendered sporozoites unable to traverse mouse cells, yet they still invaded hepatocytes in vitro (Ishino et al., 2004). (Ishino et al., 2005, each of which is incorporated herein by reference in its entirety). When injected into rodents, sporozoites lacking SPECT1 or SPECT2 were impaired for liver infection, although a small number of sporozoites still established liver infection and resulted in subsequent patency. However, deletion of Kupffer cells enabled the mutants to establish liver infection at levels comparable to wild-type parasites (Ishino et al., 2004, Ishino et al., 2005, each of which is incorporated herein by reference in its entirety). This data suggests that traversal by sporozoites infecting rodents is important for navigating through the sinusoidal layer but not for hepatocyte invasion, malarial exoerythrocytic development, or growth within erythrocytes (Ishino et al., 2004, Ishino et al., 2005, each of which is incorporated herein by reference in its entirety).

[0145] The ortholog of SPECT2 in P. yoelii, PLP1, has been shown to play a role in cell traversal. This protein is not required for hepatocyte entry, but plays a role in release from transient vacuoles during traversal (Risco-Castillo et al., 2015, incorporated herein by reference in its entirety). Thus, sporozoites that infect rodents can traverse host cells by generating vacuoles during the entry step and can use perforin-like proteins (e.g., SPECT2 / PLP1) to escape this compartment and / or the host cell during cell egress.

[0146] Once sporozoites invade hepatocytes, they differentiate into merozoites, a replicative form of the parasite that can lyse hepatocytes after multiple rounds of replication. Within a few days, hundreds of sporozoites can become hundreds of thousands of merozoites. When infected hepatocytes rupture, they release merozoites into the bloodstream, where they invade red blood cells and initiate the asexual reproductive stage, the symptomatic stage of the disease. Within a few days, millions of merozoites can be present in the blood.

[0147] Malaria symptoms typically develop 4–8 days after initial red blood cell invasion. The merozoite replication cycle within the red blood cell continues for 36–72 hours until hemolysis, releasing merozoites for another round of red blood cell infection. Thus, in synchronous infections (infection resulting from a single infectious bite), fever occurs every 36–72 hours as infected red blood cells lyse and release large amounts of endotoxin.

[0148] Plasmodium spp. parasites gain entry into red blood cells through specific ligand-receptor interactions mediated by proteins on the parasite surface that interact with receptors on host erythrocytes (mature red blood cells) or reticulocytes (immature red blood cells). P. falciparum can invade and replicate in both erythrocytes and reticulocytes, while P. vivax and other species primarily invade reticulocytes, which are less abundant than erythrocytes. While most erythrocyte- or reticulocyte-binding proteins associated with invasion are redundant or expressed as families of variant forms, two essential erythrocyte receptors have been identified for P. falciparum: basigin and complement decay-accelerating factor (CD55).

[0149] Plasmodium vivax and Plasmodium ovale can also enter a dormant state, or hypnozoite, in the liver.

[0150] Merozoites released from red blood cells invade other red blood cells and continue replicating, or in some cases, differentiate into male or female gametocytes. Gametocytes are concentrated in skin capillaries and then taken up by mosquito vectors in another blood meal. Within the mosquito gut, each male gametocyte produces eight male gametes after three mitotic divisions, and female gametocytes mature into female gametes. The male gametes are flagellated, motile forms that seek female gametes. The male and female gametocytes fuse to form a diploid zygote, which elongates to become an ookinete. This motile form secretes chitinase to enter the peritrophic membrane, crosses the midgut epithelium to the basolateral side of the midgut, and establishes itself as an oocyst at the basement membrane. Oocysts mature over 14–15 days, undergo a replication cycle, and eventually form sporozoites that are released into the hemocoel, a sugar- and substrate-rich environment favorable for parasite survival. Thousands of sporozoites form from a single oocyst and become randomly distributed throughout the hemocoel. These sporozoites are motile, rapidly rupture the hemolymph, and only approximately 20% successfully invade the salivary glands. Following salivary gland invasion, sporozoites are reprogrammed through an unknown mechanism to prepare for liver invasion. Evidence for this reprogramming is also demonstrated by the inability of midgut sporozoites (directly from the oocyst) to invade hepatocytes and the fact that sporozoites that successfully invade salivary glands are unable to reinvade other salivary glands if presented with one. Salivary gland sporozoites alter mosquito behavior and salivary gland function due to reduced saliva production, resulting in increased mosquito probing behavior and increased transmission to a human host via mosquito bite.

[0151] Some drugs that prevent the invasion or proliferation of Plasmodium spp. in the liver have prophylactic activity, drugs that block the erythrocytic stage are necessary to treat the symptomatic phase of the disease, and compounds that inhibit gametocyte formation or its development in mosquitoes (including drugs that kill blood-feeding mosquitoes) are transmission-blocking agents (Phillips, et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety).

[0152] B. Genome Since the first sequence of the P. falciparum 3D7 genome was completed in 2002, genomic research on Plasmodium parasites has progressed rapidly. Except for a brief diploid phase following fertilization in the mosquito midgut, Plasmodium parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases and contain 14 chromosomes, a roughly 35-kb circular plasmid genome, and multiple copies of 6-kb mitochondrial DNA. Comparison of genomes from different species has shown that homologous genes are often found in composite blocks arranged in different orders among different chromosomes.

[0153] The adenine-thymine (AT) content of Plasmodium spp. can also vary greatly, ranging from approximately 80% AT in P. falciparum, P. reichenowi, and P. gallinaceum to approximately 75% AT in rodent Plasmodium parasites and approximately 60% AT in P. vivax, P. knowlesi, and P. cynomolgi. AT content is often higher in introns and intergenic noncoding regions than in protein-coding exons, averaging 80.6% AT across the P. falciparum genome compared with 86.5% in noncoding sequences. The high AT content of P. falciparum reflects numerous low-complexity regions, simple sequence repeats, and microsatellites, as well as highly skewed codon usage bias. Polymorphisms in AT-rich repeats provide rich markers for linkage mapping of drug resistance genes and tracking the evolution and structure of parasite populations.

[0154] The Plasmodium parasite genome contains a multigene family that plays important roles in parasite interactions with the host, including antigenic variation, signal transduction, protein transport, and adhesion. Among these gene families, the gene encoding P. falciparum erythrocyte membrane protein 1 (PfEMP1) has been the most extensively studied. Individual P. falciparum parasites carry a unique set of 50–150 copies of var genes within their genome, where gene expression switches can generate antigenic variation. PfEMP1 mediates cytoadhesion of infected red blood cells (iRBCs) in deep tissues, playing a key role in the pathogenesis of clinical manifestations such as cerebral malaria and placental malaria. Different PfEMP1 molecules bind to various host molecules, including α2-macroglobulin, CD36, chondroitin sulfate A (CSA), complement 1q, CR1, E-selectin and P-selectin, endothelial protein C receptor (EPCR), heparan sulfate, ICAM1, IgM, IgG, PECAM1, thrombospondin (TSP), and VCAM1. Such binding leads to the activation of various host inflammatory responses. Hemoglobin abnormalities, including hemoglobin C and hemoglobin S phenotypes, prevent the display of PfEMP1 on the knob structure of iRBCs. This insufficient display of PfEMP1 on the host cell surface provides protection against malaria by reducing cell adhesion and activation of inflammatory processes that promote the development of severe disease.

[0155] Members of the large Plasmodium interspersed repeat (pir) multigene family have different names depending on the parasite species, e.g., yir in P. yoelii, bir in P. berghei, and vir in P. vivax. Several P. falciparum gene families (stevor, rif, and PfMC-2™) are classified under pir due to their similar gene structure, characteristically containing a short first exon, a long second exon, and a third exon encoding a transmembrane domain. Recent studies have shown that the pir gene from P. chabaudi (cir) is expressed in different cellular locations within and on the surface of iRBCs, as well as in merozoites. Plasmodium parasites dedicate a large portion of their genome to gene families that ensure evasion of host immune defenses and the protection of molecular processes essential for infection. Despite the inherent difficulties in investigating these families, the importance of studying their role in parasite-host interactions and virulence is emphasized.

[0156] Additional exemplary polymorphic gene families include 14 gene families encoding proteins with six cysteines (6-Cys). These proteins are often localized on the parasite surface where they interact with host proteins and are expressed in different parasite developmental stages. 6-Cys proteins also demonstrate diverse functions, such as playing roles in parasite fertilization, mating interactions, evasion of immune responses, and invasion of hepatocytes. Proteins expressed in asexual stages are generally polymorphic and / or under selection, suggesting they may be targets of the host immune response; however, their function in parasite development remains largely unknown.

[0157] The Plasmodium genome can be highly polymorphic. Early studies demonstrated polymorphism involving tens to hundreds of kilobases, indicating that the chromosomal structure of P. falciparum is largely conserved in the central region, but that there is extensive polymorphism in both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repeat sequences and gene families.

[0158] The frequency of simple sequence repeats (microsatellites) in P. falciparum is estimated to be approximately one polymorphic microsatellite per kb of DNA. While not wishing to be bound by any one theory, this high rate may reflect the AT-rich nature of the genome. Microsatellites appear to be less frequent in other Plasmodium species, which have genomes with lower AT content. In addition to the highly polymorphic and repetitive structure of the polymorphic genome, there are also numerous single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) (Su et al., Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev. 2019 Jul 31;32(4), which is incorporated herein by reference in its entirety).

[0159] C. Plasmodium proteins Plasmodium parasites are known to express a variety of proteins at different stages of their life cycle. Exemplary malarial proteins are listed below, and SEQ ID NOs corresponding to exemplary amino acid sequences are listed in Table 2 below.

[0160] Circumsporozoite protein (CSP) is a multifunctional protein involved in the Plasmodium life cycle, as it is required for sporozoite formation in the mosquito midgut, sporozoite release from oocysts, invasion of the salivary glands, sporozoite attachment to hepatocytes in the liver, and sporozoite invasion of hepatocytes (see, e.g., Zhao et al. (2016) PLoS ONE 11(8):e0161607, which is incorporated herein by reference in its entirety). CSPs are present in all Plasmodium species, and although there is variation in amino acid sequence across species, the overall domain structure of the central repeat region and non-repetitive flanking regions is well conserved (see, e.g., Zhao et al. (2016) PLoS ONE 11(8):e0161607; Wahl et al. (2022) J. Exp. Med. 219:e20201313, each of which is incorporated by reference in its entirety). CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, A0A2L,1CF88, C6FGZ3, C6FH2, 7C6FHG7, M1V060, M1V0A3, M1V0B0, M1V0C4, M1V0E0, M1V9I4, M1VFN9, M1VKZ2, P02893, Q5EIJ9, Q5EIK2, Q5EIK8, Q5EIL3, Q5EIL5, Q5EIL8, Q5R2L2, Q7K740, Q8I9G5, Q8I9J3, Q8I9J4), and Table 1 includes exemplary sequences of CSP P. falciparum isolates from Asia, South America, and Africa. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0161] An exemplary CSP amino acid sequence is provided in SEQ ID NO:1.

[0162] RH5 is found in Plasmodium falciparum (P. falciparum) but not in other species of Plasmodium that infect humans. RH5 orthologs are also found in other species belonging to the subgenus Lavarenia, including parasites that infect chimpanzees and gorillas, demonstrating a unique role in P. falciparum invasion of human erythrocytes. See, e.g., Ragotte, et al. Trends Parasitol. 36(6) 2020, incorporated herein by reference in its entirety. RH5 is expressed during the mature schizont stage and can complex with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (Ripr) to form an elongated protein trimer on the merozoite surface that binds to the erythrocyte surface protein basigin. See, e.g., Ragotte (2020), incorporated herein by reference in its entirety.

[0163] In humans, RH5 binding to basigin plays a key role in invasion, acting downstream of membrane deformation. RH5 binding to basigin is required to induce calcium spikes within red blood cells, which is blocked when merozoites attempt invasion in the presence of anti-RH5, anti-Ripr, or anti-basigin antibodies or soluble basigin. See, e.g., Ragotte (2020), which is incorporated herein by reference in its entirety.

[0164] RH5 is a 63 kDa protein expressed at the mature schizont stage. It is cleaved into a 45 kDa form that is processed and shed by the parasite. The structure of PfRH5 reveals a kite-like architecture formed by two three-helical bundles brought together. See, e.g., Ragotte (2020), which is incorporated herein by reference in its entirety.

[0165] The RH5 sequence is known (e.g., UniProt accession numbers A0A159SK44, A0A159SK99, A0A159SKS8, A0A159SKW8, A0A159SL23, A0A159SL78, A0A159SL96, A0A159SLM7, A0A159SMC8, A0A159SMR9, A0A161FQT0, A0A1B1UZE2, A0A1B1UZE4, A0A1B1UZE5). , A0A346RCI1, A0A346RCJ0, A0A346RCJ2, A0A346RCJ3, A0A346RCJ4, A0A346RCK4, A0A346RCK5, A0A346RCK6, A0A346RCK9, B2L3N7, Q8IFM5, each of which is incorporated by reference in its entirety), an exemplary RH5 amino acid sequence is provided in SEQ ID NO: 365.

[0166] P113 is a glycosylphosphatidylinositol (GPI)-linked protein that directly interacts with the N-terminus of unprocessed RH5, providing a mechanism by which the RH5 invasion complex is tethered to the merozoite surface. See, for example, Ragotte (2020). P113 orthologs have been found in all Plasmodium species sequenced to date, suggesting common and conserved function(s) (Bullen et al. (2022) Molecular Microbiology 117:1245-1262, incorporated herein by reference in its entirety). Nevertheless, in a rodent model of malaria, P. berghei, p113 knockout parasites were viable, indicating that the protein was not essential for asexual blood-stage growth and invasion. However, knockout parasites exhibit defects in natural sporozoite transmission, leading to delayed patency in infected mice (Offeddu et al. (2014) Mol. Biochem. Parasitology 193:101-109, which is incorporated herein by reference in its entirety).

[0167] The Plasmodium P113 sequence is known (see, e.g., Uniprot accession number Q8ILP3). An exemplary P113 amino acid sequence is provided in SEQ ID NO:326.

[0168] Cysteine-rich protective antigen (CyRPA) is a 43 kDa protein with a predicted N-terminal secretion signal. CyRPA is part of a multiprotein complex that includes RH5 and Ripr, and secretes Ca. 2+ PfCyRPA is highly conserved with only a single SNP at greater than 5% prevalence, is essential for invasion (as conditional knockdown causes loss of invasion activity), and exhibits poor seroreactivity from natural exposure (see, e.g., Ragotte (2020), which is incorporated herein by reference in its entirety).

[0169] Plasmodium CyRPA sequences are known (see, e.g., Uniprot Accession Nos. A0A2S1Q7P0, A0A2S1Q7P5, A0A2S1Q7Q4, Q8IFM8, each of which is incorporated by reference in its entirety). An exemplary CyRPA amino acid sequence is provided in SEQ ID NO:329.

[0170] RH5-interacting protein (Ripr) is an approximately 120 kDa protein that localizes to micronemes during the schizont stage of the P. falciparum life cycle. The full-length 120 kDa protein is processed into two similarly sized fragments: an N-terminal fragment (containing EGF domains 1 and 2) and a C-terminal fragment (containing EGF domains 3-10). Ripr colocalizes with RH5 and CyRPA during parasite invasion at the junction between merozoites and erythrocytes. Parasites with a conditional knockout of PfRipr induce membrane deformation but are unable to complete invasion (see, e.g., Ragotte (2020), incorporated herein by reference in its entirety).

[0171] Plasmodium Ripr sequences are known (see, e.g., UniProt accession numbers A0A193PDI9, A0A193PDK3, A0A193PDK8, A0A193PDL3, A0A193PDL9, A0A193PDP4, A0A193PDQ8, A0A193PE01, A0A193PE05, A0A193PE07, O97302, A0A193PE17). An exemplary Ripr amino acid sequence is provided in SEQ ID NO:332.

[0172] E140 is found in all Plasmodium species for which genome sequences are available and is well conserved, with amino acid identity ranging from 34 to 92% between species. See, e.g., Smith, et al. PLoS One 15.5 (2020): e0232234; http: / / doi:10.1371 / journal.pone.023223; and US Patent Publication No. US2019 / 0117752, each of which is incorporated herein by reference in its entirety. E140 is also highly conserved (95 to 99%) and exhibits low mutation frequency in P. falciparum strains isolated from different locations around the world. E140 is expressed during different stages of the Plasmodium parasite life cycle (specifically, E140 has been detected in sporozoite, hepatic, and blood-stage parasites).

[0173] Protein structure algorithms predict that the E140 protein has five transmembrane domains that may span membranes of either parasite or host origin. E140 shows distinct patterns of protein expression at the mature sporozoite, late hepatic, and late schizont stages. It transports to the anterior and posterior ends of sporozoites, the parasitophorous space at the late hepatic stage, and around developing merozoites at the late schizont stage. It is also known to be expressed in mature salivary gland sporozoites and oocyst-derived sporozoites and oocysts.

[0174] The E140 sequence is known (see, e.g., UniProt Accession Nos. A0A650D649, A0A650D653, A0A650D672, A0A650D687, A0A650D690, A0A650D694, A0A650D6A3, A0A650D6B8, A0A650D6L3, A0A650D6L7, Q8I299, each of which is incorporated by reference in its entirety), and an exemplary E140 amino acid sequence is provided in SEQ ID NO:335.

[0175] CelTOS is required for sporozoite traversal through Kupfer cells during the liver invasion process. CelTOS forms pores from within the cell, allowing sporozoites to be released into the liver. Antibody epitopes have been characterized from immunized mice and infected human populations (Pf and Pv). Mouse studies have shown that immunization with CelTOS provides protection and protection against challenge. Vaccination with CelTOS can generate antibodies that bind to the extracellular domain of the pore-forming complex, block complete pore formation, and prevent sporozoite traversal to the liver. See, e.g., Jimah et al., Elife 2016 Dec 1;5:e20621.doi:10.7554 / eLife.20621, incorporated herein by reference in its entirety.

[0176] Plasmodium CelTOS sequences are known (see, e.g., Uniprot Accession Nos. M1ETJ8, Q53UB7, A0A2R4QLA5, A0A2R4QLI0, A0A2R4QLI5, A0A2R4QLJ1, A0A2R4QLJ4, M1ETJ8, Q53UB8, Q8I5P1, each of which is incorporated by reference in its entirety). An exemplary CelTOS amino acid sequence is provided in SEQ ID NO:350.

[0177] SPECT1 and SPECT2 (the latter also known as perforin-like protein 1 (PLP1)) are essential Plasmodium proteins that may play a role in cell traversal. See Yang et al., Cell Rep. 2017 Mar 28;18(13):3105-3116. doi:10.1016 / j.celrep.2017.03.017, which is incorporated herein by reference in its entirety. Targeted disruption of P. falciparum SPECT1 or SPECT2 has been shown to reduce sporozoite infectivity in the development of the hepatic stage in humanized mice. However, the mechanism of cell traversal of these two proteins has not yet been defined in P. falciparum. See Yang et al.

[0178] SPECT1 and SPECT2 are considered attractive pre-erythrocytic immune targets due to the important role they are thought to play in the crossing of Plasmodium parasites across the dermis and liver sinusoidal wall prior to hepatocyte invasion. Recombinant P. falciparum SPECT2, as well as the MACPF / CDC domain of PfSPECT2, binds Ca. 2+ PfSPECT2 has been shown to cause lysis of red blood cells in a Ca2+-dependent manner. PfSPECT2 is also involved in the Ca2+-dependent release of P. falciparum merozoites from red blood cells.

[0179] Plasmodium SPECT1 and SPECT2 sequences are known (see, e.g., UniProt accession numbers Q8IDR4 and Q9U0J9, each of which is incorporated by reference in its entirety), and exemplary amino acid sequences are provided in SEQ ID NOs: 353 and 356, respectively.

[0180] Export protein 1 (EXP1) is a single-pass transmembrane protein with an N-terminal signal peptide that is expressed during the intraerythrocytic and hepatic stages (see, e.g., Spielmann et al., Int J Med Microbiol. 2012 Oct;302(4-5):179-86, which is incorporated herein by reference in its entirety). EXP1 has been shown to initially localize to dense granules in merozoites and then transport to the parasitophorous vacuole membrane (PVM) after invasion (see, e.g., Iriko et al., Parasitol Int. 2018 Oct;67(5):637-639, which is incorporated herein by reference in its entirety). Once localized to the PVM, EXP1 forms a homo-oligomer with its N-terminus exposed to the parasitophorous vacuole lumen and its C-terminus exposed to the erythrocyte cytoplasm (see, e.g., Mesen-Ramirez et al., PLoS Biol. 2019 Sep 30;17(9):e3000473, which is incorporated herein by reference in its entirety).

[0181] EXP1 has been demonstrated to have glutathione S-transferase (GST) activity that may protect Plasmodium from oxidative damage (see, e.g., Mesen-Ramirez et al., PLoS Biol 17(9) 2019 Sep 30;17(9):e3000473, which is incorporated herein by reference in its entirety). Recently, EXP1 has been demonstrated to be important for Plasmodium survival by maintaining the correct localization of EXP2, a nutrient-permeable channel within the PVM (see, e.g., Mesen-Ramirez et al., PLoS Biol. 2019 Sep 30;17(9):e3000473, which is incorporated herein by reference in its entirety).

[0182] The P. falciparum EXP1 polypeptide sequence is known (see, e.g., UniProt Accession Nos. Q8IIF0, W7JTD3, Q25840, Q548U2, Q5VKK2, Q5VKK5, Q5WRH8, Q6V9G4, Q6V9G6, Q6V9G9, Q6V9H1, Q6V9H2, Q9U590, P04923, P04926, each of which is incorporated by reference in its entirety). An exemplary EXP1 amino acid sequence is provided in SEQ ID NO:314.

[0183] The upregulated infectious sporozoite gene 3 (UIS3) protein is a membrane-bound protein that localizes to the sporozoite parasitophorous membrane (PVM) in infected hepatocytes. UIS3 interacts with liver fatty acid-binding protein (L-FABP) and has been shown to be involved in fatty acid and / or lipid import during Plasmodium growth (see, e.g., Sharma et al. J Biol Chem. 2008 Aug 29;283(35):24077-24088; Mikolajczak et al., Int J Parasitol. 2007 Apr;37(5):483-9, each of which is incorporated herein by reference in its entirety).

[0184] Sporozoite invasion of host hepatocytes is followed by synthesis of key Plasmodium structural features (e.g., the parasitophorous vacuole membrane). During the hepatocyte stage, Plasmodium relies on host fatty acids for rapid synthesis of its membrane (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29;283(35):24077-24088, incorporated herein by reference in its entirety). UIS3 insertion into the PVM provides Plasmodium with a way to import essential fatty acids and / or lipids during the rapid sporozoite growth phase (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29;283(35):24077-24088, incorporated herein by reference in its entirety).

[0185] Immunization with UIS3-deficient Plasmodium berghei sporozoites protects against malaria in rodent malaria models (see Mueller et al., Nature. 2005 Jan 13;433(7022):164-7, which is incorporated herein by reference in its entirety). Although UIS3-deficient Plasmodium berghei are able to initiate the transformation process in the liver, they exhibit severe defects during transformation to trophozoites (see, e.g., Mueller et al., Nature. 2005 Jan 13;433(7022):164-7, which is incorporated herein by reference in its entirety). UIS3-deficient Plasmodium berghei also fails to develop into mature liver schizonts, thus aborting malaria infection within the liver itself (see, e.g., Mueller et al., Nature. 2005 Jan 13;433(7022):164-7, which is incorporated herein by reference in its entirety). Furthermore, it has previously been demonstrated that UIS3 from Plasmodium berghei and UIS3 from Plasmodium falciparum exhibit low (i.e., 34%) amino acid sequence identity (see, e.g., Mueller et al., Nature. 2005 Jan 13;433(7022):164-7, which is incorporated herein by reference in its entirety).

[0186] Plasmodium UIS3 sequences are known (see, e.g., UniProt Accession Nos. A0A509ARS3, A0A1C6YLP3, Q8IEU1, A0A384KLI1, A0A1G4H423, A0A077YB01, Q9NFU4, each of which is incorporated by reference in its entirety). An exemplary UIS3 amino acid sequence is provided in SEQ ID NO:359.

[0187] Upregulated infectious sporozoite gene 4 (UIS4) contains a single transmembrane domain and localizes to secretory organelles of sporozoites and the parasitophorous vacuole membrane (PVM) of liver stage sporozoites. UIS4 is not expressed in blood stage or early sporozoites produced in oocysts (see, e.g., Mackellar et al., Eukaryot Cell. 2010 May;9(5):784-794, which is incorporated herein by reference in its entirety).

[0188] Deletion of the UIS4 gene is associated with a block in the development of early liver disease stages (see, e.g., Vaughan and Kappe, Cold Spring Harb Perspect Med. 2017 Jun 1;7(6):a025486, incorporated herein by reference in its entirety. Recently, UIS4 has been demonstrated to be involved in Plasmodium berghei survival by evading host actin structures deployed as part of the host cytoplasmic defense (see, e.g., Bana et al., iScience. 2022 Apr 22;25(5):104281. doi:10.1016 / j.isci.2022.104281.eCollection 2022 May 20, incorporated herein by reference in its entirety). P. falciparum inhibits the growth of P. yoelii. It has an orthologue to UIS4 named ETRAMP10.3 that is unable to function as a functional complement to UIS4, indicating that it likely serves a different function in the P. falciparum life cycle (see Mackellar et al., Eukaryot. Cell 9:784-94 (2010) which is incorporated herein by reference in its entirety).

[0189] Plasmodium falciparum early transcribed membrane protein 10.3 (ETRAMP10.3) is an approximately 10 kDa protein conserved across Plasmodium species and a member of a multigene family of early transcribed membrane proteins, including proteins located in the parasitophorous vacuole. Some ETRAMP proteins are specific to P. falciparum and are not found in Plasmodium species that infect other organisms. ETRAMP10.3 is an example of an ETRAMP protein expressed in both liver and blood-stage P. falciparum parasites. ETRAMP10.3 transcription has been found to peak during the transition from the ring to the trophozoite stage of P. falciparum blood-stage infection in human hosts. ETRAMP10.3 is localized to the parasitophorous vacuole and exported to host erythrocytes during blood-stage infection. ETRAMP10.3 is sometimes referred to as upregulated in infectious sporozoite gene 4 (UIS4), but ETRAMP10.3 is understood to be an ortholog of UIS4 based on similarity and structural similarity. However, ETRAMP10.3 is not a functional ortholog of UIS4 and may play a different biological role. While the biological function of ETRAMP10.3 has not yet been fully resolved, its localization to vesicular structures within host erythrocytes suggests a role in host-parasite interactions or in the remodeling of infected erythrocytes. ETRAMP10.3 appears to play an important role in the Plasmodium life cycle. Deletion of ETRAMP10.3 can lead to aborted liver stage development and progression to the asexual blood stage in mice.

[0190] Although in the literature the terms "UIS4" and "ETRAMP10.3" are sometimes used to refer to different proteins, in the context of the present disclosure the terms "UIS4" and "ETRAMP10.3" are used interchangeably to refer to ETRAMP10.3.

[0191] The Plasmodium ETRAMP10.3 sequence is known (see, e.g., UniProt Accession No. Q8IJM9, which is incorporated herein by reference in its entirety). An exemplary ETRAMP10.3 amino acid sequence is provided in SEQ ID NO:362.

[0192] Liver-specific protein 1 (LISP-1) is expressed in hepatocytes during Plasmodium pathogenesis and localizes to the parasitophorous vacuole membrane (PVM) (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;11(9):1329-1339, incorporated herein by reference in its entirety). LISP-1 is expressed at high levels during late liver stage development and has been shown to be involved in PVM degradation and subsequent merozoite release (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;11(9):1329-1339, incorporated herein by reference in its entirety).

[0193] Intracellular Plasmodium lacking LISP-1 develop into liver merozoites and exhibit normal infectivity for erythrocytes (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;11(9):1329-1339, which is incorporated herein by reference in its entirety). However, LISP1-deficient liver-stage Plasmodium do not destroy the PVM and remain trapped within hepatocytes (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep;11(9):1329-1339, which is incorporated herein by reference in its entirety).

[0194] Plasmodium LISP-1 sequences are known (see, e.g., UniProt Accession Nos. A0A2I0C2X6, Q8ILR5, each of which is incorporated by reference in its entirety). An exemplary LISP-1 amino acid sequence is provided in SEQ ID NO:308.

[0195] Liver-specific protein 2 (LISP-2) contains a modified 6-cys domain and is expressed during Plasmodium development in hepatocytes (see, e.g., Orito et al., Mol Microbiol. 2013 Jan;87(1):66-79, which is incorporated herein by reference in its entirety). LISP-2 has been shown to be expressed by hepatic-stage Plasmodium, exported into hepatocytes, and distributed throughout the host cell, including the nucleus (see, e.g., Orito et al., Mol Microbiol. 2013 Jan;87(1):66-79, which is incorporated herein by reference in its entirety).

[0196] Intracellular Plasmodium lacking LISP2 do not mature efficiently during merozoite development (see, e.g., Orito et al., Mol Microbiol. 2013 Jan;87(1):66-79, which is incorporated herein by reference in its entirety).

[0197] Plasmodium LISP-2 sequences are known (see, e.g., UniProt accession numbers A0A2I0BZR4, Q8I1X6, Q9U0D4, each of which is incorporated by reference in its entirety). An exemplary LISP-2 amino acid sequence is provided in SEQ ID NO:311.

[0198] Thrombospondin-related adhesion proteins (TRAPs) contain an N-terminal domain, commonly referred to as the von Willebrand factor A domain, which binds Mg, required for sporozoite motility in vitro and infection in vivo. 2+It is most similar to the integrin I domain because it contains a metal ion-dependent adhesion site (MIDAS) with an ion (see, e.g., Lu et al., PLoS One. 2020; 15(1): e0216260, which is incorporated herein by reference in its entirety). The I domain is inserted into an extendable β-ribbon, followed by a thrombospondin repeat (TSR) domain, a C-terminal proline-rich segment, a single-pass transmembrane domain, and a cytoplasmic domain (see, e.g., Lu et al., PLoS One. 2020; 15(1): e0216260, which is incorporated herein by reference in its entirety). Sequence analysis of the proline-rich segment revealed the presence of an SH3 domain-binding PxxP motif in Plasmodium TRAP (see Akhouri et al., Malar J. 2008 Apr 22;7:63. doi:10.1186 / 1475-2875-7-63, which is incorporated herein by reference in its entirety).

[0199] TRAP is conserved in the micronemes and becomes surface-exposed at the apical tip of the sporozoite upon parasite contact with the host cell (see, e.g., Akhouri et al., Malar J. 2008 Apr 22;7:63. doi:10.1186 / 1475-2875-7-63, which is incorporated herein by reference in its entirety). TRAP also plays an important role in sporozoite invasion of hepatocytes by assisting sporozoites in gliding motility and recognition of host receptors on mosquito salivary glands and hepatocytes (see, Akhouri et al., Malar J. 2008 Apr 22;7:63. doi:10.1186 / 1475-2875-7-63, which is incorporated herein by reference in its entirety).

[0200] Plasmodium trap sequences are known (see, e.g., UniProt Accession Nos. A0A5Q2EXK8, A0A5Q2EZD7, A0A5Q2F1F6, A0A5Q2F2B8, A0A5Q2F2H6, A0A5Q2F4G9, O76110, P16893, Q01507, Q26020, Q76NM2, W8VNB6, each of which is incorporated by reference in its entirety), and an exemplary trap amino acid sequence is provided in SEQ ID NO:287.

[0201] Liver stage-associated protein 1 (LSAP-1) has been shown to be found primarily in the periphery of intracellular liver parasites throughout their development, but not in blood-stage parasites, and possibly in trace amounts in salivary gland sporozoites (see, e.g., Siau et al., PLoS Pathog. 2008 Aug 8;4(8):e1000121, incorporated herein by reference in its entirety). LSAP-1 is one of the most abundant transcripts in the salivary gland transcriptome, but has not been detected in proteomic studies of sporozoites. Rather, expression has been detected only in the liver stage (see, e.g., Siau et al., PLoS Pathog. 2008 Aug 8;4(8):e1000121, incorporated herein by reference in its entirety).

[0202] Plasmodium LSAP-1 sequences are known (see, e.g., UniProt Accession Nos. Q8I632, W7JR53, each of which is incorporated herein by reference in its entirety). An exemplary LSAP-1 amino acid sequence is provided in SEQ ID NO:302.

[0203] Like LSAP-1, LSAP-2 is also the most abundant transcript in the salivary gland transcriptome, but has not been detected in proteomic studies of sporozoites. LSAP-2 has shown some efficacy as a vaccine when combined with other antigens. See, e.g., Halbroth et al., Infect Immun. 2020 Jan 22;88(2):e00573-19.doi:10.1128 / IAI.00573-19.Print 2020 Jan 22, which is incorporated herein by reference in its entirety.

[0204] Plasmodium LSAP-2 sequences are known (see, e.g., UniProt Accession Nos. Q8I632, W7JR53, each of which is incorporated herein by reference in its entirety). An exemplary LSAP-2 amino acid sequence is provided in SEQ ID NO:305.

[0205] Liver-stage antigen 1 (LSA-1) is expressed after Plasmodium invades hepatocytes and accumulates in parasitophorous vacuoles (see, e.g., Tucker, K. et al., 2016, "Pre-Erythrocytic Vaccine Candidates in Malaria," in AJ Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, each of which is incorporated herein by reference in its entirety). The function of LSA-1 is currently unknown (see, e.g., Tucker, K. et al., 2016, "Pre-Erythrocytic Vaccine Candidates in Malaria," in AJ Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, each of which is incorporated herein by reference in its entirety).

[0206] LSA-1 is a 230 kDa pre-erythrocytic protein containing a large central region consisting of more than 80 repeat units of 17 amino acid residues flanked by highly conserved C- and N-terminal regions (Richie, TL and Parekh, FK (2009) Malaria, which is incorporated herein by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, ADT and Stanberry LR, eds), pp. 1309-1364, Elsevier, which is incorporated herein by reference in its entirety). LSA1 is expressed only by hepatic stage Plasmodium, not by sporozoites (Richie, TL and Parekh, FK (2009) Malaria, which is incorporated herein by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, ADT and Stanberry LR, eds), pp. 1309-1364, Elsevier, which is incorporated herein by reference in its entirety.) Repetitive regions result in significant protein variation between strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016, "Pre-Erythrocytic Vaccine Candidates in Malaria," in AJ Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety).

[0207] Plasmodium LSA-1 sequences are known (see, e.g., UniProt Accession Nos. Q25886, Q25887, Q25893, Q26028, Q9GTX5, O96125, each of which is incorporated herein by reference in its entirety). An exemplary LSA-1 amino acid sequence is provided in SEQ ID NO:290.

[0208] Liver stage antigen 3 (LSA-3) is a 200-kDa protein consisting of three non-repeat regions (NR-A, NR-B, and NR-C) flanked by two short repeat regions and one long repeat region (see, e.g., Tucker, K. et al., 2016, "Pre-Erythrocytic Vaccine Candidates in Malaria," in A.J. Rodriguez-Morales (ed.), incorporated herein by reference in its entirety; Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, incorporated herein by reference in its entirety). The non-repetitive regions are well conserved across geographically diverse strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in AJ Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety). The most significant variation is in the repetitive regions, resulting from the organization and number of repeat subunits, rather than the composition of the repetitive regions (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in AJ Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety).

[0209] Recently, in vitro data have shown that antibodies against LSA-3 (particularly the C-terminal portion of LSA-3) can provide some protection (see, e.g., Morita et al, Sci Rep. 2017 Apr 5;7:46086. doi:10.1038 / srep46086, which is incorporated herein by reference in its entirety).

[0210] Plasmodium LSA-3 sequences are known (e.g., UniProt accession numbers C7DU21, C7DU22, C7DU23, C7DU24, C7DU25, C7DU26, C7DU27, C7DU28, C7DU29, C7DU32, C7DU33, C7DU34, C7DU36, C7DU37, C7DU38, C7DU39, C7DU40, Q8I042, Q8I0A5, Q8I0D0, Q8IFR1, Q8IFR2, Q8IFR3, Q8IFR4, Q8IFR5, Q8 (See, IFR6, Q8IFR7, Q8IFR8, Q8IFR9, Q8IFS0, Q8IFS1, Q8IFS2, Q8IFS3, Q8IFS4, Q8IFS5, Q8IFS6, Q8IFS7, Q8IFS8, Q8IFS9, Q8IFT0, Q8IFT1, Q8IFT2, Q8IFT3, Q8IFT4, Q9U0N9, Q9U0P0, A0A2I0BVD6, A0PFM9, O96275, each of which is incorporated by reference in its entirety.) An exemplary LSA-3 amino acid sequence is provided in SEQ ID NO:299.

[0211] Glutamate-rich protein (GARP) is an 80 kDa protein whose name derives from its glutamate-rich amino acid sequence, which comprises 24% of all residues. GARP is primarily expressed in the ring stage and trophozoites and is a nonessential gene in cell culture, but has been shown to be highly immunogenic in animal models. Although GARP is nonessential in cell culture, its localization to the periphery of infected erythrocytes may indicate a role in the sequestration of infected erythrocytes. It has been proposed that GARP's involvement in sequestration occurs through binding to the chloride / bicarbonate anion exchanger. Antibodies to GARP have been proposed to function as a protective signature against severe malaria and have shown efficacy in experimental studies in monkeys (see, e.g., Hon et al., Trends in Paras 2020 Aug;36(8):653-655. doi:10.1016 / j.pt.2020.05.012, and Lau et al., Plos Path. 2014 10, e1004135, each of which is incorporated herein by reference in its entirety). GARP sequences are known (see, e.g., UniProt Accession Nos. Q9GTW3 and Q9U0N1, each of which is incorporated herein by reference in its entirety), and an exemplary GARP amino acid sequence is provided in SEQ ID NO: 341.

[0212] Parasite-infected erythrocyte-specific protein 2 (PIESP2) (see, e.g., UniProt Accession No. Q8I488) is a highly immunogenic protein first expressed in the trophozoite stage and thought to be important for the clinical progression of cerebral malaria. This protein is primarily found within erythrocytes, but has been shown to be present on their surface and can attach to endothelial cells of the brain vasculature. Antibodies against PIESP2 have been shown to prevent Plasmodium vascular attachment and may prove useful in preventing inflammatory responses in the brain and damage to the blood-brain barrier during cerebral malaria progression (see, e.g., Liu et al., Int J Biol Macromol. 2021 Apr 30;177:535-547. doi:10.1016 / j.ijbiomac.2021.02.145, which is incorporated herein by reference in its entirety). PIESP2 sequences are known (see, eg, UniProt Accession No. Q8I488, which is incorporated herein by reference in its entirety), and an exemplary PIESP2 amino acid sequence is provided in SEQ ID NO:344.

[0213] Schizont egress antigen-1 (SEA1) is a large 244 kDA protein lacking a transmembrane domain or known targeting signal. While the function of SEA1 is unknown, it has been shown to be effective in rodent vaccine studies and has been proposed as the target of protective antibodies found in children. SEA1 received its name after antibodies against this protein were reported to inhibit the egress of Plasmodium merozoites. SEA1 localizes closely to centromeres during nuclear division, impacting its role in essential processes of replication. To date, various studies have proposed a role for SEA1 not only in egress but also in mitotic division of replicating nuclei. (Perrin et al. 2021, incorporated herein by reference in its entirety) (see, e.g., Perrin et al, mBio. 2021 Mar 9;12(2):e03377-20. doi:10.1128 / mBio.03377-20, incorporated herein by reference in its entirety). SEA1 sequences are known (see, e.g., UniProt Accession No. A0A143ZXM2, incorporated herein by reference in its entirety), and an exemplary SEA1 amino acid sequence is provided in SEQ ID NO: 347.

[0214] D. Plasmodium Array Embodiments An exemplary wild-type CSP polypeptide amino acid sequence from Plasmoidum falciparum isolate 3D7 is presented in SEQ ID NO: 1 and includes the following: secretion signal (amino acids 1-18), N-terminal domain (amino acids 19-104), junction region (amino acids 93-104), central domain (amino acids 105-272), and C-terminal domain (amino acids 273-397). In exemplary SEQ ID NO: 1, the N-terminal domain includes the N-terminal region (amino acids 19-80), the N-terminal tail region (amino acids 81-92), and junction region (amino acids 93-104). In exemplary SEQ ID NO: 1, the junction region includes the R1 region (amino acids 93-97) and junction region (SEQ ID NO: 132) at positions 98-104. In exemplary SEQ ID NO: 1, the central domain includes the minor repeat region (amino acids 105-128) and the major repeat region (amino acids 129-272). In exemplary SEQ ID NO: 1, the minor repeat region contains three repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 102). In exemplary SEQ ID NO: 1, the major repeat region contains 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147), and the 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147) are separated into two consecutive stretches, one containing 17 repeats of the amino acid sequence NANP (SEQ ID NO: 147) and one containing 18 repeats of the amino acid sequence NANP (SEQ ID NO: 147) adjacent to the amino acid sequence of NVDP (SEQ ID NO: 144). The major repeat region contains the amino acid sequences NPNANP (SEQ ID NO: 150) and NANPNA (SEQ ID NO: 153). In exemplary SEQ ID NO: 1, the C-terminal domain contains a C-terminal region (amino acids 273-375), a serine-valine (amino acids 376-377), and a transmembrane domain (amino acids 378-397). In the exemplary SEQ ID NO: 1, the C-terminal region includes the Th2R region (amino acids 314-327) and the Th3R region (amino acids 352-363). [Table 2]

[0215] II. Plasmodium Polypeptide Constructs The present disclosure utilizes RNA technology as a modality to express one or more Plasmodium polypeptide constructs (also referred to herein as "malaria polypeptide constructs" or "malarial polypeptide constructs") comprising, among other things, one or more malarial proteins described herein, or one or more portions thereof. For example, in some embodiments, the Plasmodium polypeptide construct comprises one or more Plasmodium CSP polypeptide regions or portions thereof (e.g., immunogenic fragments of Plasmodium CSPs). The portion of a CSP polypeptide or region can be a distinctive portion of a CSP polypeptide or region. In some embodiments, the Plasmodium polypeptide construct further comprises one or more additional amino acid sequences, such as a secretion signal (e.g., a heterologous secretion signal), a transmembrane region (e.g., a heterologous transmembrane region), a helper antigen, a multimerization region, and / or a linker, as described herein.

[0216] A.CSP In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more regions or portions of a CSP, e.g., a Plasmodium CSP, e.g., a P. falciparum CSP (SEQ ID NO: 1), or a variant thereof (e.g., one or more immunogenic fragments of a CSP, e.g., a Plasmodium CSP, e.g., a P. falciparum CSP, or an immunogenic variant thereof). A region of a CSP (or CSP polypeptide region) may refer to the N-terminal region, the N-terminal end region, the junction region, the minor repeat region, the major repeat region, or the C-terminal region. A portion of a CSP (or CSP polypeptide portion) may refer to a portion of a CSP polypeptide region or a portion spanning two or more CSP polypeptide regions. In some embodiments, the CSP polypeptide portion comprises 25, 30, 35, 40, or 45 consecutive amino acids of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the Plasmodium polypeptide construct does not contain a secretory signal or transmembrane region, for example, corresponding to amino acids 19 to 375 of the amino acid sequence according to SEQ ID NO: 1, or corresponding to amino acids 19 to 376 or 19 to 377 of the amino acid sequence according to SEQ ID NO: 1, i.e., a serine or serine and valine immediately following the C-terminal region.

[0217] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a minor repeat region of CSP. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of a minor repeat region of CSP. In some embodiments, the portion of the minor repeat region of CSP is about 10, 15, 20, 21, 22, or 23 contiguous amino acids in length. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a major repeat region of CSP. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of a major repeat region of CSP. In some embodiments, the portion of the major repeat region of CSP is about 100, 110, 120, 130, 135, 140, 141, or 142 amino acids in length. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a CSP C-terminal region. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of a CSP C-terminal region. In some embodiments, the portion of the CSP C-terminal region is about 80, 90, 95, 100, 101, or 102 amino acids in length. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a CSP N-terminal region. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of the CSP N-terminal region. In some embodiments, the portion of the CSP N-terminal region is about 45, 50, 55, 60, or 61 amino acids in length. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a CSP N-terminal end region. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of the CSP N-terminal end region. In some embodiments, the portion of the CSP N-terminal end region is about 8, 9, 10, or 11 amino acids in length. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a CSP junction region. In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of the CSP junction region.In some embodiments, the portion of the CSP junction region is about 8, 9, 10, or 11 amino acids in length.

[0218] A small number of repetitive regions In some embodiments, the Plasmodium polypeptide constructs described herein comprise a minority repeat region or portion thereof of one or more Plasmodium CSPs comprising one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), and the polypeptide does not comprise the amino acid sequence of NPNA, NPNANP (SEQ ID NO: 150), or NANPNA (SEQ ID NO: 153).

[0219] In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP polypeptide regions or portions thereof comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP polypeptide regions or portions thereof comprising two or more (e.g., 2-12, or 2-10, or 2-9, or 2-8, or 4-12, or 4-10) repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP polypeptide regions or portions thereof comprising exactly three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP polypeptide regions or portions thereof that comprise exactly 8 repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102). In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP polypeptide regions or portions thereof that comprise exactly 9 repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102).

[0220] In some embodiments, the repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) are all contiguous with one another. In some embodiments, the repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102) are not all contiguous with one another. In some embodiments, the Plasmodium polypeptide constructs described herein comprise four portions of the minority repeat region of Plasmodium CSP, wherein each portion of the Plasmodium CSP polypeptide comprises two contiguous repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102).

[0221] C-terminal region In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP C-terminal regions (e.g., amino acids 273-375 of SEQ ID NO:1), or one or more portions thereof, wherein the C-terminal regions do not include the transmembrane region. In some embodiments, the Plasmodium polypeptide constructs described herein comprise exactly one Plasmodium CSP C-terminal region, wherein the Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to amino acids 273-375 of SEQ ID NO:1. In some embodiments, the Plasmodium polypeptide constructs described herein comprise two or more portions of the Plasmodium CSP C-terminal region (e.g., amino acids 273-375 of SEQ ID NO:1).

[0222] In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more portions of the Plasmodium CSP C-terminal region, each of which comprises or consists of: (i) amino acids 314-327 of SEQ ID NO:1 (or amino acids 314-327 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (ii) amino acids 352-363 of SEQ ID NO:1 (or amino acids 352-363 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (iii) amino acids 326-374 of SEQ ID NO:1 (or amino acids 326-374 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (iv) amino acids 364-377 of SEQ ID NO:1 (or amino acids 364-377 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); or (v) a combination thereof.

[0223] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a portion of the Plasmodium CSP C-terminal region, the portion comprising or consisting of: (i) amino acids 314-327 of SEQ ID NO:1 (or amino acids 314-327 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (ii) amino acids 352-363 of SEQ ID NO:1 (or amino acids 352-363 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (iii) amino acids 326-374 of SEQ ID NO:1 (or amino acids 326-374 of SEQ ID NO:Z with 1, 2, 3, 4, or 5 amino acid substitutions); (iv) amino acids 364-377 of SEQ ID NO:1 (or amino acids 364-377 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); or (v) a combination thereof.

[0224] In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more portions of the Plasmodium CSP C-terminal region, wherein the one or more portions collectively comprise or consist of: (i) amino acids 314-327 of SEQ ID NO:1 (or amino acids 314-327 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (ii) amino acids 352-363 of SEQ ID NO:1 (or amino acids 352-363 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (iii) amino acids 326-374 of SEQ ID NO:1 (or amino acids 326-374 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); (iv) amino acids 364-377 of SEQ ID NO:1 (or amino acids 364-377 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions); or (v) combinations thereof.

[0225] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a serine amino acid residue immediately following the Plasmodium CSP C-terminal region described herein, hi some embodiments, the Plasmodium polypeptide constructs described herein comprise a serine-valine amino acid sequence immediately following the Plasmodium CSP C-terminal region described herein.

[0226] joint area In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP junction regions or portions thereof. In some embodiments, the Plasmodium polypeptide constructs described herein comprise two or more Plasmodium CSP junction regions or portions thereof. In some embodiments, the Plasmodium polypeptide constructs described herein comprise exactly one Plasmodium CSP junction region. In some embodiments, the Plasmodium CSP junction region comprises or consists of amino acids 93-104 of SEQ ID NO:1 (or amino acids 93-104 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions). In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more portions of a Plasmodium CSP junction region. In some embodiments, the one or more portions of a Plasmodium CSP junction region comprise a deletion of one or more of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO:1. In some embodiments, one or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, and Q96, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, one or more portions of the Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96, and P97, where the amino acid numbering corresponds to SEQ ID NO: 1.

[0227] In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP junction region variants. In some embodiments, the Plasmodium CSP junction region variants comprise one or more amino acid substitution mutations. In some embodiments, the one or more substitution mutations comprise a K93A mutation, an L94A mutation, or both, wherein the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the Plasmodium CSP junction region variant comprises the amino acid sequence of AAKQ (SEQ ID NO: 426).

[0228] N-terminal end region In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP N-terminal end regions or portions thereof. In some embodiments, the Plasmodium polypeptide constructs described herein comprise two or more Plasmodium CSP N-terminal end regions or portions thereof. In some embodiments, the Plasmodium CSP N-terminal end region comprises or consists of amino acids 81-92 of SEQ ID NO:1 (or amino acids 81-92 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions). In some embodiments, the Plasmodium polypeptide constructs described herein do not comprise a Plasmodium CSP N-terminal end region or any portion thereof (i.e., lack or exclude the CSP N-terminal end region or any portion thereof).

[0229] N-terminal region In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP N-terminal regions or portions thereof. In some embodiments, the Plasmodium polypeptide constructs described herein comprise two or more Plasmodium CSP N-terminal regions or portions thereof. In some embodiments, the Plasmodium CSP N-terminal region comprises or consists of amino acids 19-80 of SEQ ID NO:1. In some embodiments, the Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to amino acids 19-80 of SEQ ID NO:1. In some embodiments, the Plasmodium polypeptide constructs described herein do not comprise the Plasmodium CSP N-terminal region or any portion thereof (i.e., lack or exclude the Plasmodium CSP N-terminal region or any portion thereof).

[0230] Major repeat region In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more Plasmodium CSP major repeat regions or portions thereof. In some embodiments, the Plasmodium polypeptide constructs described herein comprise exactly one Plasmodium CSP major repeat region or portion thereof, wherein the Plasmodium CSP major repeat region or portion thereof comprises a total of at least two and at most 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147). In some embodiments, the Plasmodium CSP major repeat region or portion thereof comprises two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 147), wherein the two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 147) are flanked by the amino acid sequence of NVDP (SEQ ID NO: 144). In some embodiments, the Plasmodium CSP major repeat region comprises, from N-terminus to C-terminus, 17 repeats of the amino acid sequence NANP (SEQ ID NO: 147), the amino acid sequence of NVDP (SEQ ID NO: 144), and 18 repeats of the amino acid sequence NANP (SEQ ID NO: 147). In some embodiments, a portion of the major repeat region of a Plasmodium CSP consists of up to 18 consecutive repeats of the amino acid sequence NANP (SEQ ID NO: 147). In some embodiments, a portion of the major repeat region of a Plasmodium CSP consists of two consecutive repeats of the amino acid sequence NANP (SEQ ID NO: 147). The major repeat region or portion thereof of one or more Plasmodium CSPs always contains at least one repeat of the amino acid sequence NPNANP (SEQ ID NO: 150) or NANPNA (SEQ ID NO: 153). In some embodiments, the major repeat region of a Plasmodium CSP comprises or consists of an amino acid sequence having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to amino acids 129-272 of SEQ ID NO: 1.In some embodiments, the Plasmodium polypeptide constructs described herein do not comprise the major repeat region of Plasmodium CSP or the portion of the major repeat region of Plasmodium CSP that comprises the amino acid sequence NPNA (SEQ ID NO: 141) (i.e., they lack or exclude the major repeat region of Plasmodium CSP or the portion of the major repeat region of Plasmodium CSP that comprises the amino acid sequence NPNA [SEQ ID NO: 141]).

[0231] In some embodiments, the Plasmodium polypeptide constructs described herein optionally comprise one or more of the following Plasmodium CSP polypeptide regions or portions thereof, if present, in order from N-terminus to C-terminus: (i) one or more Plasmodium CSP N-terminal regions or portions thereof, (ii) one or more Plasmodium CSP N-terminal end regions or portions thereof, (iii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof, (iv) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 102), (v) one or more Plasmodium CSP major repeat regions or portions thereof, and (vi) one or more Plasmodium CSP C-terminal regions or portions thereof.

[0232] In some embodiments, the Plasmodium polypeptide constructs described herein optionally comprise one or more of the following Plasmodium CSP polypeptide regions or portions thereof, if present, in order from N-terminus to C-terminus: (i) one Plasmodium CSP N-terminal region or portion thereof, (ii) one Plasmodium CSP N-terminal end region or portion thereof, (iii) one Plasmodium CSP junction region, portion thereof, or variant thereof, (iv) one or more Plasmodium CSP minor repeat sequences, (v) one Plasmodium CSP major repeat region or portion thereof, and (vi) one Plasmodium CSP C-terminal region or portion thereof.

[0233] B. Secretion signal In some embodiments, the Plasmodium polypeptide constructs described herein comprise a secretion signal that is functional, for example, in mammalian cells. In some embodiments, the secretion signal comprises or consists of a Plasmodium secretion signal. In some embodiments, the Plasmodium secretion signal comprises or consists of a Plasmodium CSP secretion signal. In some embodiments, the Plasmodium CSP secretion signal is from Plasmodium falciparum. In some embodiments, the Plasmodium CSP secretion signal is from Plasmodium falciparum isolate 3D7 (SEQ ID NO: 174).

[0234] In some embodiments, the secretory signal utilized is a heterologous secretory signal. In some embodiments, the heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, the heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, the viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). In some embodiments, the HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, the secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, the Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.

[0235] The present disclosure provides insight that, in some embodiments, including a viral secretion signal in a polypeptide construct encoding a parasitic antigen can have one or more improved characteristics. In some embodiments, the polypeptide construct comprises a viral secretion signal and one or more parasitic antigens. In some embodiments, the one or more parasitic antigens comprise one or more malaria antigens. In some embodiments, the one or more malaria antigens comprise one or more Plasmodium CSP polypeptide regions or portions thereof as described herein. In some embodiments, the viral secretion signal comprises an HSV secretion signal (e.g., an HSV-1 or HSV-2 secretion signal). In some embodiments, the HSV secretion signal comprises an HSV glycoprotein D (gD) secretion signal. In some embodiments, the HSV gD secretion signal comprises an HSV-1 gD secretion signal. In some embodiments, the HSV gD secretion signal comprises an HSV-2 gD secretion signal.

[0236] In some embodiments, the polypeptide construct comprises an HSV-1 gD secretion signal and one or more parasitic antigens. In some embodiments, the polypeptide construct comprises an HSV-1 gD secretion signal and one or more malaria antigens. In some embodiments, the polypeptide construct comprises an HSV-1 gD secretion signal and one or more Plasmodium CSP polypeptide regions or portions thereof as described herein.

[0237] In some embodiments, the polypeptide construct comprises an HSV-2 gD secretion signal and one or more parasitic antigens. In some embodiments, the polypeptide construct comprises an HSV-2 gD secretion signal and one or more malaria antigens. In some embodiments, the polypeptide construct comprises an HSV-2 gD secretion signal and one or more Plasmodium CSP polypeptide regions or portions thereof, as described herein.

[0238] In some embodiments, polypeptide constructs comprising a parasitic antigen and a viral secretion signal have one or more improved characteristics. In some embodiments, the improved characteristics are, for example, increased expression (e.g., increased ex vivo expression (e.g., extracellular expression) or increased in vivo expression (e.g., extracellular expression)), improved inhibition of sporozoite traversal, and / or improved sporozoite binding. In some embodiments, Plasmodium polypeptide constructs comprise a viral secretion signal and have one or more improved characteristics. In some embodiments, Plasmodium polypeptide constructs comprise an HSV secretion signal (e.g., an HSV-1 or HSV-2 secretion signal) and have one or more improved characteristics. In some embodiments, Plasmodium polypeptide constructs comprise an HSV glycoprotein D (gD) secretion signal and have one or more improved characteristics. In some embodiments, Plasmodium polypeptide constructs comprise an HSV-2 glycoprotein D (gD) secretion signal and have one or more improved characteristics. In some embodiments, the Plasmodium polypeptide construct comprises the HSV-1 glycoprotein D (gD) secretion signal and has one or more improved characteristics.

[0239] In some embodiments, Plasmodium polypeptide constructs comprising a viral secretory region have increased expression (e.g., increased ex vivo expression (e.g., extracellular expression) or increased in vivo expression (e.g., extracellular expression). For example, in some embodiments, Plasmodium polypeptide constructs comprising a heterologous secretory region have increased ex vivo expression, e.g., in mammalian cells. In some embodiments, the mammalian cells can be as described in Example 1 below (e.g., HEK293T cells).

[0240] In some embodiments, a Plasmodium polypeptide construct comprises a viral secretion signal and has increased expression in mammalian cells (e.g., HEK293T cells) compared to an otherwise identical construct having a non-viral secretion signal (e.g., a Pf secretion signal). In some embodiments, a Plasmodium polypeptide construct comprises an HSV secretion signal (e.g., an HSV-1 or HSV-2 secretion signal) and has increased expression in mammalian cells (e.g., HEK293T cells) compared to an otherwise identical construct having a non-viral secretion signal (e.g., a Pf secretion signal). In some embodiments, a Plasmodium polypeptide construct comprises an HSV glycoprotein D (gD) secretion signal and has increased expression in mammalian cells (e.g., HEK293T cells) compared to an otherwise identical construct having a non-viral secretion signal (e.g., a Pf secretion signal).

[0241] In some embodiments, Plasmodium polypeptide constructs comprising heterologous secretory domains have improved inhibition of sporozoite traversal. For example, in some embodiments, Plasmodium polypeptide constructs comprising heterologous secretory domains have improved production of antibodies that inhibit sporozoite traversal, e.g., as measured using a traversal assay, e.g., as described in Example 2 below.

[0242] In some embodiments, a Plasmodium polypeptide construct comprises a viral secretory signal and has improved inhibition of sporozoite traversal relative to an otherwise identical construct having a non-viral secretory signal (e.g., a Pf secretory signal). In some embodiments, a Plasmodium polypeptide construct comprises an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal) and has improved inhibition of sporozoite traversal relative to an otherwise identical construct having a non-viral secretory signal (e.g., a Pf secretory signal). In some embodiments, a Plasmodium polypeptide construct comprises an HSV glycoprotein D (gD) secretory signal and has improved inhibition of sporozoite traversal relative to an otherwise identical construct having a non-viral secretory signal (e.g., a Pf secretory signal).

[0243] In some embodiments, Plasmodium polypeptide constructs comprising a heterologous secretory domain have improved sporozoite binding. For example, in some embodiments, Plasmodium polypeptide constructs comprising a heterologous secretory domain improve binding to native PfCSP on PfCSP-expressing Plasmodium berghei (PbPf) sporozoites, e.g., as described in Example 2 below.

[0244] In some embodiments, Plasmodium polypeptide constructs include a viral secretory signal and have improved inhibition of sporozoite binding relative to constructs with an otherwise identical non-viral secretory signal (e.g., a Pf secretory signal). In some embodiments, Plasmodium polypeptide constructs include an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal) and have improved sporozoite binding relative to constructs with an otherwise identical non-viral secretory signal (e.g., a Pf secretory signal). In some embodiments, Plasmodium polypeptide constructs include an HSV glycoprotein D (gD) secretory signal and have improved sporozoite binding relative to constructs with an otherwise identical non-viral secretory signal (e.g., a Pf secretory signal).

[0245] In some embodiments, the secretory signal is characterized by a length of about 15 to 30 amino acids.

[0246] In many embodiments, the secretion signal is located at the N-terminus of the Plasmodium polypeptide constructs described herein. In some embodiments, the secretion signal preferably enables transport of the Plasmodium polypeptide construct with which it is associated to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment.

[0247] In some embodiments, the secretory signal is selected from the S1S2 signal peptide (aa 1-19), immunoglobulin secretory signal peptide (aa 1-22), human SPARC signal peptide, human insulin isoform secretory signals, human albumin signal peptide, etc. Those of skill in the art will recognize other secretory signals, such as those disclosed in WO2017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOS: 1-1115 and 1728, or fragment variants thereof). In some embodiments, the Plasmodium polypeptide constructs described herein do not comprise a secretory signal.

[0248] In some embodiments, the secretory signal comprises an amino acid sequence according to a SEQ ID NO: listed in Table 3, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences thereto. In some embodiments, the signal sequence is selected from those provided in Table 3 below and / or those encoded by the sequences provided in Table 4 below. [Table 3] [Table 4]

[0249] C. Transmembrane region In some embodiments, the Plasmodium polypeptide constructs described herein comprise a transmembrane region. In some embodiments, the transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, the transmembrane region utilized is one that is normally associated with CSP in nature. In some embodiments, the Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, the Plasmodium CSP GPI anchor region is from Plasmodium falciparum. In some embodiments, the Plasmodium CSP GPI anchor region is from Plasmodium falciparum isolate 3D7 (SEQ ID NO: 231), e.g., amino acids 378-397 of SEQ ID NO: 1. In some embodiments, the transmembrane region utilized is a heterologous transmembrane region.

[0250] In some embodiments, the transmembrane region is located at the N-terminus of the Plasmodium polypeptide construct. In some embodiments, the transmembrane region is located at the C-terminus of the Plasmodium polypeptide construct. In some embodiments, the transmembrane region is not located at the N-terminus or C-terminus of the Plasmodium polypeptide construct.

[0251] Transmembrane regions are known in the art, any of which can be utilized in the Plasmodium polypeptide constructs described herein. In some embodiments, the transmembrane region comprises or is a transmembrane domain of influenza virus hemagglutinin (HA), HIV-1 Env, equine infectious anemia virus (EIAV), murine leukemia virus (MLV), mouse mammary tumor virus, vesicular stomatitis virus (VSV) G protein, rabies virus, or a seven transmembrane domain receptor.

[0252] In some embodiments, the heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of an HSV transmembrane region (e.g., an HSV-1 or HSV-2 transmembrane region). In some embodiments, the HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprises or consists of the amino acid sequence of SEQ ID NO: 234.

[0253] In some embodiments, the heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, the human transmembrane region comprises or consists of a human decay-accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, the hDAF-GPI anchor region comprises or consists of the amino acid sequence of SEQ ID NO: 237.

[0254] In some embodiments, the Plasmodium polypeptide constructs described herein do not include a transmembrane region.

[0255] G. Helper antigens In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more helper antigens. Those skilled in the art will recognize a variety of potentially useful helper antigens (e.g., P2 tetanus toxoid, PADRE peptide, Hepatitis B surface antigen (HBsAg)), including, for example, those described in WO2020128031 (incorporated herein by reference in its entirety). In some embodiments, the helper antigen is a malarial protein (e.g., a malarial protein described herein), provided that the antigen is not a CSP polypeptide or portion thereof. In some embodiments, the helper antigen is Plasmodium 2-phospho-D-glycerate hydrolylase antigen, Plasmodium liver stage antigen 1(a), (LSA-1(a)), Plasmodium liver stage antigen 1(b) (LSA-1(b)), Plasmodium thrombospondin-related anonymous protein (TRAP), Plasmodium liver stage-associated protein 1 (LSAP1), Plasmodium liver stage-associated protein 2 (LSAP2), Plasmodium UIS3, Plasmodium UIS4, Plasmodium ETRAMP10.3, Plasmodium liver-specific protein 1 (LISP-1), Plasmodium liver-specific protein 2 (LISP-2), Plasmodium liver stage antigen 3 (LSA-3), Plasmodium EXP1, Plasmodium E140, Plasmodium reticulocyte-binding protein homolog 5 (Rh5), Plasmodium glutamic acid-rich protein (GARP), Plasmodium parasite-infected erythrocyte surface protein 2 (PIESP2), Plasmodium cysteine-rich protective antigen (CyRPA), Plasmodium Ripr, Plasmodium P113, or a combination thereof.

[0256] In some embodiments, the helper antigen comprises or consists of a P. falciparum 2-phospho-D-glycerate hydrolylase antigen, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 240. In some embodiments, the helper antigen comprises or consists of a P. falciparum liver stage antigen 3, e.g., comprising or consisting of the amino acid sequence according to SEQ ID NO: 243. In some embodiments, the helper antigen comprises or consists of an Anopheles antigen, e.g., Anopheles gambiae TRIO, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 246.

[0257] In some embodiments, the Plasmodium polypeptide constructs described herein include a secretion signal (eg, a secretion signal described herein), and the helper antigen immediately follows the secretion signal.

[0258] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a helper antigen located at the C-terminus.

[0259] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a linker between the CSP portion and the helper antigen.

[0260] E. Multimerization region In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more multimerization domains (e.g., heteromultimerization domains). In some embodiments, the heteromultimerization domain comprises a dimerization domain, a trimerization domain, or a tetramerization domain.

[0261] In some embodiments, the multimerization domain is one described in WO2017 / 081082 (e.g., SEQ ID NOS: 1116-1167, or a fragment or variant thereof), which is incorporated by reference in its entirety. Exemplary trimerization and tetramerization domains include, but are not limited to, engineered leucine zippers, fibritin foldon domains from enterobacteriaceae phage T4, GCN4p11, GCN4-p11, and p53.

[0262] In some embodiments, the provided Plasmodium polypeptide constructs are capable of forming trimeric complexes. For example, the provided Plasmodium polypeptide constructs may comprise a multimerization domain that allows for the formation of multimeric complexes, such as, for example, trimeric complexes of the Plasmodium polypeptide constructs described herein. In some embodiments, the multimerization domain that allows for the formation of multimeric complexes comprises a trimerization domain, such as a trimerization domain described herein. In some embodiments, the Plasmodium polypeptide construct comprises a "foldon" trimerization domain from T4-fibritin, for example, to increase its immunogenicity. In some embodiments, the Plasmodium polypeptide construct comprises a multimerization domain comprising or consisting of an amino acid sequence according to SEQ ID NO: 255.

[0263] F. Linker In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more linkers. In some embodiments, the linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the linker is or comprises about 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, or fewer amino acids. The linker can comprise any amino acid sequence and is not limited to specific amino acids. In some embodiments, the linker comprises one or more glycine (G) amino acids. In some embodiments, the linker comprises one or more serine (S) amino acids. In some embodiments, the linker comprises amino acids selected based on cleavage predictors to generate a highly cleavable linker.

[0264] In some embodiments, the linker is or comprises S-G4-S-G4-S. In some embodiments, the linker is or comprises an amino acid sequence according to SEQ ID NO: 267. In some embodiments, the linker is or comprises an amino acid sequence according to SEQ ID NO: 258. In some embodiments, the linker has an amino acid sequence according to SEQ ID NO: 261, 267, 258, 276, 279, 270, 282, 264, or 273. In some embodiments, the linker is or comprises a sequence set forth in WO2017 / 081082, the entire contents of which are incorporated herein by reference (see SEQ ID NOs: 1509-1565, or fragments or variants thereof).

[0265] In some embodiments, the Plasmodium polypeptide constructs described herein comprise a linker between the C-terminal region or portion thereof and the transmembrane region, hi some embodiments, the Plasmodium polypeptide constructs described herein comprise a linker after a small number of repeat sequences.

[0266] G. Embodiments of Plasmodium Polypeptide Constructs In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the Plasmodium CSP polypeptide domains or portions thereof described above. Exemplary combinations of domains are described below.

[0267] Full-length CSP construct In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more regions or portions of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the N-terminal region, N-terminal end region, junction region, minor repeat region, major repeat region, and a portion of the major repeat region and C-terminal region, or corresponding portions thereof, of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: N-terminal region-N-terminal end region-junction region-minor repeat region-major repeat region-C-terminal region, wherein the regions are from a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In preferred embodiments, such Plasmodium polypeptide constructs have a serine or serine and valine immediately following the C-terminal region. In a preferred embodiment, the N-terminal region or a portion thereof comprises the amino acid sequence of positions 19 to 80 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 19 to 80 of SEQ ID NO: 1. In a preferred embodiment, the N-terminal end region or a portion thereof comprises the amino acid sequence of positions 81 to 92 of SEQ ID NO: 1, or the amino acid sequence of positions 81 to 92 of SEQ ID NO: 1 with one, two, three, four, or five amino acid substitutions. In a preferred embodiment, the junction region or a portion thereof comprises the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1, or the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1 with one, two, three, four, or five amino acid substitutions. In a preferred embodiment, the minority repeat region or a portion thereof comprises the amino acid sequence of positions 105 to 128 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105 to 128 of SEQ ID NO:1.In a preferred embodiment, the major repeat region or a portion thereof comprises the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1. In a preferred embodiment, the C-terminal region or a portion thereof comprises the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1. In a preferred embodiment, the Plasmodium polypeptide construct comprises the amino acid sequence of positions 19 to 375 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 19 to 375 of SEQ ID NO:1.

[0268] Such a Plasmodium polypeptide construct containing all of the CSP regions described above and containing a serine or serine and valine immediately following the C-terminal region is referred to as a full-length CSP construct.

[0269] In some embodiments, the Plasmodium polypeptide construct may have the following structure: full-length CSP construct, sec-full-length CSP construct, full-length CSP construct-TMD, sec-full-length CSP construct-TMD, Pfsec-full-length CSP construct, Full-length CSP construct-PfTMD, Pfsec-full-length CSP construct-PfTMD, HSV-1gDsec-full-length CSP construct, Full-length CSP construct-HSV-1TMD, HSV-1gDsec-full-length CSP construct-HSV-1TMD, Pfsec-full-length CSP construct-HSV-1TMD, HSV-1gDsec-full-length CSP construct-PfTMD, Heterogeneous sec-full-length CSP construct, Full-length CSP construct-heterogeneous TMD, heterogeneous sec-full-length CSP construct-heterogeneous TMD, Full-length CSP constructs - multimerization, sec - full-length CSP construct - multimerization, Full-length CSP construct-TMD-multimerization, sec-full-length CSP construct-TMD-multimerization, Pfsec-full-length CSP construct-multimerization, Full-length CSP construct-PfTMD-multimerization, Pfsec-full-length CSP construct-PfTMD-multimerization, HSV-1gDsec-full-length CSP construct-multimerization, Full-length CSP construct-HSV-1TMD multimerization, HSV-1gDsec-full-length CSP construct-HSV-1TMD-multimerization, Pfsec-full-length CSP construct-HSV-1 TMD-multimerization, HSV-1gDsec-full-length CSP construct-PfTMD-multimerization, Heterologous sec-full-length CSP construct-multimerization, Full-length CSP construct-heterologous TMD-multimerization, or Heterogeneous sec-full-length CSP construct-heterogeneous TMD-multimerization.

[0270] CSP constructs with non-contiguous small repeat regions In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the N-terminal end region, junction region, minor repeat region, major repeat region, and C-terminal region of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7, or corresponding portions thereof.

[0271] In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: N-terminal end region-junction region-[minor repeat region-major repeat region portion] x-minor repeat region-C-terminal region, where the [minor repeat region-major repeat region portion] is repeated x times, and the region is from a CSP from Plasmodium falciparum, preferably Plasmodium falciparum isolate 3D7. In some embodiments, x is 2 to 5 (i.e., the [minor repeat region-major repeat region portion] is repeated 2 to 5 times). In preferred embodiments, such a Plasmodium polypeptide construct has two repeats of the [minor repeat region-major repeat region portion], such that the Plasmodium polypeptide constructs described herein have the structure: N-terminal end region-junction region-minor repeat region-major repeat region portion-minor repeat region-major repeat region portion-minor repeat region-C-terminal region. In preferred embodiments, the N-terminal end region or portion thereof comprises the amino acid sequence of positions 81-92 of SEQ ID NO: 1, or the amino acid sequence of positions 81-92 of SEQ ID NO: 1 with 1, 2, 3, 4, or 5 amino acid substitutions. In preferred embodiments, the junction region comprises the R1 region (amino acids 93-97) of SEQ ID NO: 1. In preferred embodiments, the junction region or a portion thereof comprises the amino acid sequence of positions 93-104 of SEQ ID NO: 1, or the amino acid sequence of positions 93-104 of SEQ ID NO: 1 with one, two, three, four, or five amino acid substitutions. In preferred embodiments, the minority repeat region or a portion thereof comprises the amino acid sequence of positions 105-128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105-128 of SEQ ID NO: 1. In some embodiments, the major repeat region portion comprises at least four repeats, at least five repeats, at least six repeats, or at least seven repeats of the sequence NANP (SEQ ID NO: 147). In preferred embodiments, the major repeat region portion comprises the sequence NANPNANPNANPNANPNANPNANP (SEQ ID NO: 437).In preferred embodiments, the C-terminal region or a portion thereof comprises the amino acid sequence of positions 273-375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273-375 of SEQ ID NO: 1. A Plasmodium polypeptide construct comprising all of the above CSP regions or their corresponding portions, and comprising non-contiguous minor repeat regions (i.e., minor repeat region-major repeat region portion-minor repeat region-major repeat region portion-minor repeat region), is referred to as a 3xMR CSP construct. In some embodiments, a Plasmodium polypeptide construct may have the following structure: 3xMR CSP construct, sec-3xMR CSP construct, 3xMR CSP construct-TMD, sec-3xMR CSP construct-TMD, Pfsec-3xMR CSP construct, 3xMR CSP construct-PfTMD, Pfsec-3xMR CSP construct-PfTMD, HSV-1gDsec-3xMR CSP construct, 3xMR CSP construct-HSV-1TMD, HSV-1gDsec-3xMR CSP construct-HSV-1TMD, HSV-1gDsec-3xMR CSP construct-PfTMD, Pfsec-3xMR CSP construct-HSV-1TMD, Heterogeneous sec-3xMR CSP construct, 3 × MR CSP construct-heterogeneous TMD, or Heterogeneous sec-3xMR CSP construct-heterogeneous TMD.

[0272] N-terminal region deleted CSP construct In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the N-terminal end region, junction region, minor repeat region, major repeat region, and C-terminal region, or corresponding portions thereof, of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: N-terminal end region-junction region-minor repeat region-major repeat region-C-terminal region, where the region is from a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In preferred embodiments, such Plasmodium polypeptide constructs have a serine or serine and valine immediately following the C-terminal region. In preferred embodiments, the N-terminal end region or portion thereof comprises the amino acid sequence of positions 81-92 of SEQ ID NO:1, or the amino acid sequence of positions 81-92 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions. In a preferred embodiment, the junction region or a portion thereof comprises the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1, or the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1 with one, two, three, four, or five amino acid substitutions. In a preferred embodiment, the minor repeat region or a portion thereof comprises the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1. In a preferred embodiment, the major repeat region or a portion thereof comprises the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1. In a preferred embodiment, the C-terminal region or a portion thereof comprises the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1.Such a Plasmodium polypeptide construct containing all of the aforementioned CSP regions or corresponding portions thereof except the N-terminal region or portion thereof, and containing a serine or serine and valine immediately following the C-terminal region, is referred to as a dNT CSP construct. In some embodiments, the Plasmodium polypeptide construct may have the following structure: dNT CSP construct, sec-dNT CSP construct, dNT CSP construct-TMD, sec-dNT CSP construct-TMD, Pfsec-dNT CSP construct, dNT CSP construct-PfTMD, Pfsec-dNT CSP construct-PfTMD, HSV-1gDsec-dNT CSP construct, dNT CSP construct-HSV-1TMD, HSV-1gDsec-dNT CSP construct-HSV-1TMD, HSV-1gDsec-dNT CSP construct-PfTMD, Pfsec-dNT CSP construct-HSV-1TMD, Heterologous sec-dNT CSP constructs, dNT CSP construct-heterologous TMD, or Heterologous sec-dNT CSP construct-heterologous TMD.

[0273] N-terminal and major repeat region deleted CSP constructs In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the N-terminal end region, junction region, one or more minor repeat regions, and C-terminal region, or corresponding portions thereof, of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: N-terminal end region-junction region-one or more minor repeat regions-C-terminal region, where the region is from a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In preferred embodiments, such Plasmodium polypeptide constructs have a serine or a serine and a valine immediately following the C-terminal region. In preferred embodiments, the N-terminal end region or a portion thereof comprises the amino acid sequence of positions 81-92 of SEQ ID NO: 1, or the amino acid sequence of positions 81-92 of SEQ ID NO: 1 with 1, 2, 3, 4, or 5 amino acid substitutions. In a preferred embodiment, the junction region or a portion thereof comprises the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1, or the amino acid sequence of positions 93 to 104 of SEQ ID NO: 1 with one, two, three, four, or five amino acid substitutions. In a preferred embodiment, the minority repeat region or a portion thereof comprises the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1. In a preferred embodiment, the C-terminal region or a portion thereof comprises the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273 to 375 of SEQ ID NO: 1. In some embodiments, such Plasmodium polypeptide constructs have two or more minor repeat regions, for example, three minor repeat regions.Such a Plasmodium polypeptide construct containing all of the aforementioned CSP regions or corresponding portions thereof except the N-terminal region and major repeat region, or corresponding portions thereof, and having one or more minor repeat regions and containing a serine or serine and valine immediately following the C-terminal region, is referred to as a dND-d major CSP construct. In some embodiments, the Plasmodium polypeptide construct may have the following structure: dNT-d major CSP construct, sec-dNT-d major CSP construct, dNT-d major CSP construct-TMD, sec-dNT-dmajor CSP construct-TMD, Pfsec-dNT-d major CSP construct, dNT-dMajor CSP construct-PfTMD, Pfsec-dNT-d major CSP construct-PfTMD, HSV-1gDsec-dNT-d major CSP construct, dNT-d major CSP construct-HSV-1TMD, HSV-1gDsec-dNT-dmajor CSP construct-HSV-1TMD, HSV-1gDsec-dNT-dmajor CSP construct-PfTMD, Pfsec-dNT-dmajor CSP construct-HSV-1TMD, Heterologous sec-dNT-d major CSP construct, dNT-dMajor CSP construct-heterologous TMD, Heterologous sec-dNT-d major CSP construct-heterologous TMD, dNT-d major CSP construct-helper antigen, sec-dNT-d primary CSP construct, CSP construct helper antigen, dNT-d major CSP construct-TMD-helper antigen, sec-dNT-d major CSP construct-TMD-helper antigen, Pfsec-dNT-d major CSP construct helper antigen, dNT-dMajor CSP construct-PfTMD-Helper antigen, Pfsec-dNT-d major CSP construct-PfTMD-helper antigen, HSV-1gDsec-dNT-d major CSP construct-helper antigen, dNT-d major CSP construct-HSV-1 TMD-helper antigen, HSV-1gDsec-dNT-d major CSP construct-HSV-1TMD-helper antigen, HSV-1gDsec-dNT-d major CSP construct-PfTMD-helper antigen, Pfsec-dNT-d major CSP construct-HSV-1 TMD-helper antigen, Heterologous sec-dNT-d major CSP construct-helper antigen, dNT-dMajor CSP construct-heterologous TMD-helper antigen, or Heterologous sec-dNT-d major CSP construct-heterologous TMD-helper antigen.

[0274] N-terminal domain deleted CSP construct In some embodiments, the Plasmodium polypeptide constructs described herein comprise one or more of the junction region, one or more minor repeat regions, the major repeat region, and the C-terminal region, or corresponding portions thereof, of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: junction region-one or more minor repeat regions-major repeat region-C-terminal region, where the region is from a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In preferred embodiments, such Plasmodium polypeptide constructs have a serine or a serine and a valine immediately following the C-terminal region. In preferred embodiments, the junction region or portion thereof comprises the amino acid sequence of positions 93-104 of SEQ ID NO:1, or the amino acid sequence of positions 93-104 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions. In a preferred embodiment, the minor repeat region or a portion thereof comprises the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105 to 128 of SEQ ID NO: 1. In a preferred embodiment, the major repeat region or a portion thereof comprises the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 129 to 272 of SEQ ID NO: 1. In preferred embodiments, the C-terminal region or portion thereof comprises the amino acid sequence of positions 273-375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273-375 of SEQ ID NO: 1. In some embodiments, such Plasmodium polypeptide constructs have two or more minor repeat regions, for example, three minor repeat regions.Such Plasmodium polypeptide constructs containing all of the aforementioned CSP regions or corresponding portions thereof, or portions thereof, excluding the N-terminal domain (i.e., excluding the N-terminal and N-terminal tail regions), and having one or more small repeat regions and including a serine or serine and valine immediately following the C-terminal region, are referred to as dND CSP constructs. In some embodiments, the Plasmodium polypeptide construct may have the following structure: dND CSP constructs, sec-dND CSP construct, dND CSP construct-TMD, sec-dND CSP construct-TMD, Pfsec-dND CSP construct, dND CSP construct-PfTMD, Pfsec-dND CSP construct-PfTMD, HSV-1gDsec-dND CSP construct, dND CSP construct-HSV-1TMD, HSV-1gDsec-dND CSP construct-HSV-1TMD, HSV-1gDsec-dND CSP construct-PfTMD, Pfsec-dND CSP construct-HSV-1TMD, Heterologous sec-dND CSP constructs, dND CSP constructs - heterologous TMD, or Heterologous sec-dND CSP construct-heterologous TMD.

[0275] N-terminal domain and major repeat region deleted CSP constructs In some embodiments, the Plasmodium polypeptide constructs described herein comprise the junction region, one or more minor repeat regions, and C-terminal region, or corresponding portions thereof, of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described herein have the structure: junction region-one or more minor repeat regions-C-terminal region, where the region is from a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In preferred embodiments, such Plasmodium polypeptide constructs have a serine or a serine and a valine immediately following the C-terminal region. In preferred embodiments, the junction region or portion thereof comprises the amino acid sequence of positions 93-104 of SEQ ID NO:1, or the amino acid sequence of positions 93-104 of SEQ ID NO:1 with 1, 2, 3, 4, or 5 amino acid substitutions. In preferred embodiments, the minority repeat region or portion thereof comprises the amino acid sequence of positions 105-128 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 105-128 of SEQ ID NO: 1. In preferred embodiments, the C-terminal region or portion thereof comprises the amino acid sequence of positions 273-375 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 273-375 of SEQ ID NO: 1. In some embodiments, such Plasmodium polypeptide constructs have two or more minority repeat regions, for example, three minority repeat regions. Such a Plasmodium polypeptide construct comprising all of the aforementioned CSP regions or corresponding portions thereof excluding the N-terminal domain (i.e., excluding the N-terminal region and N-terminal end region), and the major repeat region or corresponding portions thereof, with one or more minor repeat regions, and containing a serine or serine and valine immediately following the C-terminal region, is referred to as a dND-d major CSP construct.In some embodiments, the Plasmodium polypeptide construct may have the following structure: dND-d major CSP construct, sec-dND-d major CSP construct, dND-d major CSP construct-TMD, sec-dND-d major CSP construct-TMD, Pfsec-dND-d major CSP construct, dND-d major CSP construct-PfTMD, Pfsec-dND-d major CSP construct-PfTMD, HSV-1gDsec-dND-d major CSP construct, dND-d major CSP construct-HSV-1TMD, HSV-1gDsec-dND-dmajor CSP construct-HSV-1TMD, HSV-1gDsec-dND-dmajor CSP construct-PfTMD, Pfsec-dND-dmajor CSP construct-HSV-1TMD, Heterologous sec-dND-d major CSP construct, dND-d major CSP construct-heterologous TMD, or Heterologous sec-dND-d major CSP construct-heterologous TMD.

[0276] N-terminal domain and major repeat region deleted CSP constructs with junction region variants or portions In some embodiments, the Plasmodium polypeptide constructs described herein comprise a junction region variant or portion thereof, one or more minor repeat regions, and a C-terminal region, or corresponding portions thereof, of a CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. In some embodiments, the Plasmodium polypeptide constructs described her...

Claims

1. 1. A polyribonucleotide encoding a polypeptide comprising one or more Plasmodium CSP polypeptide regions or portions thereof, said polypeptide comprising one or more repeats of the amino acid sequence of NANPNVDP, and said polypeptide does not comprise the amino acid sequence of NPNA.

2. 1. A polyribonucleotide encoding a polypeptide comprising one or more Plasmodium CSP polypeptide regions or portions thereof, said polypeptide comprising five or more repeats of the amino acid sequence NANPNVDP.

3. 3. The polyribonucleotide of claim 1, wherein each of said one or more Plasmodium CSP polypeptide regions or portions thereof comprises 25 or more consecutive amino acids of an amino acid sequence according to SEQ ID NO:

1.

4. 4. The polyribonucleotide of claim 1 or 3, wherein the polypeptide comprises two or more repeats of the amino acid sequence of NANPNVDP.

5. The polyribonucleotide of any one of claims 1 to 4, wherein the polypeptide comprises one or more Plasmodium CSP C-terminal regions or portions thereof.

6. The polyribonucleotide of any one of claims 1 to 5, wherein the polypeptide comprises one or more Plasmodium CSP junction regions or portions thereof.

7. 7. The polyribonucleotide of claim 6, wherein at least one of the one or more Plasmodium CSP junction regions or portions thereof comprises a deletion of K93, L94, K95, and Q96, and the amino acid numbering corresponds to SEQ ID NO:

1.

8. The polyribonucleotide of any one of claims 1 to 5, wherein the polypeptide comprises one or more Plasmodium CSP junction region variants.

9. 9. The polyribonucleotide of claim 8, wherein at least one of the one or more Plasmodium CSP junction region variants comprises a K93A mutation, a L94A mutation, or both, and wherein the amino acid numbering corresponds to SEQ ID NO:

1.

10. The polyribonucleotide of any one of claims 1 to 9, wherein the polypeptide comprises one or more Plasmodium CSP N-terminal end regions or portions thereof.

11. The polyribonucleotide according to any one of claims 1 to 9, wherein the polypeptide does not contain the Plasmodium CSP N-terminal end region or any part thereof.

12. The polyribonucleotide of any one of claims 1 to 11, wherein the polypeptide comprises one or more Plasmodium CSP N-terminal regions or portions thereof.

13. The polyribonucleotide of any one of claims 1 to 11, wherein the polypeptide does not contain the Plasmodium CSP N-terminal region or any part thereof.

14. The polyribonucleotide of any one of claims 2 to 13, wherein the polypeptide comprises one or more Plasmodium CSP major repeat regions or portions thereof.

15. 15. The polyribonucleotide of claim 14, wherein the major repeat region or portion thereof of one or more Plasmodium CSPs comprises the amino acid sequence NANPNA or NPNANP.

16. 16. The polyribonucleotide of claim 14 or 15, wherein the polypeptide comprises exactly one Plasmodium CSP major repeat region or portion thereof, and the Plasmodium CSP major repeat region or portion thereof comprises a total of at least two and at most 35 repeats of the amino acid sequence NANP.

17. 16. The polyribonucleotide of claim 14 or 15, wherein the polypeptide comprises at least two major repeat region portions of Plasmodium CSP, each of which comprises at least four and at most seven repeats of the amino acid sequence NANP.

18. 18. The polyribonucleotide of claim 17, wherein the polypeptide comprises at least two Plasmodium CSP minority repeat regions, each Plasmodium CSP minority repeat region comprising three repeats of the amino acid sequence NANPNVDP.

19. 14. The polyribonucleotide of any one of claims 2 to 13, wherein the polypeptide does not contain the major repeat region of Plasmodium CSP comprising the amino acid sequence NPNA or any part thereof.

20. The one or more Plasmodium CSP polypeptide regions or portions thereof, if present, are, in order from N-terminus to C-terminus: (i) one or more Plasmodium CSP N-terminal regions or portions thereof; (ii) one or more Plasmodium CSP N-terminal end regions or portions thereof; (iii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iv) one or more repeats of the amino acid sequence of NANPNVDP; (v) one or more Plasmodium CSP major repeat regions or portions thereof; and (vi) one or more Plasmodium CSP C-terminal regions or parts thereof. The polyribonucleotide according to any one of claims 1 to 19.

21. The polyribonucleotide of any one of claims 1 to 20, wherein the polypeptide comprises one or more helper antigens.

22. The one or more helper antigens are selected from the group consisting of Plasmodium 2-phospho-D-glycerate hydrolylase antigen, Plasmodium liver stage antigen 1(a), (LSA-1(a)), Plasmodium liver stage antigen 1(b) (LSA-1(b)), Plasmodium thrombospondin-related anonymous protein (TRAP), Plasmodium liver stage-associated protein 1 (LSAP1), Plasmodium liver stage-associated protein 2 (LSAP2), Plasmodium UIS3, Plasmodium UIS4, Plasmodium ETRAMP10.3, Plasmodium liver-specific protein 1 (LISP-1), Plasmodium liver-specific protein 2 (LISP-2), Plasmodium liver stage antigen 3 (LSA-3), Plasmodium EXP1, Plasmodium E140, Plasmodium reticulocyte-associated protein homolog 5 (Rh5), Plasmodium glutamic acid-rich protein (GARP), Plasmodium parasite-infected erythrocyte surface protein 2 (PIESP2), Plasmodium cysteine-rich protective antigen (CyRPA), Plasmodium Ripr, Plasmodium P113, P114, P115, P116, P117, P118, P119, P120, P121, P122, P123, P124, P125, P126, P127, P128, P129, P130, P131, P132, P133, P134, P135, P136, P137, P1

23. 23. The polyribonucleotide of claim 22, wherein the one or more helper antigens comprise an Anopheles antigen, preferably wherein the one or more helper antigens comprise Anopheles gambiae TRIO.

24. The polyribonucleotide of any one of claims 1 to 23, wherein the polypeptide comprises a multimerization region.

25. The polyribonucleotide of any one of claims 1 to 24, wherein the polypeptide comprises a secretion signal.

26. 26. The polyribonucleotide of claim 25, wherein the secretion signal comprises or consists of a Plasmodium secretion signal, preferably a Plasmodium CSP secretion signal.

27. 26. The polyribonucleotide of claim 25, wherein the secretion signal comprises or consists of a heterologous secretion signal.

28. 28. The polyribonucleotide of claim 27, wherein the heterologous secretory signal comprises or consists of a non-human secretory signal.

29. The heterologous secretory signal comprises or consists of a viral secretory signal, preferably the viral secretory signal is (a) an HSV-1 or HSV-2 secretory signal, even more preferably wherein said viral secretory signal comprises or consists of the HSV glycoprotein D (gD) secretory signal, or 28. The polyribonucleotide of claim 27, comprising or consisting of (b) an Ebola virus secretory signal, even more preferably wherein the viral secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.

30. 30. The polyribonucleotide of any one of claims 1 to 29, wherein the polypeptide comprises a transmembrane region.

31. 31. The polyribonucleotide of claim 30, wherein the transmembrane region comprises or consists of a Plasmodium transmembrane region, preferably wherein the Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region.

32. The transmembrane region comprises or consists of a heterologous transmembrane region, preferably the heterologous transmembrane region comprises: (a) does not contain a hemagglutin transmembrane domain; (b) comprising or consisting of a viral transmembrane domain, preferably wherein said viral transmembrane domain comprises or consists of an HSV-1 or HSV-2 transmembrane domain, and even more preferably wherein said HSV transmembrane domain comprises or consists of an HSV gD transmembrane domain, or (c) a human transmembrane domain comprising or consisting of a human decay-accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor domain;

33. The polyribonucleotide of any one of claims 1 to 24 and 30 to 32, wherein the polypeptide does not contain a secretory signal.

34. 30. The polyribonucleotide of any one of claims 1 to 29, wherein the polypeptide does not include a transmembrane region.

35. 35. The polyribonucleotide of any one of claims 1 to 34, wherein the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

33.

36. 35. The polyribonucleotide of any one of claims 1 to 34, wherein the polypeptide comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

81.

37. The polyribonucleotide according to any one of claims 1 to 36, wherein the Plasmodium is Plasmodium falciparum.

38. 38. The polyribonucleotide of any one of claims 1 to 37, wherein the one or more Plasmodium CSP polypeptide regions or portions thereof are one or more P. falciparum CSP polypeptide regions or portions thereof, and preferably the Plasmodium falciparum is Plasmodium falciparum isolate 3D7.

39. The polyribonucleotide according to any one of claims 1 to 38, wherein the polyribonucleotide is an isolated polyribonucleotide.

40. 40. The polyribonucleotide of any one of claims 1 to 39, wherein the polyribonucleotide is an engineered polyribonucleotide.

41. The polyribonucleotide according to any one of claims 1 to 40, wherein the polyribonucleotide is a codon-optimized polyribonucleotide.

42. In the order 5' to 3', (i) a 5'UTR comprising or consisting of a modified human alpha-globin 5'-UTR; (ii) a polyribonucleotide according to any one of claims 1 to 41; (iii) a 3′UTR comprising or consisting of a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; and (iv) an RNA construct comprising a polyA tail sequence.

43. 43. The RNA construct of claim 42, further comprising a 5' cap.

44. A composition comprising one or more polyribonucleotides according to any one of claims 1 to 39, or one or more RNA constructs according to claim 42 or 43.

45. further comprising a lipid nanoparticle, polyplex (PLX), lipidated polyplex (LPLX), or liposome; 45. The composition of claim 44, wherein the one or more polyribonucleotides or the one or more RNA constructs are fully or partially encapsulated within the lipid nanoparticle, the polyplex (PLX), the lipidated polyplex (LPLX), or the liposome.

46. 46. ​​A pharmaceutical composition comprising the composition of claim 44 or 45 and at least one pharmaceutically acceptable excipient.

47. A method for treating or preventing a malaria infection, comprising administering to a subject a polyribonucleotide according to any one of claims 1 to 41, an RNA construct according to claim 42 or 43, a composition according to claim 44 or 45, or a pharmaceutical composition according to claim 46.

48. 47. The pharmaceutical composition of claim 46 for use in the treatment or prevention of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

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