Compositions for delivery of plasmodium antigens and related methods

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for delivery of Plasmodium protein antigens and related technologies (e.g., components thereof and / or methods relating thereto). Among other things, the present disclosure provides combinations comprising a first pharmaceutical composition comprising a first polyribonucleotide and a second pharmaceutical composition comprising a second polyribonucleotide. In some embodiments, a first polyribonucleotide encodes a first polypeptide that comprises one or more Plasmodium T-cell antigens. In some embodiments, a second polyribonucleotide encodes a second polypeptide that comprises one or more Plasmodium polypeptide (e.g., CSP) or antigenic portions thereof.

Description

COMPOSITIONS FOR DELIVERY OF PLASMODIUM ANTIGENS AND RELATED METHODS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Serial Nos.63 / 515,330, filed July 24, 2023, 63 / 580,303, filed September 01, 2023, 63 / 570,777, filed March 27, 2024, 63 / 634,381, filed April 15, 2024, and 63 / 641,939, filed May 02, 2024, the entirety of each of which is incorporated herein by reference. BACKGROUND

[0002] Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the Plasmodium genus. According to the World Health Organization, an estimated 3.4 billion people in 92 countries are at risk of being infected with the malaria parasite and developing disease. SUMMARY

[0003] The present disclosure provides technologies (e.g., compositions, methods, etc.) for delivery of Plasmodium antigens.

[0004] For instance, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for delivering particular Plasmodium polypeptide constructs to a subject (e.g., a patient) and related technologies (e.g., methods). As described further herein, Plasmodium polypeptide constructs can comprise one or more Plasmodium polypeptide or antigenic portions thereof. In particular, the present disclosure provides Plasmodium vaccine compositions and related technologies (e.g., methods).

[0005] In some embodiments, the present disclosure provides particular pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) formats including, for example, polyribonucleotides comprising particular elements and / or sequences useful for delivery of Plasmodium antigens.

[0006] The present disclosure provides a variety of insights and technologies related to such Plasmodium antigen constructs and vaccine (e.g., RNA vaccine) compositions. For example, the present disclosure provides technologies for preventing, characterizing, treating, and / or monitoring malaria outbreaks and / or infections including, as noted, various nucleic acid constructs and encoded proteins, as well as agents (e.g., antibodies) that bind to such proteins, and compositions that comprise and / or deliver them.

[0007] In some aspects, provided herein are technologies (e.g., compositions and methods) for augmenting, inducing, promoting, enhancing and / or improving an immune response against a Plasmodium parasite. In some embodiments, technologies described herein are designed to act as immunological boost to a primary vaccine, such as a vaccine directed to antigen(s) and / or epitope(s) of a Plasmodium parasite.

[0008] The present disclosure further recognizes that an approach comprising the delivery of one or more Plasmodium polypeptides or antigenic portions thereof that are present and / or exposed at various time points in a Plasmodium life cycle may elicit a more robust immune response. In particular, it can be beneficial to deliver one or more Plasmodium polypeptides or antigenic portions thereof that will generate an immune response that targets Plasmodium parasites at an early life cycle stage, e.g., (1) in the asymptomatic infection stage from the deposition of the parasites in the skin until the infection of hepatocytes (see FIG.1, 1 – Sporozoite), and / or (2) after infection of a subject’s hepatocytes (see FIG.1, 2 – Liver Stage), and / or (3) after egress from hepatocytes but before infecting a subject’s erythrocytes (see FIG.1, 3 – Pre-invasion). Preventing the symptomatic phase of a Plasmodium parasite, e.g., killing the parasites prior to infecting erythrocytes, can help prevent disease and death of a subject, as well as mitigate onward transmission. When such an approach is applied to a large enough proportion of a target population(e.g., constituting an infectious reservoir), the approach may enable interruption of transmission and aid in malaria elimination.

[0009] Thus, the present disclosure provides, among other things, combinations comprising more than one Plasmodium polypeptide construct that each include one or more Plasmodium polypeptides or antigenic fragments thereof, wherein the more than one Plasmodium polypeptide constructs comprise Plasmodium polypeptides or antigenic fragments thereof that are expressed at different stages of the asymptomatic phase of infection, such as the initial sporozoite stage and the liver stage of infection. In some embodiments, a combination of two or more Plasmodium polypeptide constructs that include Plasmodium polypeptides or antigenic fragments thereof that are expressed at different stages within the asymptomatic phase (such as sporozoite and liver stage) may prevent erythrocyte infection by parasites and thereby prevent symptomatic malaria disease and disrupting onwards transmission. In some embodiments, Plasmodium polypeptide constructs described herein provide a combination that acts as a pre-erythrocytic vaccine. In some embodiments, targeting two or more stages of the parasitic infection (such as sporozoite and liver stage) can provide additional layers of immune protection for a subject, e.g., fewer Plasmodium sporozoites invading the liver, leading to fewer infected hepatocytes that need to be eliminated. In some embodiments, a combination as described herein includes two or more Plasmodium polypeptide constructs, wherein the Plasmodium polypeptide constructs comprise Plasmodium polypeptides or antigenic fragments thereof that are expressed at different stages within the asymptomatic phase (such as sporozoite and liver stage). In some embodiments, the two or more Plasmodium polypeptide constructs do not interfere with each other (e.g., the elicitation of an immune response by one Plasmodium polypeptide construct does not interfere with the elicitation of an immune response by another of the Plasmodium polypeptide constructs. For example, in some embodiments, the presence of two or more Plasmodium polypeptide constructs in a combination does not abolish an immunogenic effect of one or more antigens on a first Plasmodium polypeptide constructs that is otherwise present in the absence of a second Plasmodium polypeptide constructs. In some embodiments, the lack of interference by one Plasmodium polypeptide construct with another is observed regardless of whether there are three or more (e.g., three, four, five, six, etc.) present in a combination.

[0010] In some embodiments, a combination of two or more Plasmodium polypeptide constructs elicits a humoral and / or cellular immune response. In some embodiments, in a combination of two or more Plasmodium polypeptide constructs, each Plasmodium polypeptide construct elicits a humoral and / or cellular immune response. In particular, the present disclosure provides the insight that antigens or epitopes that are present on the surface of cells, particularly the Plasmodium cell surface, may be able to induce a B cell response, while antigens or epitopes that are not surface exposed or are minimally exposed may still be able to induce a T cell response. The resulting combined B cell and T cell response may be important for developing strong immune protection against current and future Plasmodium infections. As such, the present disclosure provides compositions for delivering antigens or epitopes that can be useful prophylactically or therapeutically.

[0011] The present disclosure also provides the insight that certain, multi-prong approaches can be useful for providing protection against Plasmodium infections (e.g., malaria). For example, a combination as described herein can comprise a first pharmaceutical composition comprising a first polyribonucleotide and a second pharmaceutical composition comprising a second polyribonucleotide. In some embodiments, a first polyribonucleotide encodes a first polypeptide that comprises one or more Plasmodium T-cell antigens. In some embodiments, a secondpolyribonucleotide encodes a second polypeptide that comprises one or more Plasmodium polypeptides or antigenic portions thereof.

[0012] In some embodiments, a first polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, and 221; and a second polyribonucleotide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 5, 8, 10, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105 , 107-112, 117, 122, 125, 130, 135, 138, and 141.

[0013] In some embodiments, a first polypeptide comprises (i) an antigenic Plasmodium CSP polypeptide fragment, (ii) an antigenic Plasmodium TRAP polypeptide fragment, (iii) an antigenic Plasmodium UIS3 polypeptide fragment, (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment, and (v) an antigenic Plasmodium LSAP2 polypeptide fragment. In some embodiments, a second polyribonucleotide comprises (i) a secretory signal, (ii) a Plasmodium CSP N-terminal end region, (iii) a Plasmodium CSP junction region, (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (v) a Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof, (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof, (vii) a linker, and (viii) a transmembrane region, and wherein the second polypeptide does not comprise any of (a) a Plasmodium CSP N-terminal region or portion thereof, and (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polyribonucleotide comprises (i) a secretory signal, (ii) a Plasmodium CSP N-terminal end region, (iii) a Plasmodium CSP junction region, (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (v) a Plasmodium CSP C-terminal region, (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, (vii) a linker, and (viii) a transmembrane region, and wherein the second polypeptide does not comprise any of (a) a Plasmodium CSP N-terminal region or portion thereof, and (b) an amino acid sequence of NPNA (SEQ ID NO: 228).

[0014] In some embodiments, a first polypeptide comprises (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment, (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment, (iii) an antigenic Plasmodium LISP-2 polypeptide fragment, and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a second polyribonucleotide comprises (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junction region, (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C- terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof, and a transmembrane region. In some embodiments, a first polypeptide comprises (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment, (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment, (iii) an antigenic Plasmodium LISP-2 polypeptide fragment, and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a second polyribonucleotide comprises (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junction region, (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C- terminal region, and a transmembrane region.

[0015] In some embodiments, a combination includes a first pharmaceutical composition and a second pharmaceutical composition. In some embodiments, a first pharmaceutical composition comprises a firstpolyribonucleotide. In some embodiments, a first polyribonucleotide encodes a first polypeptide. In some embodiments, a first polypeptide comprises one or more Plasmodium T-cell antigens. In some embodiments, a second pharmaceutical composition comprises a second polyribonucleotide. In some embodiments, a second polyribonucleotide encodes a second polypeptide. In some embodiments, a second polypeptide comprises one or more Plasmodium polypeptide or antigenic portions thereof. In some embodiments, a combination includes: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide encodes a first polypeptide, and a first polypeptide comprises one or more Plasmodium T-cell antigens and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium polypeptide or antigenic portions thereof.

[0016] In some embodiments, a first polypeptide comprises at least 10 amino acids and at most 1100 amino acids. In some embodiments, a first polypeptide comprises at least 10 amino acids and at most 500 amino acids. In some embodiments, one or more Plasmodium T-cell antigens comprised in a first polypeptide comprise at least 2 Plasmodium T-cell antigens. In some embodiments, one or more Plasmodium T-cell antigens comprised in a first polypeptide comprise at most 10 Plasmodium T-cell antigens. In some embodiments, one or more Plasmodium T-cell antigens comprised in a first polypeptide comprise at least 2 and at most 10 Plasmodium T-cell antigens.

[0017] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium CSP polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-1 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium TRAP polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSAP2 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium UIS3 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium ETRAMP10.3 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LISP-2 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-3 polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-1(a) polypeptide fragment. In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-1(b) polypeptide fragment.

[0018] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise two or more of: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium LSA-1 polypeptide fragment; (iii) an antigenic Plasmodium TRAP polypeptide fragment; (iv) an antigenic Plasmodium LSAP2 polypeptide fragment; (v) an antigenic Plasmodium UIS3 polypeptide fragment; (vi) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (vii) an antigenic Plasmodium LISP-1 polypeptide fragment; (viii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (ix) an antigenic Plasmodium LSA-3 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 179.

[0019] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LSA-3 polypeptide fragment; (vii) an antigenic Plasmodium LSA-1(a) polypeptide fragment; and (viii) an antigenic Plasmodium LSA-1(b) polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 182.

[0020] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LSA- 1(a) polypeptide fragment; (vii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (viii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (ix) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 188.

[0021] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LSA- 1(a) polypeptide fragment; (vii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; and (viii) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 191.

[0022] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LISP- 2 polypeptide fragment; and (vii) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 194.

[0023] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LSA- 1(b) polypeptide fragment; and (vii) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 197.

[0024] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; (v) an antigenic Plasmodium LSAP2 polypeptide fragment; (vi) an antigenic Plasmodium LSA- 1(a) polypeptide fragment; (vii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (viii) an antigenic Plasmodium LISP-2 polypeptide fragment; (ix) an antigenic Plasmodium LISP-1 polypeptide fragment; and (x) an antigenic Plasmodium LSA-3 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 200.

[0025] In some embodiments, one or more Plasmodium T cell antigens comprised in the first polypeptide comprise: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; (iv) an antigenic Plasmodium LISP- 1 polypeptide fragment; and (v) an antigenic Plasmodium LSA-3 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 212.

[0026] In some embodiments, one or more Plasmodium T cell antigens comprised in the first polypeptide comprise: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 209.

[0027] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium CSP polypeptide fragment, and wherein the antigenic Plasmodium CSP polypeptide fragment comprises a Plasmodium CSP N-terminal region. In some embodiments, an antigenic Plasmodium CSP polypeptide fragment further comprises a Plasmodium CSP N-terminal end region. In some embodiments, an antigenic Plasmodium CSP polypeptide fragment further comprises a Plasmodium CSP junction region. In some embodiments, an antigenic Plasmodium CSP polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 437.

[0028] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide do not comprise an antigenic Plasmodium berghei CSP polypeptide fragment.

[0029] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-1(a) polypeptide fragment, and wherein the antigenic Plasmodium LSA-1(a) polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 447.

[0030] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-1(b) polypeptide fragment, and wherein the antigenic Plasmodium LSA-1(b) polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 457.

[0031] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium TRAP polypeptide fragment, and wherein the antigenic Plasmodium TRAP polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 472.

[0032] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSAP2 polypeptide fragment, and wherein the antigenic Plasmodium LSAP2 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 497.

[0033] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium UIS3 polypeptide fragment, and wherein the antigenic Plasmodium UIS3 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 510.

[0034] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium ETRAMP10.3 polypeptide fragment, and wherein the antigenic Plasmodium ETRAMP10.3 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 516.

[0035] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LISP-1 polypeptide fragment, and wherein the antigenic Plasmodium LISP-1 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 252.

[0036] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LISP-2 polypeptide fragment, and wherein the antigenic Plasmodium LISP-2 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 533.

[0037] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide comprise an antigenic Plasmodium LSA-3 polypeptide fragment, and wherein the antigenic Plasmodium LSA-3 polypeptide fragment comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 543.

[0038] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide each comprise one or more T cell epitopes.

[0039] In some embodiments, a first polypeptide further comprises an MHC class I trafficking signal (MITD). In some embodiments, a MITD is located at the C-terminal end comprised in the first polypeptide. In some embodiments, a MITD comprises or consists of an amino acid sequence according to SEQ ID NO: 561.

[0040] In some embodiments, a first polypeptide comprises a secretory signal. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 332. In some embodiments, a secretory signal comprises or consists of a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV-1 or HSV-2 secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 314. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 320. In some embodiments, a viral secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal. In some embodiments, an Ebola virus SGP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 335. In some embodiments, a secretory signal is located at the N-terminus of the polypeptide. In some embodiments, a first polypeptide does not comprise a secretory signal.

[0041] In some embodiments, a polypeptide comprises one or more linkers. In some embodiments, one or more linkers comprise one or more glycine-serine linkers. In some embodiments, one or more linkers comprise at least one linker comprising an amino acid sequence according to SEQ ID NO: 404. In some embodiments, one or more linkers comprise at least one linker comprising an amino acid sequence according to SEQ ID NO: 411. In some embodiments, one or more linkers comprise at least one linker comprising an amino acid sequence according to SEQ ID NO: 408. In some embodiments, one or more linkers comprise at least one linker comprising an amino acid sequence according to SEQ ID NO: 412. In some embodiments, a polypeptide comprises a linker between two Plasmodium T-cell antigens.

[0042] In some embodiments, a polypeptide comprises a transmembrane region. In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 385. In some embodiments, a transmembrane region comprises or consists of aheterologous transmembrane region. In some embodiments, a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 379. In some embodiments, a transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, a hDAF-GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 382. In some embodiments, a polypeptide does not comprise a transmembrane region.

[0043] In some embodiments, a polypeptide does not comprise an antigenic fragment of a bacterial polypeptide. In some embodiments, a polypeptide does not comprise an antigenic bacillus Calmette-Guérin (BCG) polypeptide fragment, optionally wherein the antigenic BCG polypeptide fragment comprises an amino acid sequence according to SEQ ID NO: 416. In some embodiments, a polypeptide does not comprise an antigenic tetanus toxin (TT) polypeptide fragment, optionally wherein the antigenic TT polypeptide fragment comprises an amino acid sequence according to SEQ ID NO: 417.

[0044] In some embodiments, one or more Plasmodium T cell antigens comprised in a first polypeptide do not comprise an antigenic Plasmodium sporozoite threonine–asparagine-rich protein (STARP) polypeptide fragment, and optionally wherein the antigenic Plasmodium STARP polypeptide fragment comprises an amino acid sequence according to SEQ ID NO: 418.

[0045] In some embodiments, one or more Plasmodium polypeptide or antigenic portions thereof comprised in a second polypeptide are one or more Plasmodium CSP polypeptide regions or antigenic portions thereof. In some embodiments, each of the one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise 10 or more contiguous amino acids of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), and wherein the second polypeptide does not comprise the amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise five or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise between two and twelve repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In someembodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise between four and twelve repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223).

[0046] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly eight repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223).

[0047] In some embodiments, repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223) are all contiguous with each other. In some embodiments, repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223) are not all contiguous with each other.

[0048] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise three portions of a Plasmodium CSP polypeptide, wherein each portion comprises three contiguous repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), and wherein each of the portions are not contiguous with each other. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise four portions of a Plasmodium CSP polypeptide, and wherein each portion comprises two contiguous repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223).

[0049] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise two to eighteen repeats of the amino acid sequence of NANP. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise: (i) at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (ii) two to eighteen repeats of the amino acid sequence of NANP; and (iii) a Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise, in N-terminal to C-terminal order: (i) at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (ii) two to eighteen repeats of the amino acid sequence of NANP; and (iii) a Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof.

[0050] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP C-terminal regions or antigenic portions thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP C-terminal region variants, or antigenic portions thereof.

[0051] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly one Plasmodium CSP C-terminal region. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly one Plasmodium CSP C-terminal region, and wherein the Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 235.

[0052] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP C-terminal region. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region variant. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or more antigenic portions of a Plasmodium CSP C-terminal region.

[0053] In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 261, wherein X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 262, wherein X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 263, wherein X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 264, wherein X1X2 is EK or KE, X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 265, wherein X1X2 is EK or KE, X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 266, wherein X1X2 is EK or KE, X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 267. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region or a Plasmodium CSP C-terminal region variant comprises an amino acid sequence according SEQ ID NO: 992. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region or a Plasmodium CSP C-terminal region variant comprises an amino acid sequence according SEQ ID NO: 993.

[0054] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region variant or antigenic portion thereof. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, insertions, or deletions.

[0055] In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise S301N, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise K317E, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise E318Q, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C- terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise N321K, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise E357Q, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant orantigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise A361E, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise S301N K317E, E318Q, N321K, E357Q, A361E, or any combination thereof, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions comprise S301N, K317E, E318Q, N321K, E357Q, and A361E, wherein the amino acid numbering is relative to SEQ ID NO: 1.

[0056] In some embodiments, a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 994.

[0057] In some embodiments, a second polypeptide comprises one or more portions of the Plasmodium CSP C- terminal region, wherein each of the one or more portions comprise or consist of an amino acid sequence according to SEQ ID NO: 244. In some embodiments, a second polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprise or consist of an amino acid sequence according to SEQ ID NO: 248. In some embodiments, a second polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprise or consist of an amino acid sequence according to SEQ ID NO: 262. In some embodiments, a second polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprise or consist of an amino acid sequence according to SEQ ID NO: 256. In some embodiments, a second polypeptide comprises one or more portions of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprise or consist of: (i) an amino acid sequence according to SEQ ID NO: 244; (ii) an amino acid sequence according to SEQ ID NO: 248; (iii) an amino acid sequence according to SEQ ID NO: 262; (iv) an amino acid sequence according to SEQ ID NO: 256; or (v) a combination thereof.

[0058] In some embodiments, a second polypeptide comprises one portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of an amino acid sequence according to SEQ ID NO: 244. In some embodiments, a second polypeptide comprises one portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of an amino acid sequence according to SEQ ID NO: 248. In some embodiments, a second polypeptide comprises one portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of an amino acid sequence according to SEQ ID NO: 262. In some embodiments, a second polypeptide comprises one portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of an amino acid sequence according to SEQ ID NO: 256. In some embodiments, a second polypeptide comprises one portion of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of: (i) an amino acid sequence according to SEQ ID NO: 244; (ii) an amino acid sequence according to SEQ ID NO: 248; (iii) an amino acid sequence according to SEQ ID NO: 262; (iv) an amino acid sequence according to SEQ ID NO: 256; or (v) a combination thereof. In some embodiments, a second 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) an amino acid sequence according to SEQ ID NO: 244; (ii) an amino acid sequence according to SEQ ID NO: 248; (iii) an amino acid sequence according to SEQ ID NO: 262; and (iv) an amino acid sequence according to SEQ ID NO: 256.

[0059] In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 259.

[0060] In some embodiments, a second polypeptide comprises a serine immediately following a Plasmodium CSP C-terminal region. In some embodiments, a second polypeptide comprises a serine-valine sequence immediately following a Plasmodium CSP C-terminal region. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP junction regions, portions thereof, or variants thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or more Plasmodium CSP junction regions or portions thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise exactly one Plasmodium CSP junction region. In some embodiments, one Plasmodium CSP junction region consists of an amino acid sequence according to SEQ ID NO: 272.

[0061] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more portions of a Plasmodium CSP junction region.

[0062] In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of K93, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of L94, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of K95, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of Q96, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of P97, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96, P97, or a combination thereof, and wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, and Q96, and wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96 and P97, and wherein the amino acid numbering is relative to SEQ ID NO: 1.

[0063] In some embodiments, each portion of a Plasmodium CSP junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 275. In some embodiments, each portion of a Plasmodium CSP junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 277. In some embodiments, two or more Plasmodium CSP junction regions consist of an amino acid sequence according to SEQ ID NO: 272.

[0064] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or more portions of a Plasmodium CSP junction region. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of K93, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of L94, wherein the amino acid numbering is relative to SEQ ID NO: 1. In someembodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of K95, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of Q96, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of P97, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96, P97, or a combination thereof, and wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, and Q96, and wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, two or more portions of a Plasmodium CSP junction region comprise a deletion of K93, L94, K95, Q96 and P97, and wherein the amino acid numbering is relative to SEQ ID NO: 1.

[0065] In some embodiments, each portion of a Plasmodium CSP junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 275. In some embodiments, each portion of a Plasmodium CSP junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 277.

[0066] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP junction region variants. In some embodiments, a Plasmodium CSP junction region variant comprises one or more substitution mutations. In some embodiments, one or more substitution mutations comprise a K93A mutation, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more substitution mutations comprise a L94A mutation, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more substitution mutations comprise a K93A mutation, an L94A mutation, or both, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, each Plasmodium CSP junction region variant comprises the amino acid sequence of AAKQ.

[0067] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP N-terminal end regions or portions thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide 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 an amino acid sequence according to SEQ ID NO: 285.

[0068] In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N-terminal end region or any portion thereof.

[0069] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP N-terminal regions or portions thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or more Plasmodium CSP N-terminal regions or portions thereof.

[0070] In some embodiments, each Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 288.

[0071] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise an antigenic portion of a Plasmodium CSP N-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region is a Plasmodium CSP N-terminal startregion. In some embodiments, a Plasmodium CSP N-terminal start region comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 1010.

[0072] In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N-terminal region or any portion thereof.

[0073] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise one or more Plasmodium CSP major repeat regions or portions thereof. In some embodiments, one or more Plasmodium CSP major repeat regions or portions thereof comprise the amino acid sequence NANPNA or NPNANP. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise at least two Plasmodium CSP major repeat region portions. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise at least two Plasmodium CSP major repeat region portions, wherein each CSP major repeat region portion comprises at least 4 and at most 7 repeats of the sequence NANP. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or three Plasmodium CSP major repeat region portions, wherein each CSP major repeat region portion comprises 6 repeats of the sequence NANP (SEQ ID NO: 230). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise two or three Plasmodium CSP major repeat region portions, wherein each CSP major repeat region portion comprises 6 repeats of the sequence NANP (SEQ ID NO: 230). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise 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 2 and at most 35 repeats of the amino acid sequence NANP (SEQ ID NO: 230).

[0074] In some embodiments, a Plasmodium CSP major repeat region or portion thereof comprises two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 230), and wherein the two contiguous stretches of repeats of the amino acid sequence NANP (SEQ ID NO: 230) flank an amino acid sequence of NVDP (SEQ ID NO:229). In some embodiments, a Plasmodium CSP major repeat region comprises, in N-terminus to C-terminus order, 17 repeats of the amino acid sequence NANP (SEQ ID NO: 230), an amino acid sequence of NVDP (SEQ ID NO:229), and 18 repeats of the amino acid sequence NANP (SEQ ID NO: 230).

[0075] In some embodiments, a portion of a Plasmodium CSP major repeat region consists of at most 18 contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230). In some embodiments, a portion of the Plasmodium CSP major repeat region consists of 2 contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230).

[0076] In some embodiments, a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 305.

[0077] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprises an antigenic portion of a Plasmodium CSP major repeat region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises at least six repeats of the amino acid sequence of NANP.In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following of the six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 303.

[0078] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 308.

[0079] In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N-terminal region or any portion thereof. In some embodiments, a second polypeptide does not comprise a Plasmodium CSP C-terminal region or any portion thereof. In some embodiments, a second polypeptide does not comprise (i) a Plasmodium CSP N-terminal region or any portion thereof and / or (ii) a Plasmodium CSP C-terminal region or any portion thereof.

[0080] In some embodiments, a second polypeptide does not comprise a Plasmodium CSP major repeat region or a portion of a Plasmodium CSP major repeat region comprising the amino acid sequence NPNA (SEQ ID NO: 228).

[0081] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof, if present in a second polypeptide, are in the following N-terminus to C-terminus order: (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: 223); (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.

[0082] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof, if present in the second polypeptide, are in the following N-terminus to C-terminus order: (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: 223); (v) one Plasmodium CSP major repeat region or portion thereof; and (vi) one Plasmodium CSP C-terminal region or portion thereof.

[0083] In some embodiments, a second polypeptide comprises one or more helper antigens. In some embodiments, one or more helper antigens comprise a Plasmodium antigen. In some embodiments, one or more helper antigens are Plasmodium 2-phospho-D-glycerate hydro-lyase 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 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.

[0084] In some embodiments, one or more helper antigens comprise or consist of a P. falciparum 2-phospho- D-glycerate hydro-lyase antigen. In some embodiments, a P. falciparum 2-phospho-D-glycerate hydro-lyase antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 388.

[0085] In some embodiments, one or more helper antigens comprise or consist of a P. falciparum liver-stage antigen 3. In some embodiments, a P. falciparum liver-stage antigen 3 comprises or consists of an amino acid sequence according to SEQ ID NO: 391.

[0086] In some embodiments, one or more helper antigens comprise an Anopheles antigen. In some embodiments, a helper antigen comprises or consists of an Anopheles gambiae TRIO. In some embodiments, an Anopheles gambiae TRIO comprises or consists of an amino acid sequence according to SEQ ID NO: 393.

[0087] In some embodiments, a second polypeptide comprises a secretory signal and the helper antigen immediately follows the secretory signal. In some embodiments, a second polypeptide comprises a helper antigen at the C-terminus of the second polypeptide.

[0088] In some embodiments, a second polypeptide comprises a multimerization region. In some embodiments, a multimerization region comprises or consists of a trimerization region. In some embodiments, a trimerization region comprises or consists of a fibritin region. In some embodiments, a fibritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 399. In some embodiments, a second polypeptide comprises a multimerization region at the N-terminus of the second polypeptide.

[0089] In some embodiments, a second polypeptide comprises a self-aggregation region. In some embodiments, a self-aggregation region comprises or consists of a ferritin region. In some embodiments, a ferritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 402. In some embodiments, a polypeptide comprises a self-aggregation region at the N-terminus of the polypeptide.

[0090] In some embodiments, a second polypeptide comprises a secretory signal. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 332. In some embodiments, a secretory signal comprises or consists of a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV-1 or HSV-2 secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 314. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 320. In some embodiments, a viral secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, a Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal. In some embodiments, a Ebola virus SGP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 335. In some embodiments, a secretory signal is located at the N-terminus of a second polypeptide. In some embodiments, a second polypeptide does not comprise a secretory signal.

[0091] In some embodiments, a second polypeptide comprises a transmembrane region. In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In someembodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 385. In some embodiments, a transmembrane region comprises or consists of a heterologous transmembrane region. In some embodiments, a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a viral transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 379. In some embodiments, a heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, a hDAF-GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 382. In some embodiments, a second polypeptide does not comprise a transmembrane region.

[0092] In some embodiments, a second polypeptide comprises one or more linkers. In some embodiments, one or more linkers comprise one or more glycine-serine linkers. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 404. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 411. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 408. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 412.

[0093] In some embodiments, a second polypeptide comprises a linker between the C-terminal region or portion thereof and the transmembrane region. In some embodiments, a second polypeptide comprises a linker after an amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, a second polypeptide comprises a secretory signal. In some embodiments, a second polypeptide comprises one or more Plasmodium CSP junction regions, portions thereof, or variants thereof. In some embodiments, a second polypeptide comprises one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, a second polypeptide comprises one or more Plasmodium CSP C-terminal regions or portions thereof. In some embodiments, a second polypeptide comprises a transmembrane region. In some embodiments, a second polypeptide does not comprise an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N-terminal region or portion thereof. In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) one or more Plasmodium CSP C- terminal regions or portions thereof; and (v) a transmembrane region, and a second polypeptide does not comprise: (a) an amino acid sequence of NPNA (SEQ ID NO: 228); and (b) a Plasmodium CSP N-terminal region or portion thereof. In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N-terminal end region. In some embodiments, a second polypeptide comprises one or more Plasmodium CSP N-terminal end regions or portions thereof.

[0094] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); and (iv) one or more Plasmodium CSP C-terminal regions or portions thereof.

[0095] In some embodiments, a second polypeptide comprises a secretory signal. In some embodiments, a second polypeptide comprises three or more Plasmodium CSP junction regions, portions thereof, or variants thereof. In some embodiments, a second polypeptide comprises three or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223). In some embodiments, a second polypeptide comprises two or more Plasmodium CSP major repeat region portions. In some embodiments, a second polypeptide does not comprise a Plasmodium CSP N- terminal region or portion thereof. In some embodiments, a second polypeptide does not comprise a Plasmodium CSP C-terminal region or portion thereof. In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) three or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) three or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); and (iv) two or more Plasmodium CSP major repeat region portions, and a second polypeptide does not comprise: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) a Plasmodium CSP C-terminal region or portion thereof. In some embodiments, a second polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence of SEQ ID NO: 108.

[0096] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) one or more Plasmodium CSP C-terminal regions or portions thereof; and (v) a transmembrane region.

[0097] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) one or more Plasmodium CSP C-terminal regions or portions thereof; and (v) a transmembrane region. In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) three or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) three or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) two or more Plasmodium CSP major repeat region portions; and (v) a transmembrane region, and a second polypeptide does not comprise: (a) a Plasmodium CSP N- terminal region or portion thereof; and (b) a Plasmodium CSP C-terminal region or portion thereof. In some embodiments, a second polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence of SEQ ID NO: 107 or 109.

[0098] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) three or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) three or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); and (iv) two or more Plasmodium CSP major repeat region portions, wherein a second polypeptide does not comprise: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) a Plasmodium CSP C-terminal region or portion thereof. In some embodiments, a second polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence of SEQ ID NO: 110 or 111.

[0099] In some embodiments, a second polypeptide comprises one or more helper antigens.

[0100] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; and (vi) five antigenic repeat regions, wherein each antigenic repeat region comprises: (A) a linker; and (B) a helper antigen, and a second polypeptide does not comprise any of: (a) an amino acid sequence of NPNA (SEQ ID NO: 228); (b) a Plasmodium CSP N-terminal region or portion thereof; and (c) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 36.

[0101] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a helper antigen; (iii) a linker; (iv) a Plasmodium CSP N-terminal end region; (v) a Plasmodium CSP junction region; (vi) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vii) a Plasmodium CSP C-terminal region; (viii) a serine- valine sequence immediately following the Plasmodium CSP C-terminal region; (ix) a linker; and (x) a transmembrane region, and a second polypeptide does not comprise any of: (a) an amino acid sequence of NPNA (SEQ ID NO: 228); and (b) a Plasmodium CSP N-terminal region or portion thereof. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 39.

[0102] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a portion of a Plasmodium CSP junction region; (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 57.

[0103] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a portion of a Plasmodium CSP junction region; (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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; (c) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 60.

[0104] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junction region; (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 63.

[0105] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junction region; (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 66.

[0106] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junction region variant; (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 69.

[0107] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junction region variant; (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 72.

[0108] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a portion of a Plasmodium CSP junction region; (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 75.

[0109] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a portion of a Plasmodium CSP junction region; (iii) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vi) a linker; and (vii) a transmembrane region, and a second 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) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 78.

[0110] In some embodiments, second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 81.

[0111] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal end region; (iii) a Plasmodium CSP junction region variant; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 84.

[0112] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; and (vi) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 102.

[0113] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; and (vi) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 105.

[0114] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) two or more Plasmodium CSP neutralizing region repeats, wherein each Plasmodium CSP neutralizing region repeat comprises or consists of: (a) a Plasmodium CSP N-terminal end region; (b) a Plasmodium CSP junction region; (c) two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); and (d) a linker; (iii) a portion of a Plasmodium CSP major repeat region; (iv) a Plasmodium CSP C-terminal region; (v) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vi) a linker; and (vii) a transmembrane region, and a second polypeptide does not comprise a Plasmodium CSP N-terminal region or portion thereof. In some embodiments, a second polypeptide comprises exactly four Plasmodium CSP neutralizing region repeats. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 87.

[0115] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) one Plasmodium CSP junction region; (iii) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (iv) a Plasmodium CSP major repeat region; (v) one Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) a Plasmodium CSP N-terminal end region or portion thereof. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 30.

[0116] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a portion of a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a serine immediately following the Plasmodium CSP C- terminal region, and a second polypeptide does not comprise a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 27.

[0117] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a serine immediately following the Plasmodium CSP C-terminalregion, and a second polypeptide does not comprise a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 8.

[0118] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a serine immediately following the Plasmodium CSP C-terminal region, and a second polypeptide does not comprise a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 24.

[0119] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, and a second polypeptide does not comprise a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 99.

[0120] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 33.

[0121] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (ix) a linker; and (x) a multimerization region, and a second polypeptide does not comprise a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 42.

[0122] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; (viii) a serine immediately following the Plasmodium CSP C-terminalregion; (ix) a linker; and (x) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 48.

[0123] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (ix) a linker; and (x) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 90.

[0124] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; (viii) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (ix) a linker; and (x) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 90.

[0125] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; (viii) a serine immediately following the Plasmodium CSP C-terminal region; and (ix) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 21.

[0126] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) an antigenic portion of a Plasmodium CSP major repeat region; (iii) a Plasmodium CSP C-terminal region; and (iv) a serine-valine immediately following the Plasmodium CSP C-terminal region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 51.

[0127] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) an antigenic portion of a Plasmodium CSP major repeat region; (iii) a Plasmodium CSP C-terminal region; (iv) a serine immediately following the Plasmodium CSP C-terminal region; (v) a linker; and (vi) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 54.

[0128] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise in N-terminus to C-terminus order: (i) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); (ii) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230); (iii) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); (iv) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230); and (v) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223). In some embodiments, a second polypeptide further comprises: (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 Plasmodium CSP C-terminal regions or portions thereof; or (v) a combination thereof.

[0129] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in a second polypeptide comprise in N-terminus to C-terminus order: (i) a Plasmodium CSP N-terminal end region or portion thereof; (ii) a Plasmodium CSP R1 region or portion thereof; (iii) a Plasmodium CSP junction region or portion thereof; (iv) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); (v) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230); (vi) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); (vii) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230); (viii) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); and (ix) a Plasmodium CSP C-terminal region or portion thereof.

[0130] In some embodiments, a second polypeptide further comprises a Plasmodium CSP GPI domain. In some embodiments, a Plasmodium CSP C-terminal region or portion thereof comprised in the second polypeptide comprises a substitution at a fucosylation site.

[0131] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises one or more Plasmodium CSP junction regions, portions thereof, or variants thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N- terminus to C-terminus order three repeating domains, wherein each repeating domain comprises: (i) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (ii) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); and (iii) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230).

[0132] In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises one or more Plasmodium R1 regions or portions thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises one or more Plasmodium CSP junction regions, portions thereof, or variants thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230). In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof comprised in the second polypeptide comprise in N-terminus to C-terminus order three repeating domains, wherein each repeating domain comprises: (i) one or more Plasmodium R1 regions or portions thereof; (ii) one or more Plasmodium CSP junction regions, portions thereof, or variants thereof; (iii) three contiguous repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223); and (iv) six contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 230).

[0133] In some embodiments, a combination further comprises one or more linker sequences. In some embodiments, a linker is a glycine-serine linker. In some embodiments, a second polypeptide comprises a linker sequence following each six contiguous repeats of the amino acid sequence NANP.

[0134] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junctional region; (v) a Plasmodium CSP minor repeat region; (vi) an antigenic portion of a Plasmodium CSP major repeat region; and (vii) a Plasmodium CSP C-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 117. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises of six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 122.

[0135] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junctional region; (v) a Plasmodium CSP minor repeat region; (vi) an antigenic portion of a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 112.

[0136] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a linker; (v) a Plasmodium CSP junctional region; (vi) a Plasmodium CSP minor repeat region; (vii) an antigenic portion of a Plasmodium CSP major repeat region; (viii) a Plasmodium CSP C-terminal region; (ix) a linker; and (x) a transmembrane region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 125.

[0137] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junctional region; (v) a Plasmodium CSP minor repeat region; (vi) a Plasmodium CSP major repeat region; (vii) a linker; and (viii) a transmembrane region. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 130.

[0138] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junctional region; (iii) a Plasmodium CSP minor repeat region; (iv) an antigenic portion of a Plasmodium CSP major repeat region; (v) a linker; (vi) an antigenic portion of a Plasmodium CSP C-terminal region; (vii) a serine-valine; (viii) a linker; and (ix) a multimerization region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises of six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 259. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 135.

[0139] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junctional region; (iii) a Plasmodium CSP minor repeat region; (iv) an antigenic portion of a Plasmodium CSP major repeat region; (v) a linker; (vi) an antigenic portion of a Plasmodium CSP C-terminal region; (vii) a serine-valine; (viii) a linker; and (ix) a self-aggregation region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises of six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine- alanine positioned immediately following the six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 259. In some embodiments, a second polypeptidecomprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 138.

[0140] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP junctional region; (iii) a Plasmodium CSP minor repeat region; (iv) an antigenic portion of a Plasmodium CSP major repeat region; (v) a linker; (vi) an antigenic portion of a Plasmodium CSP C-terminal region; (vii) a serine-valine; (viii) a linker; and (ix) a transmembrane region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises of six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine- alanine positioned immediately following the six repeats of the amino acid sequence of NANP (SEQ ID NO: 230). In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 259. In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 141.

[0141] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) a Plasmodium CSP N- terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junctional region; (v) a Plasmodium CSP minor repeat region; (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region variant; and (viii) a transmembrane region. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 332. In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 385. In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 989. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 989. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 991. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 991.

[0142] In some embodiments, a second polypeptide comprises: (i) a secretory signal; (ii) an antigenic portion of a Plasmodium CSP N-terminal region; (iii) a first linker; (iv) a Plasmodium CSP N-terminal end region; (v) a Plasmodium CSP junctional region; (vi) a Plasmodium CSP minor repeat region; (vii) an antigenic portion of a Plasmodium CSP major repeat region; (viii) a Plasmodium CSP C-terminal region; (ix) a serine-valine sequence; (x) a second linker; and (xi) a transmembrane region. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists ofan amino acid sequence according to SEQ ID NO: 332. In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 379. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of according to SEQ ID NO: 1010. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP. In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 997. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 997. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 999. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 999.

[0143] In some embodiments, second polypeptide comprises: (i) a secretory signal; (ii) an antigenic portion of a Plasmodium CSP N-terminal region; (iii) a first linker; (iv) a Plasmodium CSP N-terminal end region; (v) a Plasmodium CSP junctional region; (vi) a Plasmodium CSP minor repeat region; (vii) an antigenic portion of a Plasmodium CSP major repeat region; (viii) a second linker; (ix) an antigenic portion of a Plasmodium CSP C-terminal region; (x) a serine-valine sequence; (xi) a third linker; and (xii) a transmembrane region. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 332. In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 379. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of according to SEQ ID NO: 1010. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 259. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP. In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 1000. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 1000. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1002. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 1002. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region furthercomprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP. In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 1003. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 1003. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1005. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 1005. In some embodiments, when present, the features are in a second polypeptide in numerical order from the C-terminus to the N-terminus.

[0144] In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment. In some embodiments, a combination comprises a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal region; (iii) a Plasmodium CSP N- terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region. In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment, and a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region.

[0145] In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 203.

[0146] In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 33.

[0147] In some embodiments, a combination further comprises a third pharmaceutical composition comprising a third polyribonucleotide, wherein the third polyribonucleotide encodes a third polypeptide, and the third polypeptide comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment.

[0148] In some embodiments, a third polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 209.

[0149] In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment. In some embodiments, a combination comprises a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C- terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium CSP polypeptide fragment; (ii) an antigenic Plasmodium TRAP polypeptide fragment; (iii) an antigenic Plasmodium UIS3 polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment, and a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228).

[0150] In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 203.

[0151] In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 81.

[0152] In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a combination comprises a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region. In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment, and a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal region; (iii) a Plasmodium CSP N-terminal end region; (iv) a Plasmodium CSP junction region; (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (vi) a Plasmodium CSP major repeat region; (vii) a Plasmodium CSP C-terminal region; and (viii) a transmembrane region.

[0153] In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 209.

[0154] In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 33.

[0155] In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment. In some embodiments, a combination comprises a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228). In some embodiments, a combination comprises a first polypeptide that comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment; (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment; (iii) an antigenic Plasmodium LISP-2 polypeptide fragment; and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment, and a second polypeptide that comprises: (i) a secretory signal; (ii) a Plasmodium CSP N-terminal end region; (iii) a Plasmodium CSP junction region; (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223); (v) a Plasmodium CSP C-terminal region; (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region; (vii) a linker; and (viii) a transmembrane region, and a second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof; and (b) an amino acid sequence of NPNA (SEQ ID NO: 228).

[0156] In some embodiments, a first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 209.

[0157] In some embodiments, a second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 81.

[0158] In some embodiments, Plasmodium is Plasmodium falciparum. In some embodiments, one or more Plasmodium CSP polypeptide regions or portions thereof are one or more P. falciparum CSP polypeptide regions or portions thereof.

[0159] In some embodiments, one or more Plasmodium T-cell antigens are one or more P. falciparum T-cell antigens. In some embodiments, Plasmodium falciparum is Plasmodium falciparum isolate 3D7.

[0160] In some embodiments, a first polyribonucleotide and / or second polyribonucleotide is an isolated polyribonucleotide. In some embodiments, a third polyribonucleotide is an isolated polyribonucleotide. In someembodiments, a first polyribonucleotide and / or second polyribonucleotide is an engineered polyribonucleotide. In some embodiments, a third polyribonucleotide is an engineered polyribonucleotide.

[0161] In some embodiments, a first polyribonucleotide and / or second polyribonucleotide is a codon-optimized polyribonucleotide. In some embodiments, a third polyribonucleotide is a codon-optimized polyribonucleotide.

[0162] The present disclosure further provides RNA constructs. In some embodiments, a first polyribonucleotide is comprised in a first RNA construct, wherein the first RNA construct comprises in 5' to 3' order: (i) a 5' UTR; (ii) the first polyribonucleotide; (iii) a 3' UTR; and (iv) a polyA tail sequence. In some embodiments, second polyribonucleotide is comprised in a second RNA construct, wherein the second RNA construct comprises in 5' to 3' order: (i) a 5' UTR; (ii) the second polyribonucleotide; (iii) a 3' UTR; and (iv) a polyA tail sequence.

[0163] In some embodiments, a 5' UTR of a first and / or second RNA construct comprises or consists of a modified human alpha-globin 5'-UTR. In some embodiments, a 5' UTR of a first and / or second RNA construct consists of a ribonucleic acid sequence according to SEQ ID NO: 565.

[0164] In some embodiments, a 3' UTR of a first and / or second RNA construct comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA. In some embodiments, a 3' UTR of a first and / or second RNA construct consists of a ribonucleic acid sequence according to SEQ ID NO: 567.

[0165] In some embodiments, a 5' UTR of a first and / or second RNA construct comprises or consists of a modified human alpha-globin 5'-UTR and a 3' UTR of a first and / or second RNA construct comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0166] In some embodiments, a polyA tail sequence of a first and / or second RNA construct is a split polyA tail sequence. In some embodiments, a split polyA tail sequence consists of a ribonucleic acid sequence according to SEQ ID NO: 569.

[0167] In some embodiments, a first and / or second RNA construct further comprise a 5' cap. In some embodiments, a first and / or second RNA construct comprise a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide. In some embodiments, a 5' cap comprises or consists of m7(3’OMeG)(5')ppp(5')(2'OMeA1)pG2, wherein A1 is position +1 of the polyribonucleotide, and G2 is position +2 of the polyribonucleotide. In some embodiments, a cap proximal sequence comprises A1and G2of the Cap1 structure, and a sequence comprising: A3A4U5(SEQ ID NO: 571) at positions +3, +4 and +5 respectively of the polyribonucleotide.

[0168] In some embodiments, a first and / or second RNA construct includes modified uridines in place of all uridines. In some embodiments, modified uridines are each N1-methyl-pseudouridine.

[0169] The present disclosure further provides pharmaceutical compositions, including pharmaceutical compositions for use in combinations described herein. In some embodiments, a first and / or second pharmaceutical composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes. In some embodiments, a first and / or second polyribonucleotide is fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0170] In some embodiments, a first and / or second pharmaceutical composition further comprises lipid nanoparticles. In some embodiments, a first polyribonucleotide is encapsulated within the lipid nanoparticles. In some embodiments, a second polyribonucleotide is encapsulated within the lipid nanoparticles.

[0171] In some embodiments, a first and / or second pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

[0172] In some embodiments, a combination is for use in the treatment of a malaria infection. In some embodiments, a combination is for use in the prevention of a malaria infection.

[0173] The present disclosure also provides methods. methods. In some embodiments, a method comprises administering a combination described herein to a subject. In some embodiments, a method described herein is a method of treating a malaria infection. In some embodiments, a method described herein is a method of preventing a malaria infection.

[0174] In some embodiments, a subject has or is at risk of developing a malaria infection. In some embodiments, a subject is a human.

[0175] In some embodiments, administration induces an anti-malaria immune response in a subject.

[0176] Provided herein are also uses. In some embodiments, use of a combination as described herein can be for treatment of a malaria infection. In some embodiments, use of a combination as described herein can be for prevention of a malaria injection. BRIEF DESCRIPTION OF THE DRAWING

[0177] FIG.1 presents an overview of the Plasmodium life cycle.

[0178] FIGS.2A-2B show antigenic fragments of malarial proteins. Specifically, FIG.2A depicts epitopes observed from MAS1 and MAS2 strings mapped onto MAS3a and MAS4f. FIG.2B shows antigenic fragments of malarial protein LSA-3.

[0179] FIG.3 presents a schematic representation of exemplary malarial T cell peptide string constructs containing antigens, as described herein.

[0180] FIG.4 depicts transfection of a combination including MAS3a and MAS4f into cells to generate detectable protein product. Relative protein expression 24 h after co-transfecting 2.5 μg each drug product into HEK293T cell lines are shown.

[0181] FIGS.5A-5F depict activation of T-cells, as assessed by secretion of IFN-γ. FIGS.5A-5D show an assessment of IFN-γ secretion using isolated splenocytes (from mice immunized with different T cell peptide string constructs) incubated with construct specific antigen peptide pools (15mers, 11aa overlap across antigen). FIG.5E depicts a comparison of isolated splenocytes (from mice in group 2 and 3, and splenocytes isolated from mice in group 4) response to specific antigen peptide pools. FIG.5F depicts a comparison of isolated splenocytes (from mice in group 2 and splenocytes isolated from mice in group 1) response to specific antigen peptide pools.

[0182] FIGS.6A-6H depict activation of T-cells, as assessed by secretion of IFN-γ. FIGS.6A-6H show an assessment of IFN-γ secretion using isolated splenocytes (from mice immunized with different T cell peptide string constructs) incubated with construct specific antigen peptide pools (15mers, 11aa overlap across antigen).

[0183] FIGS.7A-7B depict assessment of activation of T-cells, as assessed by secretion of IFN-γ using isolated splenocytes (from mice immunized with T cell peptide string constructs individually or with T cell strings constructs in combination).

[0184] FIGS.8A-8B depict assessment of activation of T-cells, as assessed by secretion of IFN-γ using isolated splenocytes (from mice immunized with shorter T cell peptide string constructs or a longer T cell peptide string with the same antigenic content).

[0185] FIGS.9A-9B depict in vitro expression of non-formulated RNA constructs encoding different malarial polypeptide constructs in HEK293T cells. FIG.9A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.9B shows total expression as measured by median fluorescence intensity of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non-permeabilized cells show only surface expressed protein (grey bar, surface staining). Each sample was stained in triplicate, bar is a representation of mean with SD; NT, non-transfected.

[0186] FIGS.10A-10C depict in vitro expression of formulated RNA constructs in HEK293T cells. FIG.10A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.10B shows total expression as measured by median fluorescence intensity of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non-permeabilized cells show only surface expressed protein (grey bar, surface staining). Each sample was stained in triplicate, bar is a representation of mean with SD. FIG.10C shows amount of protein detected in culture supernatant where each data point represents a triplicate repeat; NT, non-transfected.

[0187] FIGS.11A-11B depict immunogenicity induced in mice by formulated RNA constructs. FIG.11A shows antibodies to Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG.11B shows antibodies to PfCSP C-terminal domain (“PfCSP-C term”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0188] FIGS.12A-12B depict binding of antibodies generated from mice immunized with formulated RNA constructs to various epitopes. FIG.12A shows a visual summary of the data in form of a heatmap. FIG.12B shows bars that are representative of the area under the curve (AUC) created when plotting dilution steps versus ECL signal.

[0189] FIGS.13A-13B depict binding specificity of antibodies generated from mice immunized with formulated RNA constructs to CSP protein in Plasmodium falciparum sporozoite lysates. FIG.13A shows binding between antibodies and CSP protein in the sporozoite (spz) lysates, represented as area under the curve (AUC), as assessed by luminescence. FIG.13B shows binding of murine anti-Pfs25 mAb32F81 used as negative control and murine anti-CSP mAb3SP2 used as a positive control.

[0190] FIGS.14A-14B depict assessment of antibodies generated from mice immunized with formulated RNA constructs for ability to inhibit P. falciparum sporozoite traversal. FIG.14A shows results as the dilution at which the % of traversal is reduced by 50% (mean with SEM). FIG.14B shows the percentage of traversed cells for the negative control (serum from vehicle mice) and the percentage of inhibition of traversal for the positive control (mAb317, an antibody that binds to NANP (SEQ ID NO: 230) repeats of the major repeat region and is known to inhibit traversal), with 002, 003, 005, 012, 014, and 018 indicating different experimental runs.

[0191] FIGS.15A-15B depict assessment of antibodies generated from mice immunized with formulated RNA constructs for ability to inhibit P. falciparum sporozoite infection of primary human hepatocytes. FIG.15A shows results as percentage of inhibition of infection activity (mean with SEM) in comparison to the average of the vehicle control, which was set as 0% inhibition for three different dilutions of the immune serum. FIG.15B shows the percentage of infected cells from negative control (serum from vehicle mice) and the percentage of inhibition of the positive control (mAb317, an antibody known to inhibit hepatocyte infection).

[0192] FIG.16 depicts activation of T-cells, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0193] FIG.17 depicts activation of T-cells, as assessed by secretion of TNF-α. TNF-α secretion was assessed using isolated splenocytes (from mice immunized formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0194] FIG.18 depicts activation of T-cells, as assessed by secretion of IL-2. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0195] FIG.19 depicts activation of T-cells, as assessed by secretion of IL-2 and IFN-γ. IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0196] FIG.20 depicts activation of T-cells, as assessed by secretion of TNF-α and IFN-γ. TNF-α and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0197] FIG.21 depicts activation of T-cells, as assessed by secretion of TNF-α and IL-2. TNF-α and IL-2 secretion was assessed using splenocytes (isolated from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control wasmeasured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0198] FIG.22 depicts activation of T-cells, as assessed by secretion of TNF-α, IL-2 and IFN-γ. TNF-α, IL-2 and IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (PfCSP_FL_pep), peptides of epitopes predicted to be presented on MHC-I, on MHC-II, or controls (e.g., negative control: gp70-AH1 (SPSYVYHQF [SEQ ID NO: 595]), 4 μg / mL; positive control: concanavalin A, 2 μg / mL). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes; ve, vehicle.

[0199] FIGS.23A-23C depict protection of mice immunized with formulated RNA constructs against a challenge with PfCSP-expressing P. berghei sporozoites as well as immunogenicity induced by this immunization. FIG.23A depicts percentage of protected mice up to 11 days after challenge with PfCSP-expressing P. berghei sporozoites, for mice immunized with formulated RNA constructs, vehicle, or positive control. Mice that received 100 μg of the 2A10 monoclonal antibody 24 h before the challenge were used as positive control (2A10, a monoclonal antibody that targets the major repeats). FIG.23B and FIG.23C depict endpoint titers against full length PfCSP two weeks after the boost (day 35, FIG.23B) and one day before the challenge (day 49, FIG.23C) for mice immunized with formulated RNA constructs and mice injected with the vehicle only. Mean ± SEM and individual animal values are shown.

[0200] FIGS.24A-24B depict protection of mice immunized with formulated RNA constructs against a challenge with PfCSP-expressing P. berghei sporozoites as well as immunogenicity induced by this immunization from three separate experiments. FIG.24A depicts percentage of protected mice up to 11 days after challenge with PfCSP-expressing P. berghei sporozoites, for mice immunized with formulated RNA constructs, vehicle (saline), or positive control. Mice that received 100 μg of the 2A10 monoclonal antibody 24 h before the challenge were used as positive control in Experiment 1 and mice immunized with Mosquirix were used as positive control in Experiment 2 and 3. FIG.24B depicts endpoint titers against full length PfCSP two weeks after the boost (day 35) and one day before the challenge (day 49) for mice immunized with formulated RNA constructs and mice injected with the vehicle only. Mean ± SEM and individual animal values are shown. Mice immunized twice IM with 5 μg of Mosquirix were used as positive control in Experiments 2 and 3. Mice injected with the vehicle were used as negative controls in all experiments.

[0201] FIGS.25A-25J depict assessment of antibodies generated from mice immunized with a formulated RNA construct for ability to recognize native PfCSP on sporozoites and inhibit sporozoite viability and motility. FIG. 25A shows log of anti-sporozoite endpoint titers using fixed PfCSP-expressing P. berghei sporozoites. Symbols represent the mean ± SEM using serum from individual mice. FIG.25B shows inhibition of sporozoite gliding speed. Circles represent the mean ± SEM of duplicates of pooled serum samples from each group. FIG.25C shows estimated length of the 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 serum from individual mice. FIG.25D shows cytotoxicity of serum samples from immunized mice against sporozoites in suspension (PBS). Symbols represent the mean ± SEM using serum from individual mice. FIG.25E shows cytotoxicity in 3D (Matrigel). Symbols represent the mean ± SEM using serum from individual mice. FIG.25F depicts 3 experiments and shows log of anti-sporozoite endpoint titers using fixed PfCSP-expressing P. bergheisporozoites. Bars represent the mean ± SEM using serum from individual mice. 2A10, positive antibody control; Mos, Mosquirix® positive control; IFA, Immunofluorescence assay. FIG.25G depicts assessment of antibodies generated from mice immunized with formulated RNA of 5 priority constructs for ability to inhibit sporozoite gliding motility. Each graph represents a different experiment and shows sporozoite gliding speed (μm / s). Bars represent the mean ± SEM of duplicates (in Experiment 1) or single replicates (Experiments 2 and 3) of pooled serum samples from each group. 2A10, positive antibody control; Mos, Mosquirix® positive control; Veh, vehicle. FIG.25H depicts assessment of antibodies generated from mice immunized with formulated RNA for 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% immune sera as measured by flow cytometry (Forward Scatter Width (FSC-W)). Symbols represent the mean ± SEM using serum from individual mice. 2A10, positive antibody control; Mos, Mosquirix® positive control; Veh, vehicle. FIG.25I depicts the cytotoxicity of 17% immune sera measured against PfCSP-expressing P. berghei sporozoites in suspension and is presented as percentage of viable sporozoites. FIG.25J depicts cytotoxicity of 17% immune sera against PfCSP-expressing P. berghei sporozoites measured in a 3D Matrigel and normalized to vehicle group (viability = 100%). For FIGS.25F- 25J, 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, positive antibody control; Mosquirix® positive control; Veh, vehicle; PBS, phosphate buffer saline.

[0202] FIGS.26A- 26B depict in vitro expression of non-formulated RNA constructs encoding different malarial peptide constructs in HEK293T cells. FIG.26A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.26B shows total expression as measured by median fluorescence intensity of the total HEK293T population for both transfected and non-transfected cells.

[0203] FIGS.27A-27B depict in vitro expression of formulated RNA constructs encoding different malarial peptide constructs in HEK293T cells. FIG.27A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.27B shows total expression as measured by median fluorescence intensity of the total HEK293T population for both transfected and non-transfected cells.

[0204] FIGS.28A-28B depict immunogenicity induced in mice by formulated RNA constructs at day 21 after immunization. FIG.28A shows antibodies to an exemplary Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG.28B shows antibodies to an exemplary PfCSP C-terminal domain (“PfCSP-C term (3D7)”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0205] FIGS.29A-29B depict immunogenicity induced in mice by formulated RNA constructs at day 35 after immunization. FIG.29A shows antibodies to an exemplary Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG.29B shows antibodies to an exemplary PfCSP C-terminal domain (“PfCSP-C term (3D7)”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0206] FIGS.30A-30K depict binding of antibodies generated from mice immunized with formulated RNA constructs to various epitopes. FIG.30A shows a visual summary of the data in FIGS.30B-30K in the form of a heatmap. FIGS.30B-30K show bars that are representative of the area under the curve (AUC) created when plotting dilution steps versus ECL signal.

[0207] FIGS.31A-31B depict binding specificity of antibodies generated from mice immunized with formulated RNA constructs to CSP protein in Plasmodium falciparum sporozoite lysates. FIG.31A shows bindingbetween antibodies in the sera of immunized mice and CSP protein in the sporozoite (spz) lysates represented as area under the curve (AUC) created when plotting dilution steps versus luminescence signal. FIG.31B shows binding of murine anti-CSP mAb3SP2 used as a positive control.

[0208] FIGS.32A-32C depict activation of T-cells, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.32A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 32B); positive control: concanavalin A, 2 μg / mL (FIG.32C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0209] FIGS.33A-33C depict activation of T-cells, as assessed by secretion of IL-2. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.33A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 33B); positive control: concanavalin A, 2 μg / mL (FIG.33C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0210] FIGS.34A-34C depict activation of T-cells, as assessed by secretion of TNF-α. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.34A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 34B); positive control: concanavalin A, 2 μg / mL (FIG.34C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0211] FIGS.35A-35C depict activation of T-cells, as assessed by secretion of both IFN-γ and IL-2. IFN-γ+IL- 2 secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.35A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.35B); positive control: concanavalin A, 2 μg / mL (FIG.35C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0212] FIGS.36A-36C depict activation of T-cells, 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 formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.36A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.36B); positive control: concanavalin A, 2 μg / mL (FIG.36C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0213] FIGS.37A-37C depict activation of T-cells, as assessed by secretion of both IL-2 and TNF-α. IL-2+ TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs)treated with overlapping peptide pools covering a full length CSP protein (FIG.37A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.37B); positive control: concanavalin A, 2 μg / mL (FIG.37C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0214] FIGS.38A-38C depict activation of T-cells, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN- γ+IL-2+TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.38A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.38B); positive control: concanavalin A, 2 μg / mL (FIG.38C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0215] FIGS.39A-39D depict activation of CD4 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.39A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.39B); positive control: concanavalin A, 2 μg / mL (FIG.39C); medium control (FIG.39D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0216] FIGS.40A-40D depict activation of CD4 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.40A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 40B); positive control: concanavalin A, 2 μg / mL (FIG.40C); medium control (FIG.40D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0217] FIGS.41A-41D depict activation of CD4 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.41A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.41B); positive control: concanavalin A, 2 μg / mL (FIG.41C); medium control (FIG.41D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0218] FIGS.42A-42D depict activation of CD4 T cells only, as assessed by secretion of both IFN-γ and IL-2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.42A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.42B); positive control: concanavalin A, 2 μg / mL (FIG.42C); medium control (FIG.42D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0219] FIGS.43A-43D depict activation of CD4 T cells only, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.43A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.43B); positive control: concanavalin A, 2 μg / mL (FIG.43C); medium control (FIG. 43D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0220] FIGS.44A-44D depict activation of CD4 T cells only, as assessed by secretion of both IL-2 and TNF-α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.44A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.44B); positive control: concanavalin A, 2 μg / mL (FIG.44C); medium control (FIG.44D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0221] FIGS.45A-45D depict activation of CD4 T cells only, as assessed by secretion of IFN-γ, IL-2 and TNF- α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.45A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.45B); positive control: concanavalin A, 2 μg / mL (FIG.45C); medium control (FIG. 45D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0222] FIGS.46A-46D depict activation of CD8 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.46A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.46B); positive control: concanavalin A, 2 μg / mL (FIG.46C); medium control (FIG.46D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0223] FIGS.47A-47D depict activation of CD8 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.47A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 47B); positive control: concanavalin A, 2 μg / mL (FIG.47C); medium control (FIG.47D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0224] FIGS.48A-48D depict activation of CD8 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.48A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.48B); positive control: concanavalin A, 2 μg / mL (FIG.48C); medium control (FIG.48D)). Pooledsamples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0225] FIGS.49A-49D depict activation of CD8 T cells only, as assessed by secretion of both IFN-γ and IL-2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.49A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.49B); positive control: concanavalin A, 2 μg / mL (FIG.49C); medium control (FIG.49D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0226] FIGS.50A-50D depict activation of CD8 T cells only, as assessed by secretion of both IFN-γ and TNF- α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.50A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.50B); positive control: concanavalin A, 2 μg / mL (FIG.50C); medium control (FIG. 50D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0227] FIGS.51A-51D depict activation of CD8 T cells only, as assessed by secretion of both IL-2 and TNF-α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.51A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.51B); positive control: concanavalin A, 2 μg / mL (FIG.51C); medium control (FIG.51D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0228] FIGS.52A-52D depict activation of CD8 T cells only, as assessed by secretion of IFN-γ, IL-2 and TNF- α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with each of the formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.52A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.52B); positive control: concanavalin A, 2 μg / mL (FIG.52C); medium control (FIG. 52D). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0229] FIGS.53A-53C depict in vitro expression of ERMA 23-7 RNA construct in HEK293T cells. FIG.53A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.53B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells. FIG.53C shows percentage of viable cells that are positive for presence of expressed protein, with non-transfected cells serving as a control.

[0230] FIGS.54A-54B depict titers of antibodies elicited against PfCSP after immunization of mice with a pharmaceutical composition comprising ERMA 23-7. FIG.54A shows endpoint titers against full-length PfCSP on day 21, pre-boost. FIG.54B shows endpoint titers against full-length PfCSP on day 35 after boost.

[0231] FIGS.55A-55B depict epitope specificity of antibodies elicited upon immunization of mice with a pharmaceutical composition comprising ERMA 23-7. FIG.55A shows a diagram depicting localization of peptidesusing in multiplex assay in central region of PfCSP sequence. FIG.55B shows binding to epitopes by calculating AUC of 8-point dilution of immune serum samples.

[0232] FIGS.56A-56C depict pro-inflammatory response from T cells after immunization of mice with a pharmaceutical composition comprising ERMA 23-7. FIG.56A shows production of IFNɣ in mouse splenocytes after immunization with a pharmaceutical composition comprising ERMA 23-7 and stimulation with PfCSP peptides. FIG. 56B shows production of IFNɣ in combination with IL-2 in mouse splenocytes after immunization with a pharmaceutical composition comprising ERMA 23-7 and stimulation with PfCSP peptides. FIG.56C shows production of IFNɣ in combination with IL-2 and TNFα in mouse splenocytes after immunization with a pharmaceutical composition comprising ERMA 23-7 and stimulation with PfCSP peptides.

[0233] FIGS.57A-57B depict titers of antibodies elicited against PfCSP after immunization of mice with a pharmaceutical composition comprising ERMA 23-7 or with Composition 1, 2, and 3 comprising ERMA 23-7, MAS3a and MAS4f. FIG.57A shows endpoint titers (reciprocal serum titer) against full-length PfCSP pre-boost on Day 21. FIG.57B shows endpoint titers (reciprocal serum titer) against full-length PfCSP after boost on Day 35.

[0234] FIG.58 depicts epitope specificity of antibodies elicited upon immunization of mice with a pharmaceutical composition comprising ERMA 23-7 or with Compositions 1, 2 and 3 comprising ERMA 23-7, MAS3a and MAS4f.

[0235] FIG.59 depicts T-cell induction following immunization with Composition 3 comprising 1 μg ERMA 23- 7, 2 μg MAS3a and 2 μg MAS4f.

[0236] FIGS.60A-60B depict T-cell induction following immunization. FIG.60A shows the results for a combination of two T-cell string constructs by comparing the effects of a pharmaceutical composition comprising 2 μg MAS3a and 2 μg MAS4f (black dots) and Composition 3 comprising 1 μg ERMA 23-7, 2 μg MAS3a and 2 μg MAS4f (red dots). FIG.60B shows the results for a CSP construct by comparing the effects of a pharmaceutical composition comprising 1 μg ERMA 23-7 (black dots) and Composition 3 comprising 1 μg ERMA 23-7, 2 μg MAS3a and 2 μg MAS4f (red dots).

[0237] FIGS.61A-61K depict an assessment of antibodies generated from mice immunized with formulated RNA constructs for their ability to inhibit P. falciparum sporozoite infection of primary human hepatocytes. Percentage of inhibition of infection activity (mean with SEM) in comparison to a control (medium only) is shown for a 1:40 dilution (FIGS.61A and 61E), a 1:160 dilution (FIGS.61B and 61F), a 1:640 dilution (FIGS.61C and 61G), and a 1:2560 dilution (FIGS.61D and 61H). Inhibition of antibodies in sera of immunized mice in ILSDA is represented as area under the curve (AUC) created when plotting dilution steps versus percentage inhibition of infection (FIGS. 61I and 61J). ILSDA results for a positive control (mAb317, an antibody reported to inhibit hepatocyte infection) are also shown (FIG.61K).

[0238] FIGS. 62A-62F depict an assessment of binding and disassociation of antibodies generated from mice immunized with formulated RNA constructs and exposed to full length PfCSP, a peptide with a junction region and minor repeats (Junction + Minor repeats), or a peptide with major repeats (Major repeats). Serum samples from all treated mice were pooled prior to analysis. Level of antibody binding to a respective binding partner is shown in resonance units (RU) (FIGS.62A, 62C, and 62E). Percentage of antibody:antigen complexes still measurable after 15 minutes of dissociation (Residual Response) is calculated from initial binding (FIGS.62B, 62D, and 62F).

[0239] FIGS.63A-63D depict T-cell induction following immunization with 1 μg of Mas3a or a codon- optimized version (Mas3a-2, Mas3a-3) and 1 μg Mas4f or a codon-optimized version (Mas4f-2, Mas4f-3). Antigenspecific T-cell induction 7 days after immunization is shown for the combination of 1 μg Mas3a and 1 μg Mas4f (FIG. 63A), and the combination of 1 μg Mas3a-3 and 1 μg Mas4f-3 (FIG.63B). Antigen specific T-cell induction 35 days after immunization is shown for the combination of 1 μg Mas3a and 1 μg Mas4f (FIG.63C), and the combination of 1 μg Mas3a-3 and 1 μg Mas4f-3 (FIG.63D).

[0240] FIGS.64A-64D depict antigen-specific IFNɣ and IL-2 T-cell responses following immunization with 1 μg of Mas3a or a codon-optimized version (Mas3a-2, Mas3a-3) and 1 μg Mas4f or a codon-optimized version (Mas4f- 2, Mas4f-3). Antigen-specific IFNɣ responses after immunization for the combination of 1 μg Mas3a and 1 μg Mas4f is compared to the combination of 1 μg Mas3a-3 and 1 μg Mas4f-3 (FIG.64A) and Mas3a-2 and 1 μg Mas4f-2 (FIG. 64C). Antigen-specific IL-2 responses after immunization for the combination of 1 μg Mas3a and 1 μg Mas4f is compared to the combination of 1 μg Mas3a -3 and 1 μg Mas4f-3 (FIG.64B) and Mas3a-2 and 1 μg Mas4f-2 (FIG. 64D).

[0241] FIGS.65A-65H depict assessment of antibodies generated from mice immunized with formulated RNA constructs for ability to inhibit P. falciparum sporozoite traversal in HC-04 hepatoma cells. Percentage of inhibition of traversal activity (mean with SEM) in comparison to a medium control, which was set as 0% inhibition, is shown for a 1:20 dilution (FIG.65A), a 1:40 dilution (FIG.65B), a 1:80 dilution (FIG.65C), a 1:160 dilution (FIG. 65D), a 1:320 dilution (FIG.65E), and a 1:640 dilution (FIG.65F). Inhibition of antibodies in sera of immunized mice in traversal assays is represented as area under the curve (AUC) created when plotting dilution steps versus % inhibition of traversal (FIG.65G). Traversal assay results for a positive control (mAb317, an antibody known to inhibit sporozoite traversal) are also shown (FIG.65H).

[0242] FIGS.66A-66G depict assessment of antibodies generated from mice immunized with formulated RNA constructs for ability to inhibit P. falciparum sporozoite traversal in HC-04 hepatoma cells. Percentage of inhibition of traversal activity (mean with SEM) in comparison to a medium control, which was set as 0% inhibition, is shown for a 1:20 dilution (FIG.66A), a 1:40 dilution (FIG.66B), a 1:80 dilution (FIG.66C), a 1:160 dilution (FIG.66D), a 1:320 dilution (FIG.66E), and a 1:640 dilution (FIG.66F). Inhibition of antibodies in sera of immunized mice in traversal assays is represented as area under the curve (AUC) created when plotting dilution steps versus % inhibition of traversal (FIG.66G).

[0243] FIGS.67A-67B depict in vitro expression of non-formulated RNA constructs 91, 100 and 104 encoding different Plasmodium polypeptides in HEK293T cells. FIG.67A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.67B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non-permeabilized cells show only surface expressed protein (grey bar, surface staining). Protein was detected using anti-PfCSP L9 antibody. Each sample was stained in triplicate, bar is a representation of mean with SD; NT, non-transfected.

[0244] FIGS.68A-68B depict in vitro expression of non-formulated RNA constructs 87 and 88 encoding different Plasmodium polypeptides in HEK293T cells. FIG.68A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.68B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non-transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non-permeabilized cells show only surface expressed protein (grey bar, surface staining). Protein was detected using anti-PfCSP L9 antibody. Each sample was stained in triplicate, bar is a representation of mean with SD; NT, non-transfected.

[0245] FIGS.69A-69B depict in vitro expression of formulated RNA constructs 87, 88, 91, 100 and 104 encoding different Plasmodium polypeptides in HEK293T cells. FIG.69A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG.69B shows total expression as measured by median fluorescence of the total HEK293T population for both transfected and non- transfected cells. Permeabilized cells show total protein expressed (black bar, intracellular staining) and non- permeabilized cells show only surface expressed protein (grey bar, surface staining). Protein was detected using anti- PfCSP L9 antibody. Each sample was stained in triplicate, bar is a representation of mean with SD; NT, non- transfected.

[0246] FIGS.70A-70B depict immunogenicity induced in mice by formulated RNA constructs 87, 88, 91, 100 and 104. FIG.70A shows antibodies to Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG.70B shows antibodies to PfCSP C-terminal domain (“PfCSP-C term (3D7)”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0247] FIG.71 depicts binding of antibodies generated from mice immunized with formulated RNA constructs 87, 88, 91, 104, and 100 during challenge studies to various epitopes in a heatmap format.

[0248] FIGS.72A-72J depict binding of antibodies generated from mice immunized with formulated RNA constructs to various epitopes. FIGS.72A-72J each show bars that are representative of the area under the curve (AUC) created when plotting dilution steps versus ECL signal.

[0249] FIGS.73A-73C depict activation of T-cells, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.73A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.73B); positive control: concanavalin A, 2 μg / mL (FIG.73C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0250] FIGS.74A-74C depicts activation of T-cells, as assessed by secretion of IL-2. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.74A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.74B); positive control: concanavalin A, 2 μg / mL (FIG.74C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0251] FIGS.75A-75C depicts activation of T-cells, as assessed by secretion of TNF-α. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.75A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.75B); positive control: concanavalin A, 2 μg / mL (FIG.75C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0252] FIGS.76A-76C depicts activation of T-cells, as assessed by secretion of both IFN-γ and IL-2. IFN- γ+IL-2 secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs)treated with overlapping peptide pools covering the full length CSP protein (FIG.76A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.76B); positive control: concanavalin A, 2 μg / mL (FIG.76C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0253] FIGS.77A-77C depicts activation of T-cells, 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 formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.77A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.77B); positive control: concanavalin A, 2 μg / mL (FIG.77C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0254] FIGS.78A-78C depicts activation of T-cells, as assessed by secretion of both IL-2 and TNF-α. IL-2+ TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.78A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.78B); positive control: concanavalin A, 2 μg / mL (FIG.78C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0255] FIGS.79A-79C depicts activation of T-cells, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN- γ+IL-2+TNF-α secretion was assessed using isolated splenocytes (from mice immunized with formulated RNA constructs) treated with overlapping peptide pools covering the full length CSP protein (FIG.79A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.79B); positive control: concanavalin A, 2 μg / mL (FIG.79C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice. ve, vehicle.

[0256] FIGS.80A-80D depicts activation of CD4 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.80A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.80B); positive control: concanavalin A, 2 μg / mL (FIG.80C); medium control (FIG.80D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0257] FIGS.81A-81D depicts activation of CD4 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.81A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.81B); positive control: concanavalin A, 2 μg / mL (FIG.81C); medium control (FIG.81D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot- forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0258] FIGS.82A-82D depicts activation of CD4 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.82A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.82B); positive control: concanavalin A, 2 μg / mL (FIG.82C); medium control (FIG.82D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0259] FIGS.83A-83D depicts activation of CD4 T cells only, as assessed by secretion of both IFN-γ and IL- 2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.83A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.83B); positive control: concanavalin A, 2 μg / mL (FIG.83C); medium control (FIG.83D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0260] FIGS.84A-84D depicts activation of CD4 T cells only, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.84A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.84B); positive control: concanavalin A, 2 μg / mL (FIG.84C); medium control (FIG.84D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0261] FIGS.85A-85D depicts activation of CD4 T cells only, as assessed by secretion of both IL-2 and TNF- α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.85A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.85B); positive control: concanavalin A, 2 μg / mL (FIG.85C); medium control (FIG.85D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0262] FIGS.86A-86D depicts activation of CD4 T cells only, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.86A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.86B); positive control: concanavalin A, 2 μg / mL (FIG.86C); medium control (FIG.86D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells; ve, vehicle.

[0263] FIGS.87A-87D depicts activation of CD8 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.87A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.87B); positive control: concanavalin A, 2 μg / mL (FIG.87C); medium control (FIG.87D)). Pooled sampleswere measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0264] FIGS.88A-88D depicts activation of CD8 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.88A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.88B); positive control: concanavalin A, 2 μg / mL (FIG.88C); medium control (FIG.88D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot- forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0265] FIGS.89A-89D depicts activation of CD8 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.89A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.89B); positive control: concanavalin A, 2 μg / mL (FIG.89C); medium control (FIG.89D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0266] FIGS.90A-90D depicts activation of CD8 T cells only, as assessed by secretion of both IFN-γ and IL- 2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.90A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.90B); positive control: concanavalin A, 2 μg / mL (FIG.90C); medium control (FIG.90D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0267] FIGS.91A-91D depicts activation of CD8 T cells only, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.91A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.91B); positive control: concanavalin A, 2 μg / mL (FIG.91C); medium control (FIG.91D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0268] FIGS.92A-92D depicts activation of CD8 T cells only, as assessed by secretion of both IL-2 and TNF- α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering the full length CSP protein (FIG.92A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.92B); positive control: concanavalin A, 2 μg / mL (FIG.92C); medium control (FIG.92D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0269] FIGS.93A-93D depicts activation of CD8 T cells only, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubatedwith overlapping peptide pools covering the full length CSP protein (FIG.93A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.93B); positive control: concanavalin A, 2 μg / mL (FIG.93C); medium control (FIG.93D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells; ve, vehicle.

[0270] FIG.94 shows binding between antibodies in the sera of immunized mice and CSP protein in the sporozoite (spz) lysates represented as area under the curve (AUC) created when plotting dilution steps versus luminescence signal. DEFINITIONS

[0271] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents each of which are hereby incorporated by reference.

[0272] Unless otherwise stated, structures depicted herein are meant 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, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0273] Unless otherwise indicated, 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 replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.

[0274] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0275] Agent: As used herein, the term “agent,” may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.

[0276] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more polypeptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In someembodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non- natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring polypeptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0277] Antigen: The term “antigen”, as used herein, refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In the context of the present disclosure, the terms “malaria antigen” and “Plasmodium antigen” are understood to refer to an antigen from a Plasmodium species, where the Plasmodium species can cause malaria in a subject.

[0278] Anti-malaria immune response: The term “anti-malaria immune response”, as used herein, refers to an immune response produced through pre-exposure to one or more Plasmodium antigens, e.g., through administration (e.g., vaccination) of the constructs as described herein directed to Plasmodium.

[0279] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0280] C-terminal domain: The term “C-terminal domain”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 273-397 of wild-type CSP sequence of Plasmodium falciparum (isolate 3D7) (SEQ ID NO: 1).

[0281] C-terminal region: The term “C-terminal region”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 273-375 of wild-type CSP sequence (SEQ ID NO: 1). In some embodiments, a serine follows immediately after the C-terminal region. In some embodiments, a serine and a valine follow immediately after the C-terminal region.

[0282] C-terminal region variant: The term “C-terminal region variant”, as used herein, refers to a C- terminal region that comprises one or more mutations as compared to amino acids 273-375 of wild-type CSP sequence (SEQ ID NO: 1). In some embodiments, one or more mutations are one or more substitution mutations. In some embodiments, one or more mutations comprise an indel.

[0283] Central domain: The term “central domain”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 105-272 of wild-type CSP sequence (SEQ ID NO: 1).

[0284] Characteristic portion: As used herein, the term “characteristic portion”, in the broadest sense, refers to a portion of a polypeptide or region thereof whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the polypeptide or region thereof. In some embodiments, a characteristic portion of a polypeptide or region thereof is a portion that is found in the polypeptide or region thereof and in related polypeptide or region thereof that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact polypeptide or region thereof. For example, in some embodiments, a “characteristic portion” of a polypeptide or region thereof is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of the polypeptide or region thereof. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a polypeptide or region thereof (e.g., CSP, its N terminal domain, its major repeat region etc.) is one that, in addition to the sequence and / or structural identity specified above, shares at least one functional characteristic with the relevant intact polypeptide or region thereof. In some embodiments, a characteristic portion may be biologically active. In some embodiments, a fragment as described herein can be a portion. Accordingly, in some embodiments, a characteristic fragment can be a “characteristic portion.”

[0285] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0286] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0287] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptide and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0288] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” (or “therapeutic regimen”) may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

[0289] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigenrefers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0290] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0291] Helper antigen: As used herein, the term “helper antigen” refers to an antigen that is included in a polypeptide comprising one or more CSP polypeptide regions or portion thereof, where the antigen is not derived from a CSP polypeptide.

[0292] Heterologous: As used herein, the term “heterologous”, with respect to secretory signal or transmembrane region, refers to a secretory signal or transmembrane region from a virus or an organism other than Plasmodium.

[0293] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0294] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0295] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0296] In order: As used herein with reference to a polynucleotide or polyribonucleotide, “in order” refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, “in order” refers to the order of features moving from the N-terminal-most of the features to the C- terminal-most of the features along the polypeptide. “In order” does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being “in order, Feature A, Feature B, and Feature C,” this description does not exclude, e.g., Feature D being located between Features A and B.

[0297] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0298] Junction: The term “junction”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 98-104 of wild-type CSP sequence (SEQ ID NO: 1).

[0299] Junction region: The term “junction region”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 93-104 of wild-type CSP sequence (SEQ ID NO: 1). Typically, a junction region includes an R1 region (amino acids 93-97) and a junction (SEQ ID NO: 277) at positions 98-104.

[0300] Junction region variant: The term “junction region variant”, as used herein, refers to a junction region that comprises one or more mutations as compared to amino acids 93-104 of wild-type CSP sequence (SEQ ID NO: 1). In some embodiments, one or more mutations are one or more substitution mutations. In some embodiments, one or more mutations comprise an indel.

[0301] Linker: As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0302] Lipid: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0303] Major repeat region: As used herein, the term “major repeat region” refers to a region of a CSP polypeptide that corresponds to amino acids 129-272 of wild-type CSP sequence (SEQ ID NO: 1) and contains 35 repeats of the amino acid sequence NANP (SEQ ID NO: 230). The 35 repeats of the amino acid sequence NANP (SEQ ID NO: 230) are separated into two contiguous stretches, the first stretch containing 17 repeats of the amino acid sequence NANP (SEQ ID NO: 230) and second stretch containing 18 repeats of the amino acid sequence NANP (SEQ ID NO: 230) which flank an amino acid sequence of NVDP (SEQ ID NO: 229). A portion of the major repeat region contains at least the amino acid sequence NPNA (SEQ ID NO: 228). Preferably a portion of the major repeat region contains at least the amino acid sequences NANPNA (SEQ ID NO: 232) and NPNANP (SEQ ID NO: 231). As used herein, “repeat” in reference to sequence A refers to sequence A being present once, and “one or more repeats” of sequence A refers to sequence A being present one or more times.

[0304] 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.

[0305] Minor repeat region: As used herein, the term “minor repeat region” refers to a region of a CSP polypeptide that corresponds to amino acids 105-128 of wild-type CSP sequence (SEQ ID NO: 1) and contains 3 repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223). A minor repeat region does not contain the amino acid sequence NPNA (SEQ ID NO: 228), and does not contain the amino acid sequence NANPNA (SEQ ID NO: 232) or NPNANP (SEQ ID NO: 231). As used herein, “repeat” in reference to sequence A refers to sequence A being present once, and three repeats of sequence A refers to sequence A being present three times.

[0306] Multimerization region: As used herein, the term “multimerization region” refers to a region that directs assembly of multimers into a complex, where each multimer comprises a polypeptide associated with the multimerization region.

[0307] N-terminal domain: As used herein, the term “N-terminal domain” refers to a region of a CSP polypeptide that corresponds to amino acids 19-104 of wild-type CSP sequence (SEQ ID NO: 1).

[0308] N-terminal start region: As used herein, the term “N-terminal start region” refers to a region of a CSP polypeptide that corresponds to amino acids 19-31 of wild-type CSP sequence (SEQ ID NO:1).

[0309] N-terminal end region: As used herein, the term “N-terminal end region” refers to a region of a CSP polypeptide that corresponds to amino acids 81-92 of wild-type CSP sequence (SEQ ID NO: 1).

[0310] N-terminal region: As used herein, the term “N-terminal region” refers to a region of a CSP polypeptide that corresponds to amino acids 19-80 of wild-type CSP sequence (SEQ ID NO: 1).

[0311] R1: The term “R1”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 93-97 of wild-type CSP sequence (SEQ ID NO: 1).

[0312] RNA lipid nanoparticle: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer- conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0313] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a parasite such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0314] Nucleic acid / Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least 10-nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises polypeptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more polypeptide bonds, e.g., as in a “polypeptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non- natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-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, intercalated bases, andcombinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0315] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., malaria), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., malaria). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., malaria) and / or interrupting or reversing the progress of the disease (e.g., malaria). In some embodiments, a desired reaction in a treatment of a disease (e.g., malaria) may be or comprise delay or prevention of the onset of a disease (e.g., malaria) or a condition (e.g., a malaria associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., malaria) or a condition (e.g., a malaria associated condition) to be treated, the severity of such a disease (e.g., malaria) or a condition (e.g., a malaria associated condition), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0316] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In someembodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptide that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptide. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptide within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptide are reference polypeptide for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptide within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a polypeptide is a Plasmodium polypeptide construct described herein. A Plasmodium polypeptide construct is a polypeptide that includes one or more Plasmodium proteins, or one or more portions thereof. In some embodiments, a Plasmodium polypeptide construct described herein includes at least one region of Plasmodium CSP or a portion thereof. In some embodiments, a Plasmodium polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), a helper antigen, a multimerization region, and / or a linker, as described herein.

[0317] Prevent: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. In some embodiments, prevention refers to reducing the risk of developing clinical malaria.

[0318] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstancesto those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0319] Ribonucleic acid (RNA) or Polyribonucleotide: As used herein, the term “ribonucleic acid,” “RNA,” or “polyribonucleotide” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods). In some embodiments, a polyribonucleotide encodes a polypeptide, which is preferably is a Plasmodium polypeptide construct.

[0320] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0321] Secretory signal: As used herein, the term “secretory signal” refers to an amino acid sequence motif that targets associated polypeptide for translocation to a secretory pathway.

[0322] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In preferred embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject displays one or more non- specific symptoms of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., malaria and / or a malaria- associated condition). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0323] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0324] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., malaria and / or a malaria-associated condition) may not have been diagnosed with the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria- associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will not develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition).

[0325] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a therapeutic agent or therapy is a medical intervention that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0326] Transmembrane region: As used herein, the term “transmembrane region” refers to a region of a polypeptide that spans a biological membrane, such as the plasma membrane of a cell.

[0327] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., malaria and / or a malaria- associated condition).

[0328] Variant: As used herein, the term “variant” refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS I. Malaria

[0329] Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et al. Malaria. Nat Rev Dis Primers 3, 17050, 2017, which is incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is taken in containing malaria parasites. The infected mosquito can then subsequently bite a non-infected subject, infecting the subject.

[0330] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many malaria parasites have developed resistance to available therapeutics. According to Malaria Eradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination.

[0331] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as “RTS,S,” a milestone in malaria vaccine development. In 2019, the World Health Organization launched pilot programs that provide RTS,S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS,S / AS01 is an adjuvanted protein subunit vaccine that consists of a portion of the major repeat region and the C- terminus of CSP from Plasmodium falciparum fused to the Hepatitis B surface antigen (HBsAg). The vaccine is a mix of this PfCSP-HBsAg compound with HBsAg that forms virus-like particles (RTS,S / AS01; Mosquirix™). RTS,S is administered according to a regimen that requires four doses: an initial 3-dose schedule given at least 1 month apart, and a 4th dose 15-18 months after dose 3 (see, for example, Vandoolaeghe & Schuerman Expert Rev Vaccines. 15:1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, each of which is incorporated herein by reference in its entirety). Reports indicate that RTS,S protects approximately 30% to 50% of children from clinical disease over 18 months. RTS,S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T cell responses (see, for example, Moris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). Phase III studies of RTS,S delivered as a three-dose series with a booster after 1 yr (year) showed moderate vaccine efficacy in children aged 5 to 17 months preventing 36% of clinical malaria cases over the full study period with a median follow-up of 4 yrs, with a range of 20% in high to 66% in low transmission settings. Furthermore, published literature suggests that protection wanes over time including reports of potential negative efficacy after 5 yrs in children with high malaria exposure (Olotu et al. 2016, N. Engl. J. Med. 374:2519-29, which is incorporated herein by reference in its entirety). Thus, an effective malaria vaccine remains an unmet medical need of critical importance for global health. A. Life Cycle

[0332] During a blood meal, infected mosquitos inject, along with their anticoagulating saliva, sporozoites known as the liver stage of Plasmodium spp. Sporozoites journey through the skin to the lymphatics and into hepatocytes of the liver. This journey happens very quickly; it can be complete within only a few minutes (Sinnis et al., Parasitol Int. 2007 Sep;56(3):171-8, which is incorporated herein by reference in its entirety). This is a time known to be a bottleneck of Malaria infection most favorable for therapeutic intervention, as only a small number (thought to be a few hundred at maximum) of sporozoites are injected by the mosquito, with only fraction of that number establishing infection in the liver and developing into mature live-stage parasites (Flores-Garcia et al., mBio. 2018 Nov 20;9(6):e02194-18, which is incorporated herein by reference in its entirety). Thus, a subject whose immune system is primed to clear sporozoites before they enter hepatocytes can efficiently clear an infection.

[0333] One particular challenge associated with clearing a malarial infection during this bottleneck is that the most abundant and immunogenic protein on the sporozoite surface, the circumsporozoite protein (CSP), is only exposed to the immune system in small quantities and for short duration of time due to the variably low inoculum from the mosquito and the kinetics of hepatocyte infection after inoculation. After liver infection is established, the parasite differentiates into a stage which no longer expresses CSP and instead has a different mosaic of surface antigens. Furthermore, due to the density and close proximity of neighboring CSPs on the surface of the parasite coupled with the bi-valency of antibodies, binding of antibodies to CSP can produce a phenomenon referred to as CSP precipitation reaction, whereby antibodies can crosslink neighboring CSP and cause them to precipitate and shed from the parasite surface, leaving a trail of precipitated antibody bound CSP that the parasite can replace through its normal CSP translocation process (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).

[0334] When moving from an inoculation site in the skin to the liver, sporozoites traverse host cells (Mota et al., Science 2001 Jan 5;291(5501):141-4). Sporozoites traverse different types of host cells at the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe.2008 Feb 14;3(2):88-96, which is incorporated herein by reference in its entirety), and the liver sinusoidal barrier, containing liver endothelial cells and Kupffer cells (Frevert et al., PLoS Biol 3(6): 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), in order to gain access to hepatocytes. Sporozoites preferentially traverse cells with low-sulfated heparin sulfate proteoglycans (HSPGs) but preferentially invade cells with high-sulfated HSPGs (Coppi et al., Cell Host & Microbe 2, 316–327, November 2007, which is incorporated herein by reference in its entirety).

[0335] Cell traversal was first observed as non-phagocytic entry of P. berghei sporozoites into macrophages followed by “escape” from these cells (Vanderberg et al., J. Euk. Microbiol.37:528-536, 1990, which is incorporated herein by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being explored. However, it has been suggested by electron microscopy that host cell rupture occurs upon entry and exit from the host cell (Mota et al., 2001; Tavares et al., 2013, each of which is incorporated herein by reference in its entirety). It has also been shown that P. yoelii sporozoites can enter hepatocytes via a transient vacuole and that host membrane rupture occurs upon cell exit rather than cell entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov 11;18(5):593-603, which is incorporated herein by reference in its entirety).

[0336] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is incorporated herein by reference in its entirety). Several possibilities emerged as to why this occurs. The first hypothesis suggested that migration through hepatocytes primes parasites for invasion by activating apicalexocytosis (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 neighboring 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). Lastly, other studies suggest that it takes some time for sporozoites to switch off the machinery for traversal and activate invasion machinery (Amino et al., 2008, Coppi et al., 2007, each of which is incorporated herein by reference in its entirety), and that traversal primarily functions to penetrate cell barriers and avoid phagocytosis en route to the liver (Amino et al., 2008, Coppi et al., 2007, Tavares et al., 2013, each of which is incorporated herein by reference in its entirety).

[0337] Although it has been shown that sporozoites traverse human cells (Behet et al., Malar J 2014 Apr 5;13:136; Cha et al., J Exp Med 2015 Aug 24;212(9):1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6):e0129623; van Schaijk et al., PLoS ONE, 3 (10). e35492008, each of which is incorporated herein by reference in its entirety), the molecular basis for the traversal process is largely unstudied. Antibodies against circumsporozoite protein (CSP) impair traversal (Dumoulin et al., 2015, which is incorporated herein by reference in its 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, which is incorporated herein by reference in its entirety). Furthermore, antibodies induced by chloroquine prophylaxis with sporozoites interfere with cell traversal, and these may also target CSP (Behet et al., 2014, which is incorporated herein by reference in its entirety). Recently it was shown that glyceraldehyde 3- phosphate dehydrogenase (GAPDH) on the parasite surface interacts 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).

[0338] In rodent malaria parasites such as P. berghei, two sporozoite microneme proteins have been identified that appear to be essential for cell traversal: sporozoite microneme protein essential for cell traversal 1 (SPECT1) (Ishino et al., PLoS Biol., 2 (2004), pp. 77-84, which is incorporated herein by reference in its entirety) and sporozoite microneme protein essential for cell traversal 2 (SPECT2) (Ishino et al., Cell. Microbiol., 7 (2005), pp. 199- 208, which is incorporated herein by reference in its entirety). SPECT2 can also be called perforin-like protein 1 (PLP1) (Kaiser et al., Mol. Biochem. Parasitol., 133 (2004), pp.15-26, which is incorporated herein by reference in its entirety). Even though genetic disruption of SPECT1 or SPECT2 rendered sporozoites unable to traverse murine cells, sporozoites 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, but a small number of sporozoites could still establish liver infection that resulted in subsequent patency. However, depletion of Kupffer cells allowed mutants to establish liver infection at levels comparable with 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 rodent-infecting sporozoites is important for navigating through the sinusoidal layer, but not for hepatocyte invasion, malarial exoerythrocytic forms development, or growth within erythrocytes (Ishino et al., 2004, Ishino et al., 2005, each of which is incorporated herein by reference in its entirety).

[0339] The ortholog of SPECT2 in P. yoelii, PLP1, has been shown to play a role in cell traversal. Although this protein is not required for hepatocyte entry, it plays a role in egress from transient vacuoles during traversal (Risco- Castillo et al., 2015, each of which is incorporated herein by reference in its entirety). Thus, sporozoites that infect rodents can traverse host cells by generating a vacuole at the entry step and use a perforin-like protein (e.g., SPECT2 / PLP1) to escape from this compartment and / or a host cell, during cell exit.

[0340] Once sporozoites have invaded liver cells, they differentiate into merozoites, a replicative form of the parasite capable of lysing hepatocytes after multiple rounds of replication. Within a few days, a few hundred sporozoites can become hundreds of thousands of merozoites. When infected liver cells rupture, they release the merozoites into the bloodstream, where they invade red blood cells and begin the asexual reproductive stage, which is the symptomatic stage of the disease. Within a small number of days, millions of merozoites can be present in blood.

[0341] Malaria symptoms typically develop 4-8 days after initial red blood cell invasion. Replication cycle of merozoites within the red blood cells continues for 36-72 hours, until hemolysis, releasing the merozoites for another round of red blood cell infection. Thus, in synchronous infections (infections that originate from a single infectious bite), fever occurs every 36–72 hours, when infected red blood cells lyse and release endotoxins en masse.

[0342] Plasmodium spp. parasites gain entry into red blood cells through specific ligand–receptor interactions mediated by proteins on the surface of the parasite that interact with receptors on the host erythrocyte (mature red blood cell) or reticulocyte (immature red blood cell), whereas P. falciparum can invade and replicate in erythrocytes and reticulocytes, P. vivax and other species predominantly invade reticulocytes, which are less abundant than erythrocytes. Most of the erythrocyte-binding proteins or reticulocyte-binding proteins that have been associated with invasion are redundant or are expressed as a family of variant forms; however, for P. falciparum, two essential red blood cell receptors (basigin and complement decay-accelerating factor (also known as CD55)) have been identified.

[0343] Plasmodium vivax and Plasmodium ovale can also enter a dormant state in the liver, the hypnozoite.

[0344] Merozoites released from red blood cells can invade other red blood cells and continue to replicate, or in some cases, they differentiate into male or female gametocytes. Gametocytes concentrate in skin capillaries and are then taken up by the mosquito vector in another blood meal. In the gut of the mosquito, each male gametocyte produces eight microgametes after three rounds of mitosis; the female gametocyte matures into a macrogamete. Male microgametes are motile forms with flagellae and seek the female macrogamete. The male and female gametocytes fuse, forming a diploid zygote, which elongates into an ookinete; this motile form secretes a chitinase in order to enter the peritrophic membrane and traverse the midgut epithelium to the basal lateral side of the midgut, establishing itself in the basal lamina as an oocyst. Oocysts mature over 14-15 days, undergoing cycles of replication to form sporozoites that are ultimately liberated into the hemocoel, an environment rich in sugars and substrates beneficial to the parasite’s survival. Thousands of sporozoites can form from a single oocyst and become randomly distributed throughout the hemocoel. These sporozoites are motile and rapidly destroy the hemolymph, with only approximately 20% successfully invading the salivary gland. Following invasion of the salivary gland, sporozoites are re-programmed via an unknown mechanism to prepare for liver invasion. Evidence of this reprogramming has been demonstrated by the inability of midgut sporozoites (directly from oocysts) to invade hepatocytes, and also by the fact that sporozoites which have successfully invaded a salivary gland are unable to do re-invade another salivary gland if presented one. Salivary gland sporozoites alter mosquito behavior and salivary gland function, as less saliva is produced resulting in an increase in mosquito probing behavior, increasing the chances of transmission to a human host via a mosquito bite.

[0345] Some drugs that prevent Plasmodium spp. invasion or proliferation in the liver have prophylactic activity, drugs that block the red blood cell stage are required for the treatment of the symptomatic phase of the disease, and compounds that inhibit the formation of gametocytes or their development in the mosquito (includingdrugs that kill mosquitoes feeding on blood) are transmission-blocking agents (Phillips, et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety).

[0346] Some proteins that are expressed exclusively by the parasite in specific phases of its life cycle, while other proteins are expressed and may play a role in multiple life cycle phases. B. Genome

[0347] Since completion of the first sequence of P. falciparum 3D7 genome in 2002, genomic research on malaria parasites has rapidly advanced. Except for a short diploid phase after fertilization in the mosquito midgut, Plasmodium parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases, contain 14 chromosomes, a circular plastid genome of approximately 35 kilobases, and multiple copies of a 6 kilobase mitochondrial DNA. Comparison of genomes from different species showed that homologous genes are often found in synthetic blocks arranged in different orders among different chromosomes.

[0348] The adenine-thymine (AT) content of Plasmodium spp. can also be very different, e.g., ^80% AT in P. falciparum, P. reichenowi, and P. gallinaceum; ^75% AT in rodent malaria parasites; and ^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, with an average of 80.6% AT for the whole P. falciparum genome versus 86.5% for noncoding sequences. The high AT content of P. falciparum reflects large numbers of low-complexity regions, simple sequence repeats, and microsatellites, as well as a highly skewed codon usage bias. Polymorphisms of AT-rich repeats provide abundant markers for linkage mapping of drug resistance genes and for tracing the evolution and structure of parasite populations.

[0349] Malaria parasite genomes carry multigene families that serve important roles in parasite interactions with their hosts, including, for example, antigenic variation, signaling, protein trafficking, and adhesion. Among the gene families, genes encoding P. falciparum erythrocyte membrane protein 1 (PfEMP1) have been studied most extensively. Each individual P. falciparum parasite carries a unique set of 50 to 150 copies of the var gene in its genome, where switches of gene expression can produce antigenic variation. PfEMP1 plays an important role in the pathogenesis of clinical developments such as in cerebral and placental malaria, in which it mediates the cytoadherence of infected red blood cells (iRBCs; infected erythrocytes) in the deep tissues. Different PfEMP1 molecules bind to various host molecules, including α2-macroglobulin, CD36, chondroitin sulfate A (CSA), complement 1q, CR1, E-selectins and P-selectins, endothelial protein C receptor (EPCR), heparan sulfate, ICAM1, IgM, IgG, PECAM1, thrombospondin (TSP), and VCAM1. Such binding leads to activation of various host inflammatory responses. Hemoglobinopathies, including the hemoglobin C and hemoglobin S trait conditions, interfere with PfEMP1 display in knob structures of the iRBCs. This poor display of PfEMP1 on the host cell surface offers protection against malaria by reducing the cytoadherence and activation of inflammatory processes that promote the development of severe disease.

[0350] Members of the large Plasmodium interspersed repeat (pir) multigene family are named differently by parasite species, for example, yir in P. yoelii, bir in P. berghei, vir in P. vivax. Several P. falciparum gene families (stevor, rif, and PfMC-2TM) are classified with pir by their similar gene structures, which characteristically include a short first exon, a long second exon, and a third exon encoding a transmembrane domain. In a recent study, the pir genes from P. chabaudi (cir) were shown to be expressed in different cellular locations, within and on the surface of iRBCs, and in merozoites. Malaria parasites devote large portions of their genomes to gene families that ensure evasion of host immune defenses and protection of molecular processes essential to infection. These familiesemphasize the importance of research on their roles in parasite-host interactions and virulence, despite the difficulties inherent to their investigation.

[0351] An additional, exemplary polymorphic gene family comprises a group of 14 genes encoding proteins with six cysteines (6-Cys). These proteins often localize on the parasite surface interacting with host proteins and are expressed at different parasite developmental stages.6-Cys proteins also demonstrate diverse functions and have been shown to play roles in, for example, parasite fertilization, mating interactions, evasion of immune responses, and invasion of hepatocytes. The proteins expressed in asexual stages are generally polymorphic and / or under selection, suggesting that they could be targets of the host immune response; however, their functions in parasite development remain largely unknown.

[0352] Plasmodium genomes can be highly polymorphic. Early studies demonstrated polymorphisms involving tens to hundreds of kilobases and that the chromosome structure in P. falciparum is largely conserved in central regions but extensively polymorphic is both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repetitive sequences and families of genes.

[0353] The frequency of simple sequence repeats (microsatellites) in P. falciparum is estimated to be approximately one polymorphic microsatellite per kb DNA. Without wishing to be bound by any one theory, this high rate may reflect the AT-rich nature of the genome. Microsatellites seem to be less frequent in other Plasmodium species that have genomes with lower AT contents. In addition to the highly polymorphic and repetitive structure of Plasmodium genomes, there are also large numbers of 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). C. Plasmodium Proteins

[0354] Plasmodium parasites are known to express various proteins at different stages of their lifecycles. Exemplary Plasmodium proteins are described below, and exemplary amino acid sequences are provided in Table 2.

[0355] Circumsporozoite protein (CSP) is a multifunctional protein that is involved in Plasmodium life cycle, as it is required for the formation of sporozoites in the mosquito midgut, the release of sporozoites from the oocyst, invasion of salivary glands, attachment of sporozoites 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). CSP is present in all Plasmodium species, and although variation exists in the amino acid sequence across species, the overall domain structure of a central repeat region and nonrepeat 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 herein by reference in its entirety). CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, A0A2L,1CF88, C6FGZ3, C6FH2,7 C6FHG7, 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 for CSP P. falciparum isolates from Asia, South America and Africa. Table 1: Exemplary Sequences Encoding CSP P. falciparum isolates from Asia, South America and Africa

[0356] An exemplary wild-type CSP polypeptide amino sequence from Plasmodium falciparum isolate 3D7 is presented in Table 2 as SEQ ID NO: 1, and includes the following: a secretory signal (amino acids 1-18); an N- terminal domain (amino acids 19-104); a junction region (amino acids 93-104), a central domain (amino acids 105- 272); and a C-terminal domain (amino acids 273-397). In exemplary SEQ ID NO: 1, the N-terminal domain includes an N-terminal region (amino acids 19-80); an N-terminal end region (amino acids 81-92); and a junction region (amino acids 93-104). In exemplary SEQ ID NO: 1, the junction region includes an R1 region (amino acids 93-97) and a junction (SEQ ID NO: 277) at positions 98-104. In exemplary SEQ ID NO: 1, the central domain includes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272). In exemplary SEQ ID NO: 1, the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 223). In exemplary SEQ ID NO: 1, the major repeat region includes 35 repeats of the amino acid sequence NANP (SEQ ID NO: 230), wherein 35 repeats of the amino acid sequence NANP (SEQ ID NO: 230) are separated into two contiguous stretches, and wherein one stretch includes 17 repeats of the amino acid sequence NANP (SEQ ID NO: 230) and one includes 18 repeats of the amino acid sequence NANP (SEQ ID NO: 230) which flank an amino acid sequence of NVDP (SEQ ID NO: 229). The major repeat region includes the amino acid sequences NPNANP (SEQ IDNO: 231) and NANPNA (SEQ ID NO: 232). In exemplary SEQ ID NO: 1, the C-terminal domain includes a C-terminal region (amino acids 273-375), a serine-valine (amino acids 376-377), and a transmembrane domain (amino acids 378-397). In exemplary SEQ ID NO: 1, the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363). Exemplary CSP amino acid sequence is provided in Table 2.

[0357] Upregulated in infective sporozoites gene 3 (UIS3) is a membrane-bound protein localized to sporozoite parasitophorous vacuolar membrane (PVM) in infected hepatocytes. UIS3 was shown to interact with liver fatty acid- binding protein (L-FABP) and be involved in fatty acid and / or lipid import during phases of 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).

[0358] After sporozoite invasion of host liver cells, there is synthesis of vital Plasmodium structural features (e.g., parasitophorous vacuolar membrane). During hepatocytic stages, the Plasmodium relies on host fatty acids for rapid synthesis of its membranes (see, e.g., Sharma et al., J Biol Chem.2008 Aug 29; 283(35): 24077–24088, which is incorporated herein by reference in its entirety). UIS3 insertion in the PVM provides Plasmodium a method to import essential fatty acids and / or lipids during rapid sporozoites growth phases (see, e.g., Sharma et al., J Biol Chem. 2008 Aug 29; 283(35): 24077–24088, which is incorporated herein by reference in its entirety).

[0359] Immunization with UIS3-deficient Plasmodium berghei sporozoites protected against malaria in rodent malaria model (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 can start the transformation process in the liver; however, they show severe defects during transformation into 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 are also unable to develop into mature liver schizonts and therefore abort 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). Further, it was previously demonstrated that UIS3 derived from Plasmodium berghei and UIS3 derived from Plasmodium falciparum exhibited a 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).

[0360] Plasmodium UIS3 sequences are known (see, e.g., UniProt accession number A0A509ARS3, A0A1C6YLP3, Q8IEU1, A0A384KLI1, A0A1G4H423, A0A077YB01, Q9NFU4, each of which is incorporated herein by reference in its entirety). Exemplary UIS3 amino acid sequence is provided in Table 2.

[0361] Plasmodium falciparum early transcribed membrane protein 10.3 (ETRAMP10.3) is an approximately 10 kDa protein and member of the early transcribed membrane proteins multigene family, a family which is conserved across Plasmodium species and includes proteins located in the parasitophorous vacuole. Several ETRAMP proteins are specific to P. falciparum and not found in Plasmodium species that infect other organisms. ETRAMP10.3 is one example, which is expressed in both liver and blood stage P. falciparum parasites. ETRAMP10.3 transcription has been found to peak during the transition from ring to trophozoite stages of P. falciparum blood stage infection in a human host. ETRAMP10.3 localizes to the parasitophorous vacuole and is exported to a host erythrocyte during blood stage infection. Although ETRAMP10.3 is sometimes referred to as Upregulated in Infectious Sporozoites gene 4 (UIS4), ETRAMP10.3 is understood to be an ortholog of UIS4 on the basis of synteny and structural similarity. However, ETRAMP10.3 is not a functional ortholog of UIS4 and may play a different biological role. Although the biological function of ETRAMP10.3 has not yet been completely resolved, localization to vesicular structures in the host erythrocyte suggests a role in host-parasite interaction or in remodeling of infected erythrocyte. ETRAMP10.3appears to play a key role in the Plasmodium life cycle. When ETRAMP10.3 is deleted, the deletion can lead to the disruption of liver-stage development in mice and asexual blood stage progression.

[0362] Although the terms “UIS4” and “ETRAMP10.3” in the literature are sometimes used to refer to different proteins, in context of the present disclosure, the terms “UIS4” and “ETRAMP10.3” interchangeably to refer to ETRAMP10.3.

[0363] Plasmodium ETRAMP10.3 sequences are known (see, e.g., UniProt accession number Q8IJM9, which is incorporated herein by reference in its entirety). An exemplary ETRAMP10.3 amino acid sequence is provided in Table 2.

[0364] Liver specific protein 1 (LISP-1) is expressed during Plasmodium development in hepatocytes and localized to the parasitophorous vacuolar membrane (PVM) (see, e.g., Ishino et al., Cell Microbiol.2009 Sep; 11(9): 1329–1339, which is incorporated herein by reference in its entirety). LISP-1 was shown to be expressed at high levels during late liver stages development and to be involved in PVM breakdown and subsequent merozoite release (see, e.g., Ishino et al., Cell Microbiol. 2009 Sep; 11(9): 1329–1339, which is incorporated herein by reference in its entirety).

[0365] Intracellular Plasmodium deficient in LISP-1 develop into hepatic merozoites and display normal infectivity to 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 rupture PVM and remain trapped inside hepatocytes (see, e.g., Ishino et al., Cell Microbiol.2009 Sep; 11(9): 1329–1339, which is incorporated herein by reference in its entirety).

[0366] Plasmodium LISP-1 sequences are known (see, e.g., UniProt accession number A0A2I0C2X6, Q8ILR5, each of which is incorporated herein by reference in its entirety). Exemplary LISP-1 amino acid sequence is provided in Table 2.

[0367] 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 was shown to be expressed by liver stages Plasmodium, exported to hepatocytes, and be 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).

[0368] Intracellular Plasmodium deficient in LISP2 do not mature effectively during merozoites development (see, e.g., Orito et al., Mol Microbiol.2013 Jan;87(1):66-79, which is incorporated herein by reference in its entirety).

[0369] Plasmodium LISP-2 sequences are known (see, e.g., UniProt accession number A0A2I0BZR4, Q8I1X6, Q9U0D4, each of which is incorporated herein by reference in its entirety). Exemplary LISP-2 amino acid sequence is provided in Table 2.

[0370] Thrombospondin-related adhesion protein (TRAP) contains an N-terminal domain that is commonly referred to as von Willebrand factor A domain, although it is most similar to an integrin I domain because it contains a metal ion-dependent adhesion site (MIDAS) with a bound Mg2+ion that is required for sporozoite motility in vitro and infection in vivo (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 in an extensible β-ribbon and followed by a thrombospondin repeat (TSR) domain, a proline-rich segment at the C-terminus, 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 SH3-domainbinding PxxP motifs in Plasmodium TRAPs (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).

[0371] TRAP is stored in the micronemes and becomes surface exposed at the sporozoite anterior tip when parasite comes in contact with host cells (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 liver cell invasion of sporozoites by helping sporozoites in gliding motility and in recognition of host receptors on the mosquito salivary gland and hepatocytes (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).

[0372] Plasmodium TRAP sequences are known (see, e.g., UniProt accession numbers A0A5Q2EXK8, A0A5Q2EZD7, A0A5Q2F1F6, A0A5Q2F2B8, A0A5Q2F2H6, A0A5Q2F4G9, O76110, P16893, Q01507, Q26020, Q76NM2, W8VNB6, each of which is incorporated herein by reference in its entirety), and exemplary TRAP amino acid sequence is provided in Table 2.

[0373] Liver-stage-associated protein (LSAP-1) has been shown to be found mainly at the periphery of the intracellular hepatic parasite throughout its development, but not in blood stage parasites and possibly in minor quantities in salivary gland sporozoites (see, e.g., Siau et al., PLoS Pathog.2008 Aug 8;4(8):e1000121, which is incorporated herein by reference in its entirety). LSAP-1 is among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys of sporozoites. Rather, expression has only been detected only in liver stages (see, e.g., Siau et al., PLoS Pathog. 2008 Aug 8;4(8):e1000121, which is incorporated herein by reference in its entirety).

[0374] Plasmodium LSAP-1 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53, each of which is incorporated herein by reference in its entirety). Exemplary LSAP-1 amino acid sequence is provided in Table 2.

[0375] Like LSAP-1, LSAP-2 is also among the most abundant transcripts in the salivary gland transcriptome but has not been detected in proteomic surveys 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.

[0376] Plasmodium LSAP-2 sequences are known (see, e.g., UniProt accession number Q8I632, W7JR53, each of which is incorporated herein by reference in its entirety). Exemplary LSAP-2 amino acid sequence is provided in Table 2.

[0377] Liver-Stage Antigen 1 (LSA-1) is expressed after Plasmodium have invaded hepatocytes and antigen accumulates in the parasitophorous vacuole (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. 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 remains currently not known (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety).

[0378] LSA-1 is a 230 kDa preerythrocytic stage protein containing a large central region consisting of over eighty 17 amino acid residue repeat units flanked by highly conserved C- and N-terminal regions (Richie, T.L. and Parekh, F.K. (2009) Malaria, which is incorporated herein by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds), pp. 1309–1364, Elsevier, which isincorporated herein by reference in its entirety). LSA1 is expressed only by liver stage Plasmodium and not by sporozoites (Richie, T.L. and Parekh, F.K. (2009) Malaria, which is incorporated herein by reference in its entirety). In Vaccines for Biodefense and Emerging and Neglected Diseases (Barrett, A.D.T. and Stanberry L.R., eds), pp. 1309– 1364, Elsevier, which is incorporated herein by reference in its entirety). The repeat region results in significant variation of the protein between strains of Plasmodium falciparum (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety).

[0379] Plasmodium LSA-1 sequences are known (see, e.g., UniProt accession number Q25886, Q25887, Q25893, Q26028, Q9GTX5, O96125, each of which is incorporated herein by reference in its entirety). Exemplary LSA-1 amino acid sequence is provided in Table 2.

[0380] Liver stage antigen 3 (LSA-3) is a 200-kDa protein that is composed of three nonrepeating regions (NR- A, NR-B, and NR-C) flanking 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.), which is incorporated herein by reference in its entirety), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety). The nonrepeat 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 A. J. 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 repeating regions due to organization and number of repeating subunits rather than composition of the repeating regions (see, e.g., Tucker, K. et al., 2016, 'Pre-Erythrocytic Vaccine Candidates in Malaria', in A. J. Rodriguez-Morales (ed.), Current Topics in Malaria, IntechOpen, London. 10.5772 / 65592, which is incorporated herein by reference in its entirety).

[0381] Recently, in vitro data has shown that antibodies against LSA-3 (in particular, the C-terminal portion of LSA-3) may 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).

[0382] Plasmodium LSA-3 sequences are known (see, e.g., UniProt accession number 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, Q8IFR6, 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 herein by reference in its entirety). Exemplary LSA-3 amino acid sequence is provided in Table 2. Table 2: Exemplary amino acid sequencesII. Combinations

[0383] The present disclosure provides combinations of polyribonucleotides that can be used to express one or more Plasmodium polypeptide constructs encoding at least two Plasmodium polypeptides or antigenic portions thereof, wherein the at least two Plasmodium polypeptides or antigenic portions thereof are expressed during different stages of the Plasmodium life cycle. In some embodiments, a combination as described herein comprises one or more polyribonucleotides that encode one or more polypeptides or antigenic portions thereof that are expressed during the Plasmodium sporozoite stage (e.g., shortly after a Plasmodium parasite has entered a subject and before the Plasmodium parasite has infected a hepatocyte), and one or more polypeptides or antigenic portions thereof that are expressed during the Plasmodium liver stage (e.g., after a Plasmodium parasite has entered a hepatocyte).

[0384] In a preferred embodiment, a combination as described herein comprises one or more polyribonucleotides that encode one or more Plasmodium liver stage polypeptides or antigenic portions thereof (e.g., that elicit a T cell response) and one or more polyribonucleotides that encode one or more Plasmodium sporozoite stage (e.g., initial sporozoite stage) polypeptides or antigenic portions thereof (e.g., CSP). In some preferred embodiments, one or more Plasmodium liver stage polypeptides or antigenic portions thereof comprises between 2 to 20 liver stage polypeptides or antigenic portions thereof (e.g., antigenic portions that induce a T cell response).

[0385] In some embodiments, a combination as described herein comprises one or more polyribonucleotides that encode one or more Plasmodium T-cell string polypeptide constructs as described herein and one or more polyribonucleotides that encode one or more Plasmodium CSP polypeptide constructs as described herein.

[0386] In some embodiments, a combination as described herein comprises a first polyribonucleotide that encodes a Plasmodium T-cell string polypeptide construct as described herein and a second polyribonucleotide that encodes a Plasmodium CSP polypeptide construct as described herein. In some embodiments, a combination as described herein comprises a first polyribonucleotide that encodes a first Plasmodium T-cell string polypeptide construct as described herein, a second polyribonucleotide that encodes a second Plasmodium T-cell stringpolypeptide construct as described herein, and a third polyribonucleotide that encodes a Plasmodium CSP polypeptide construct as described herein.

[0387] In some embodiments, a combination comprises a first pharmaceutical composition and a second pharmaceutical composition. In some embodiments, a first pharmaceutical composition comprises a first polyribonucleotide, wherein the first polyribonucleotide encodes a Plasmodium T-cell string polypeptide. In some embodiments, a second pharmaceutical composition comprises a second polyribonucleotide, wherein the first polyribonucleotide encodes a Plasmodium CSP polypeptide. In some embodiments, a first pharmaceutical composition and a second pharmaceutical composition are the same pharmaceutical composition. In some embodiments, a first pharmaceutical composition and a second pharmaceutical composition are different pharmaceutical compositions.

[0388] In some embodiments, a combination comprises a first pharmaceutical composition, a second pharmaceutical composition, and a third pharmaceutical composition. In some embodiments, a first pharmaceutical composition comprises a first polyribonucleotide, wherein the first polyribonucleotide encodes a first Plasmodium T- cell string polypeptide. In some embodiments, a second pharmaceutical composition comprises a second polyribonucleotide, wherein the second polyribonucleotide encodes a second Plasmodium T-cell string polypeptide. In some embodiments, a third pharmaceutical composition comprises a third polyribonucleotide, wherein the third polyribonucleotide encodes a Plasmodium CSP polypeptide. In some embodiments, a first pharmaceutical composition, a second pharmaceutical composition, and a third pharmaceutical composition are the same pharmaceutical composition. In some embodiments, a first pharmaceutical composition, a second pharmaceutical composition, and a third pharmaceutical composition are different pharmaceutical compositions. In some embodiments, a first pharmaceutical composition and a second pharmaceutical composition are the same pharmaceutical composition. In some embodiments, (i) a first pharmaceutical composition or a second pharmaceutical composition and (ii) a third pharmaceutical composition are the same pharmaceutical composition.

[0389] Descriptions of exemplary Plasmodium T-cell string polypeptide constructs and exemplary Plasmodium CSP polypeptide constructs are provided below. A. Plasmodium T-Cell String Polypeptide Constructs

[0390] The present disclosure, among other things, utilizes RNA technologies as a modality to express one or more Plasmodium T-cell string polypeptide constructs (also referred to as “Plasmodium T-cell string polypeptides,” “malaria T-cell string polypeptide constructs” or “malarial T-cell peptide string constructs”). Plasmodium T-cell string polypeptide constructs as described herein can include one or more T-cell antigens from one or more Plasmodium polypeptide, or one or more portions thereof, (e.g., one or more antigenic fragments thereof) as described herein. In some embodiments, a Plasmodium T-cell string polypeptide construct as described herein comprises one or more Plasmodium liver stage antigens that elicit a T cell response. As is understood in the art, a “T-cell antigen” as described herein can induce a T cell response in a subject or model system. In some embodiments, a Plasmodium T- cell string polypeptide construct that targets the liver stage of a Plasmodium infection includes polypeptides or antigenic portions thereof that are expected to be both of relatively high abundance in infected hepatocytes and elicit T cell response(s). Polyribonucleotides as described herein encoding Plasmodium T-cell string polypeptide constructs, as well as Plasmodium T-cell string polypeptide constructs described herein, are designed to deliver a polypeptide to a subject, and in turn, for protein degradation and processing for presentation within the subject so that the subject raises an immune response (e.g., T cell response(s)). Methods to determine the presence of a T-cell response arewell known in the art and described in the examples. In a preferred embodiment, Plasmodium T-cell string polypeptide constructs as described herein include more than one T-cell antigen and / or epitope from Plasmodium liver stage polypeptides or one or more portions thereof. In some preferred embodiments, one or more Plasmodium liver stage polypeptides or antigenic portions thereof comprises between 2 to 20 liver stage polypeptides or antigenic portions thereof (e.g., antigenic portions that induce a T cell response). In some embodiments, a Plasmodium T-cell string polypeptide construct comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 4, 5, 6, 7, 8, 9, 10, 11 or 12 different liver stage polypeptides or antigenic portions thereof (e.g., which each are capable of eliciting a T-cell response).

[0391] For example, in some embodiments, a Plasmodium T-cell string polypeptide construct includes one or more Plasmodium T-cell antigens from CSP, LSA-1 (e.g., LSA-1(a), LSA-1(b)), TRAP, LSAP2, UIS3, ETRAMP10.3, LISP-1, LISP-2, LSA-3, EXP1, LSAP1 and / or polypeptide regions or portions thereof (e.g., one or more antigenic fragments thereof). In some embodiments, a Plasmodium T-cell string polypeptide construct comprises between about 10 amino acids and about 1200 amino acids, e.g., between about 10 amino acids and about 1100 amino acids, e.g., between about 10 amino acids and about 1000 amino acids, e.g., between about 10 amino acids and about 750 amino acids, e.g., between about 25 amino acids and about 500 amino acids. In some embodiments, a Plasmodium T-cell string polypeptide construct comprises about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, abo...

Claims

CLAIMS 1. A combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide encodes a first polypeptide, and the first polypeptide comprises one or more Plasmodium T-cell antigens; and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium polypeptides or antigenic portions thereof.

2. The combination of claim 1, wherein: (a) the first polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, and 221; and (b) the second polypeptide comprises an amino acid sequence with at least 85% identity to an amino acid sequence according to any one of SEQ ID NOs: 5, 8, 10, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 107-111, 112, 117, 122, 125, 130, 135, 138, and 141.

3. The combination of claim 1, wherein: (a) the first polypeptide comprises: (i) an antigenic Plasmodium CSP polypeptide fragment, (ii) an antigenic Plasmodium TRAP polypeptide fragment, (iii) an antigenic Plasmodium UIS3 polypeptide fragment, (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment, and (v) an antigenic Plasmodium LSAP2 polypeptide fragment; and (b) the second polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junction region, (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region, and (viii) a transmembrane region.

4. The combination of claim 3, wherein the first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO: 203.

5. The combination of claim 3 or 4, wherein the second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

33.

6. The combination of any one of claims 3-5, wherein the second polypeptide comprises an amino acid sequence with 100% sequence identity to an amino acid sequence according to SEQ ID NO:

33.

7. The combination of any one of claims 3-6, wherein the combination further comprises a third pharmaceutical composition comprising a third polyribonucleotide, wherein the third polyribonucleotide encodes a third polypeptide, and the third polypeptide comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment, (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment, (iii) an antigenic Plasmodium LISP-2 polypeptide fragment, and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment.

8. The combination of claim 7, wherein the third polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

209.

9. The combination of claim 1, wherein: (a) the first polypeptide comprises: (i) an antigenic Plasmodium CSP polypeptide fragment, (ii) an antigenic Plasmodium TRAP polypeptide fragment, (iii) an antigenic Plasmodium UIS3 polypeptide fragment, (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment, and (v) an antigenic Plasmodium LSAP2 polypeptide fragment; and (b) the second polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal end region, (iii) a Plasmodium CSP junction region, (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (v) a Plasmodium CSP C-terminal region, (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, (vii) a linker, and (viii) a transmembrane region, and wherein the second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof, and (b) an amino acid sequence of NPNA (SEQ ID NO: 228).

10. The combination of claim 9, wherein the first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

203.

11. The combination of claim 9 or 10, wherein the second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

81.

12. The combination of claim 1, wherein: (a) the first polypeptide comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment, (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment, (iii) an antigenic Plasmodium LISP-2 polypeptide fragment, and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment; and (b) the second polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junction region, (v) three repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region, and (viii) a transmembrane region.

13. The combination of claim 12, wherein the first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

209.

14. The combination of claim 12 or 13, wherein the second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

33.

15. The combination of any one of claims 12-14, wherein the second polypeptide comprises an amino acid sequence with 100% sequence identity to an amino acid sequence according to SEQ ID NO: 33.

16. The combination of claim 1, wherein: (a) the first polypeptide comprises: (i) an antigenic Plasmodium LSA-1(a) polypeptide fragment, (ii) an antigenic Plasmodium LSA-1(b) polypeptide fragment, (iii) an antigenic Plasmodium LISP-2 polypeptide fragment, and (iv) an antigenic Plasmodium LISP-1 polypeptide fragment; and (b) the second polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal end region, (iii) a Plasmodium CSP junction region, (iv) nine repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 223), (v) a Plasmodium CSP C-terminal region, (vi) a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, (vii) a linker, and (viii) a transmembrane region, and wherein the second polypeptide does not comprise any of: (a) a Plasmodium CSP N-terminal region or portion thereof, and (b) an amino acid sequence of NPNA (SEQ ID NO: 228).

17. The combination of claim 16, wherein the first polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

209.

18. The combination of claim 16 or 17, wherein the second polypeptide comprises or consists of an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence according to SEQ ID NO:

81.

19. The combination of any one of claims 1-18, wherein the one or more Plasmodium T-cell antigens are one or more P. falciparum T-cell antigens.

20. The combination of any one of claims 1-19, wherein the one or more Plasmodium CSP polypeptide regions or portions thereof are one or more P. falciparum CSP polypeptide regions or portions thereof.

21. The combination of any one of claims 1-20, wherein the first polyribonucleotide and / or second polyribonucleotide is an isolated polyribonucleotide.

22. The combination of claim 7 or 8, wherein the third polyribonucleotide is an isolated polyribonucleotide.

23. The combination of any one of claims 1-22, wherein the first polyribonucleotide and / or second polyribonucleotide is an engineered polyribonucleotide.

24. The combination of any one of claims 7, 8, or 22, wherein the third polyribonucleotide is an engineered polyribonucleotide.

25. The combination of any one of claims 1-24, wherein the first polyribonucleotide and / or second polyribonucleotide is a codon-optimized polyribonucleotide.

26. The combination of any one of claims 7, 8, 22, or 24, wherein the third polyribonucleotide is a codon- optimized polyribonucleotide.

27. The combination of any one of claims 1-26, wherein the first polyribonucleotide is comprised in a first RNA construct, wherein the first RNA construct comprises in 5' to 3' order: (i) a 5' UTR; (ii) the first polyribonucleotide; (iii) a 3' UTR; and (iv) a polyA tail sequence.

28. The combination of any one of claims 1-27, wherein the second polyribonucleotide is comprised in a second RNA construct, wherein the second RNA construct comprises in 5' to 3' order: (i) a 5' UTR; (ii) the second polyribonucleotide; (iii) a 3' UTR; and (iv) a polyA tail sequence.

29. The combination of claim 27 or 28, wherein: (i) the 5' UTR of the first and / or second RNA construct comprises or consists of a modified human alpha-globin 5'-UTR; and (ii) the 3' UTR of the first and / or second RNA construct comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

30. The combination of any one of claims 27-29, wherein the 5' UTR of the first and / or second RNA construct consists of a ribonucleic acid sequence according to SEQ ID NO:

565.

31. The combination of any one of claims 27-30, wherein the 3' UTR of the first and / or second RNA construct consists of a ribonucleic acid sequence according to SEQ ID NO: 567.

32. The combination of any one of claims 27-31, wherein the polyA tail sequence of the first and / or second RNA construct is a split polyA tail sequence.

33. The combination of claim 32, wherein the split polyA tail sequence consists of a ribonucleic acid sequence according to SEQ ID NO:

569.

34. The combination of any one of claims 27-33, wherein the first and / or second RNA construct further comprise a 5' cap.

35. The combination of claim 34, wherein the first and / or second RNA construct comprise a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide.

36. The combination of claim 34 or 35, wherein the 5' cap comprises or consists of m7(3’OMeG)(5')ppp(5')(2'OMeA1)pG2, wherein A1is position +1 of the polyribonucleotide, and G2is position +2 of the polyribonucleotide.

37. The combination of claim 35 or 36, wherein the cap proximal sequence comprises A1and G2of the Cap1 structure, and a sequence comprising: A3A4U5(SEQ ID NO: 571) at positions +3, +4 and +5 respectively of the polyribonucleotide.

38. The combination of any one of claims 1-37, wherein the first and / or second RNA construct includes modified uridines in place of all uridines.

39. The combination of claim 38, wherein modified uridines are each N1-methyl-pseudouridine.

40. The combination of any one of claims 1-39, wherein the first and / or second pharmaceutical composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

41. The combination of claim 40, wherein the first and / or second polyribonucleotide is fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

42. The combination of any one of claims 1-41, wherein the first and / or second pharmaceutical composition further comprises lipid nanoparticles.

43. The combination of any one of claims 40-42, wherein the first polyribonucleotide is encapsulated within the lipid nanoparticles.

44. The combination of any one of claims 40-43, wherein the second polyribonucleotide is encapsulated within the lipid nanoparticles.

45. The combination of any one of claims 1-44, wherein the first and / or second pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

46. The combination of any one of claims 1-45 for use in the treatment of a malaria infection.

47. The combination of any one of claims 1-46 for use in the prevention of a malaria infection.

48. A combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide encodes a first peptide that comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 203; (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second peptide that comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO: 33; and (iii) a second pharmaceutical composition comprising a third polyribonucleotide, wherein the third polyribonucleotide encodes a third peptide that comprises or consists of an amino acid sequence with at least 85% sequence identity to an amino acid sequence according to SEQ ID NO:

209.

49. A combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide encodes a first peptide that comprises or consists of an amino acid sequence with 100% sequence identity to an amino acid sequence according to SEQ ID NO: 203; (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second peptide that comprises or consists of an amino acid sequence with 100% sequence identity to an amino acid sequence according to SEQ ID NO: 33; and (iii) a second pharmaceutical composition comprising a third polyribonucleotide, wherein the third polyribonucleotide encodes a third peptide that comprises or consists of an amino acid sequence with 100% sequence identity to an amino acid sequence according to SEQ ID NO:

209.

50. A method comprising administering a combination of any one of claims 1-49 to a subject.

51. The method of claim 50, wherein the method is a method of treating a malaria infection.

52. The method of claim 50 or 51, wherein the method is a method of preventing a malaria infection.

53. The method of any one of claims 50-52, wherein the subject has or is at risk of developing a malaria infection.

54. The method of any one of claims 50-53, wherein the subject is a human.

55. The method of any one of claims 50-54, wherein administration induces an anti-malaria immune response in the subject.

56. Use of the combination of any one of claims 1-49 in the treatment of a malaria infection.

57. Use of the combination of any one of claims 1-49 in the prevention of a malaria infection.

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