Compositions and methods for treating malaria

Abstract

An anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof or vaccine for use in treating and / or preventing malaria as described herein.

Description

PATENT COMPOSITIONS AND METHODS FOR TREATING MALARIA RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No.63 / 550,715, filed February 7, 2024, the subject matter of which is incorporated herein by reference in its entirety. GOVERNMENT FUNDING

[0002] This invention was made with government support under AI143694 awarded by the National Institutes of Health, and BX001350 awarded by the Department of Veterans Affairs. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 29, 2025, is named CWR-033320WO ORD.st.26 and is 84,288 bytes in size. BACKGROUND

[0004] Half of the world’s population is at risk of malaria. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) account for most human malaria cases. Pf predominates in sub- Saharan Africa, whereas Pv accounts for 80% of malaria in Asia and the Americas. Pv causes significant disease, especially in low-middle-income countries burdened with poor nutrition, anemia, and co-infections. During the erythrocytic or blood stage, Pv merozoites infect reticulocytes, primarily at the sites of erythropoiesis in the bone marrow and spleen. Pv also forms a dormant pre-erythrocytic or liver phase (hypnozoites), causing frequent relapses, further contributing to anemia and other complications. The frequent relapses produce gametocytes that drive transmission in populations. Presently, there is no vaccine available for Pv. Therefore, targeted therapies that inhibit hepatocyte and erythrocyte invasion are crucial in reducing the overall disease burden and enabling Pv elimination.

[0005] An essential piece of the invasion machinery used by sporozoites and merozoites is the interaction between Apical Membrane Antigen 1 (AMA1) and an extracellular ß-hairpin loop in the C-terminal portion of Rhoptry Neck Protein 2 (RON2).Utilized by all members of the Apicomplexa, AMA1 originates in the parasite’s micronemes and is later translocated to the parasite’s membrane. In human hosts, AMA1 is expressed during the late merozoite and sporozoite stages. Immediately preceding invasion, the RON complex (composed of RON2, 4, 5, and 8), is secreted from the parasite’s rhoptries and embeds into the target cell’s membrane. The extracellular ß-hairpin loop near the C-terminus of RON2 can then interact with AMA1 by nestling into the hydrophobic groove of Domain 1 of AMA1. For the RON2-loop to properly engage with AMA1, a mobile loop of Domain 2 of AMA1 is displaced by the incoming ligand to reveal the entire hydrophobic groove. This protein-protein interaction is important for forming a tight junction that allows the merozoite to move across the extracellular space and into the erythrocyte through connections between the parasite surface and its myosin motor along actinfilaments. Blocking the interaction of PfAMA1 or PkAMA1 with PfRON2 or PkRON2 by antibodies or peptides inhibits invasion, confirming that the interaction of AMA1-RON2 is important for the Plasmodium life cycle. While blocking this interaction can prevent invasion, there is evidence that AMA1 interacts directly with the surface of the erythrocyte or with other receptors, which are yet to be defined.

[0006] Malaria-infected individuals acquire partial immunity to infection and disease primarily directed toward blood-stage parasites. Antibodies play a key role in this partial immunity, as demonstrated by transferring human IgG from immune adults to non-immune children, resulting in protection from malaria. The relative contribution of different malaria blood-stage antigens to producing Pv- and Pf-specific antibodies is yet to be fully understood. The antibodies may protect against malaria by one or more mechanisms: 1) blocking merozoite invasion into erythrocytes, 2) complement activation, and 3) opsonic phagocytosis by monocytes and neutrophils. Elevated antibodies to specific Plasmodium antigens, such as AMA1, are associated with protection against infection and disease.

[0007] PvAMA1, a three-domain protein, is under immune selection, posing a problem for developing vaccines and therapeutic monoclonal antibodies to this antigen. Domains 1 and 2 contain two clusters of disulfide-bonded cysteines, with the crystal structure revealing regions comprised of many long loops. Extending from the core of Domain 1, these long loops allow for significant variation and proteinflexibility. This aids parasite evasion of AMA1-specific protective human antibody responses. The loops form a scaffold for the numerous polymorphisms on the surface of AMA1. The hydrophobic RON2 binding grooveis highly conserved across Pv clinical isolates (97.8% over 110 residues). However, this groove is surrounded by highly polymorphic residues, presumably due to selective pressure from host immune responses. Since the AMA1:RON2 interaction plays a role at multiple points of the parasite’s life cycle, including sporozoite infection of the liver and merozoite invasion of erythrocytes, the latter of which is essential for gametogenesis and thus transmission, it presents an opportunity for a multi-stage target.

[0008] Recently, the development of therapeutic monoclonal antibodies to sporozoite antigens protects against malaria in endemic populations. Human monoclonals (humAbs) to Pf circumsporozoite protein (CSP) have been isolated from individuals following immunization with attenuated sporozoites. The administration of humAbs reduced the risk of Pf infection by 88% in adults and protected against illness by up to 77% in children residing in malaria-endemic areas of Africa. Thesefindings suggest that utilizing human monoclonal antibodies specific to sporozoite antigens could be a promising approach to preventing malaria infection and associated illnesses. Additionally, a human-derived monoclonal antibody targeting PfAMA1 has been identified, characterized, and shown to exhibit blocking activity at an IC50 of 35 µg / mL for blood-stage parasites in vitro. To our knowledge, no human-derived monoclonal antibody specific to PvAMA1 has been documented. SUMMARY

[0009] Embodiments described herein relate to anti-Plasmodium vivax Apical Membrane Antigen 1 (anti-PvAMA1) antibodies, antibody fragments, or antigen binding fragments thereof, vaccines including Plasmodium antigens, and their use in treating and / or preventing Plasmodium infections and / or malaria in a subject in need thereof.

[0010] We identified highly potent neutralizing human derived anti-PvAMA1 monoclonal antibodies that inhibit Plasmodium vivax sporozoite invasion of hepatocytes and merozoite invasion of reticulocytes thereby preventing and / or treating malaria caused by Pv infection or disease. The dual activity against sporozoites and merozoites is valuable because new infections and relapses from dormant liver hypnozoites drive Pv disease. Reducing blood-stage infection also attenuates gametocyte production and, thus, Pv transmission.

[0011] We further determined the crystal structure of PvAMA1 bound to an anti- PVAMA1 antibody described herein shows and found that the anti-PVAMA1 antibody partially occupies a highly conserved hydrophobic groove in PvAMA1 that binds its knownreceptor, RON2. The CDR3 of the heavy chain of the anti-PVAMA1 antibody described herein recognizes a conformational epitope that overlaps with the RON2-loop binding site in Domain 1 and displays a higher affinity for PvAMA1 than PvRON2. Previous structural studies suggested that a mobile loop of PvAMA1 Domain 2 partially obstructs the RON2 binding grove, and this mobile loop must be displaced for a successful PvA-MA1:RON2 interaction to occur. The anti-PVAMA1 antibodies described herein can bind to the Domain 2 loop, thus preventing displacement and further interfering with PvRON2 engagement of PvAMA1. The PvAMA1 contact residues that directly interact with anti-PVAMA1 antibodies described herein are conserved or possess single nucleotide polymorphisms (SNP) that do not affect the anti-PVAMA1 antibodies’ potency.

[0012] The discovery of this highly conserved epitope of PvAMA1, which can be targeted by the anti-PVAMA1 antibodies described herein and prevent AMA1-dependent sporozoite invasion into hepatocytes in vivo and merozoite invasion into reticulocytes can provide new treatments that combat Pv infection, disease, and transmission. Moreover, identifying a conserved inhibitory epitope of PvAMA1 effectively targeted by the anti- PVAMA1 antibodies described herein can guide the design of structure-based vaccines for Pv. These insights can also be applied to the PfAMA1 variant for the design of a novel bivalent Pv / Pf vaccine for treating a Pv / Pf infection or disease. The bivalent Pv / Pf vaccine can include a minimalistic antigen, focus the immune response towards the critical epitope of the RON2 binding pocket, and thereby provide a novel malaria vaccine.

[0013] In some embodiments, an anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof can specifically bind an epitope corresponding to or defined by residues Thr164 to Gln175 and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), preferably specifically bind an epitope corresponding to or defined by residues Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), and more preferably specifically bind an epitope corresponding to or defined by residues Thr116 to Gln119, Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65).

[0014] In some embodiments, an anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof can include at least one of: a)a CDR-H1 comprising the amino acid sequence of GGSVSSPGY (SEQ ID NO: 3), a CDR-H2 comprising the amino acid sequence of IYYRGSSNQN (SEQ ID NO: 4), and a CDR-H3 comprising the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5); b) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 8), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 9), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 10); c) a CDR-H1 comprising the amino acid sequence of GFTASGFSFSKAWMG (SEQ ID NO: 24), a CDR-H2 comprising the amino acid sequence of GRIKRKIEGGT (SEQ ID NO: 25), and a CDR-H3 comprising the amino acid sequence of DHPGHYDYIWGSYDLTVDS (SEQ ID NO: 26); d) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 28), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 29), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 30); or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment thereof comprising at least one of a), b), c), or d) to PvAMA1.

[0015] In some embodiments, an anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof can include a heavy chain variable region that includes the 3 CDRs of one of SEQ ID NO: 2 or SEQ ID NO: 23; or a heavy chain variable region that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment thereof comprising a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 or SEQ ID NO: 23 to PvAMA1.

[0016] In some embodiments, an anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include a heavy chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 23.

[0017] In other embodiments, an anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include a light chain variable region that includes the 3 CDRs of one of SEQ ID NO: 7 or SEQ ID NO: 27; or a light chain variable region that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment comprising a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 or SEQ ID NO: 27 to PvAMA1.

[0018] In some embodiments, an anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include a light chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 27.

[0019] In some embodiments, an anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include: a) a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7; or b) a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 27.

[0020] In other embodiments, an anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include: a) a heavy chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 and a light chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7; or b) a heavy chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 and a light chainthat includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27.

[0021] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof described herein is a monoclonal antibody, antibody fragment or antigen binding fragment thereof.

[0022] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof described herein is human, humanized, de-immunized, or chimeric.

[0023] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof is recombinant.

[0024] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof described herein is an IgG, IgM, IgA or an antigen binding fragment thereof.

[0025] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof described herein is a Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, nanobody, or a single domain antibody.

[0026] Other embodiments described herein relate to an anti-PvAMA1 nanobody that includes a CDR having the amino acid sequence of X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10(SEQ ID NO: 11); wherein, X2is R or S; X3 is S, A, or G; X4is R, H, N, or Y; X5 is G or A; X6is Y or H; X7 is F or G; X8is T, S, or N;X9is T or S; X10 is D or E; and wherein SEQ ID NO: 11 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of RSRGEGYCSFGTCYTLFYYFD (SEQ ID NO: 64).

[0027] In other embodiments, the anti-PvAMA1 nanobody can include a CDR having the amino acid sequence of CX1X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10X11W, (SEQ ID NO: 12); wherein X1is A or V; X2 is R or S; X3is S, A, or G; X4 is R, H, N, or Y; X5is G or A; X6 is Y or H; X7is F or G; X8 is T, S, or N; X9is T or S; X10is D or E; X11is Y or N; and wherein SEQ ID NO: 12 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5).

[0028] In some embodiments, the anti-PvAMA1 nanobody can include a CDR having an amino acid sequence selected from: CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5), CARAHGEGHCSGGSCYSLFYYFDYW (SEQ ID NO: 13), CARARGEAYCSGGTCYTLFYYFDYW (SEQ ID NO: 14), CARAHGEGYCSGGSCYSLFYYFDYW (SEQ ID NO: 15), CARAYGEGYCSFGTCYTLFYYFDNW (SEQ ID NO: 16), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 17), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 18),CARGRGEGYCSGGNCYTLFYYFDYW (SEQ ID NO: 19), CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 20), CASGNGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 21), CVSANGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 22), or truncated sequences thereof.

[0029] In some embodiments, the anti-PvAMA1 nanobody can include a polypeptide that has an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

[0030] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody can bind to Domain 1 and / or Domain 2 of PvAMA1.

[0031] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, or nanobody can competitively inhibiting binding of RON2 to PvAMA1.

[0032] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody can be used for use in inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax sporozoite invasion of hepatocytes.

[0033] In other embodiments, the anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody can be used for inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax merozoite invasion of erythrocytes.

[0034] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody can be used for treating and / or preventing Plasmodium infection, preferably Plasmodium vivax infection, preferably in a subject at risk of becoming infected with Plasmodium vivax.

[0035] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody can be used for treating and / or preventing malaria in a subject in need thereof.

[0036] Other embodiments described herein relate to a composition that includes a nucleic acid having a nucleotide sequence encoding an anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody as described herein.

[0037] In some embodiments, the nucleic acid encodes a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 23, and / or a light chain variable region at least about 90% identical to the amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 27.

[0038] In some embodiments, the nucleic acid can include a nucleotide sequence at least about 90%, at least about 95%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69.

[0039] In some embodiments, the nucleic acid encodes a nanobody having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

[0040] In some embodiments, the nucleic acid encoding a nanobody can include a nucleotide sequence at least about 90% identical to the nucleotide sequence of SEQ ID NO: 70.

[0041] Other embodiments described herein relate to a Plasmodium vaccine composition. The Plasmodium vaccine composition can include a vector or a combination of vectors that include a nucleic acid as described herein.

[0042] In some embodiments, the composition can be used for inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax sporozoite invasion of hepatocytes.

[0043] In some embodiments, the composition can be used for inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax merozoite invasion of erythrocytes.

[0044] In some embodiments, the composition can be used for treating and / or preventing Plasmodium infection, preferably Plasmodium vivax infection, preferably in a subject at risk of becoming infected with Plasmodium vivax.

[0045] In some embodiments, the composition can be used for treating and / or preventing malaria in a subject in need thereof.

[0046] Other embodiments described herein relate to a Plasmodium vaccine composition that can include a PvAMA1 antigen amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 75 and / or a nucleic acid encoding the PvAMA1 antigen amino acid sequence.

[0047] In some embodiments, the nucleic acid encoding the PvAMA1 antigen amino acid sequence can include a nucleotide sequence at least about at least about 90% identical to the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 74.

[0048] In some embodiments, the Plasmodium vaccine composition further includes a PfAMA1 antigen amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 38 and / or a nucleic acid encoding the PfAMA1 antigen amino acid sequence.

[0049] In some embodiments, the nucleic acid encoding the PvAMA1 antigen amino acid sequence and / or the PfAMA1 antigen amino acid sequence can include DNA or RNA.

[0050] In other embodiments, the Plasmodium vaccine composition includes a self- assembling PvAMA1 antigen amino acid sequence that comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37 and / or SEQ ID NO: 76 and / or a self-assembling PfAMA1 antigen amino acid sequence that comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 39.

[0051] Other embodiments described herein relate to a self-assembled peptide nanoparticle (SAPN) that includes a self-assembling PvAMA1 antigen amino acid sequence and / or a self-assembling PfAMA1 antigen amino acid sequence. The self-assembling PvAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 75. The self-assembling PfAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 38.

[0052] In some embodiments, the self-assembling PvAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 76.

[0053] In some embodiments, the self-assembling PfAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 39.

[0054] In some embodiments, the vaccine or SAPN can be used for inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax and / or Plasmodium falciparum sporozoite invasion of hepatocytes.

[0055] In some embodiments, the vaccine or SAPN can be used for inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax and / or Plasmodium falciparum merozoite invasion of erythrocytes.

[0056] In some embodiments, the vaccine or SAPN can be used in treating and / or preventing Plasmodium infection, preferably Plasmodium vivax and / or Plasmodium falciparum infection, preferably in a subject at risk of becoming infected with Plasmodium vivax and / or Plasmodium falciparum.

[0057] In other embodiments, vaccine or SAPN can be used in treating and / or preventing malaria in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figs.1(A-E) illustrate sequence characterization of 12 humAbs and their selectivity, avidity, and affinity towards AMA1. A) Sequences of CDR3 IGH (SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 62) and corresponding IGL or IGK (SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 63) from individual B cells from which humAbs were generated. The number of somatic hypermutations (SHM) of nucleotides that differ from germline sequences is shown for each clone. B) 67 clonal groups were identified, from which clonal groups PvAMA1-specific humAbs were isolated is indicated. C) HumAb reactivity to PvAMA1_Palo Alto, PvAMA1_PNG16, PkAMA1, PfAMA1_3D7, and TgAMA1 at varying concentrations (1.0, 0.5, 0.250, 0.125, 0.062, 0.313, 0.016, 0.008 µg / mL). D) HumAb avidity as measured by reduction in binding (MFI) to AMA1 with varying concentrations of NH4SCN (0.0, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 4.0 M). HumAbs were used at a concentration of 0.2 μg / mL. E) Affinities (KDs) of PvAMA1- specific humAbs determined using SPR single-cycle kinetics. HumAbs 800801, 804805, and 808809 affinities could not be determined (N / A) in this assay. Standard error of the mean was calculated using Prism.

[0059] Figs.2(A-C) illustrate HumAbs inhibition of blood-stage infection. A) Dose response of PvAMA1-specific humAbs against Pf-PvAMA1 transgenic parasites of humAb with lowest IC50s. Each symbol represents the average of three replicates for everyconcentration. B) The mean percentage (± SEM) of reticulocytes infected using Pv clinical isolates in short-term invasion inhibition with different humAbs at 100 μg / mL. Each dot represents a biological replicate from a different clinical isolate (n = 2–7). The flow cytometry background of target cells (reticulocytes) without parasites (mean is 9%, range 5– 15% invasion) was subtracted from each experiment. Mouse mAb, 2C3 (100 μg / mL) binds to Duffy antigen on reticulocytes, thus blocking Pv invasion of reticulocytes (positive control) (p-value = 0.9891). Only humAb 826827 significantly inhibited reticulocyte invasion compared to the negative control (p-value = <0.0001). HumAb 043038 was used as a negative control for experiments represented in panel A and B. A multi-variant one-way ANOVA and Tukey’s secondary test was used to calculate the P-values compared to the positive and negative controls using Prism. C) Dose response of humAb 826827 against four different Pv clinical isolates in short-term invasion inhibition cultures (Isolate 1, 2, 3, and 4 have IC50s of 39.96, 66.78, 25.22, and 61.04 μg / mL respectively. Note: These are different isolates than those used for Fig.2B).

[0060] Figs.3(A-B) illustrate PvAMA1-specific humAbs inhibition of sporozoite invasion of human hepatocyte HCO4 cell line and primary human hepatocytes. A) IC50 of different humAbs for Pv sporozoite invasion of human HC04 hepatocytes was performed at five concentrations (0.1–1000 μg / mL). Values represent the mean (SEM) of three biological replicates, with each biological replicate performed in duplicate. 043038 was used as a negative control (p-value = 0.0045). Murine anti-CSP 2F2 was used as a positive control (p- value = >0.999). A multi-variant one-way ANOVA and Tukey’s secondary test was used to calculate the P-values compared to the negative control using Prism. B) Percent inhibition of isolated Pv sporozoites (n = 5) into human primary hepatocytes using 826827 (white bars) at various concentrations.043038 (shaded bar) was used as a negative control and was only tested at one concentration (p-value = <0.0001 at the same concentration). A multi-variant one-way ANOVA and Tukey’s secondary test was used to calculate the P-values compared to the negative control using Prism.

[0061] Figs.4(A-B) illustrate reduction in P. vivax liver infection in FRG-humHep mice after PvAMA1 monoclonal blockaid. Mice were injected intravenously with 30 μg and 300 μg of anti-PvAMA1 (humAb 826827, N = 4 for each concentration) and 300 μg anti- tetanus toxoid (humAb 048038, N = 4) approximately 3 hours before infection with 400,000 freshly dissected P. vivax sporozoites. A) Serum concentrations of humAbs were measured 2days after the sporozoite challenge and on day 8. B) Liver sections were harvested on day 9 post-infection, weighed, homogenized, and P. vivax DNA levels were determined by RT- PCR. Each dot represents one mouse. Shown in mean ± SD. Statistics: one-tailed Mann- Whitney test.

[0062] Figs.5(A-G) illustrate crystal structure of the PvAMA1–Fab 826827 complex. A) Ribbon representation of PvAMA1–Fab 826827 complex (PDB ID: 9DX6). The heavy chain of 826827 is orange, the light chain is yellow. PvAMA1 with N-terminal extension (residues 46-62) is white, Domain 1 (residues 63-248) light blue, Domain 2 (residues 249- 385) dark blue, and Domain 3 purple (residues 386-474). Close-up views show interactions between humAb 826827 and (panel i) the mobile Domain 2 loop of PvAMA1 and (panel ii) the hydrophobic groove. For clarity, only polar interactions between side chains are indicated with dotted lines. B) Five CDR loops (L1, L2, H1, H2, and H3) are involved in PvAMA1 binding. PvAMA1, in surface representation, is colored as described in panel A. The size of the buried interaction surface is indicated. C) Comparison of PvAMA1 when bound by CDR-H3 of Fab 826827 or PvRON2 peptide (PDB ID: 5NQG). PvAMA1 is shown with transparency around the Domain 2 loop (dark blue). CDR-H3 and PvRON2 peptide (magenta) form disulfide-linked β-hairpin loops that bind to the hydrophobic groove on Domain 1. HumAb 826827 stabilizes the Domain 2 Ioop in a closed position on Domain 1. This loop is dislocated when PvRON2 peptide is bound to PvAMA1. D) Ribbon representation of overlaid PvAMA1 structures, comprising our structure of PvAMA1 in complex with Fab 826827 (blue, PDB ID: 9DX6), PvAMA1 in complex with PvRON2 peptide (light blue, PDB ID: 5NQG), and unbound PvAMA1 (gray, PDB ID: 1W8K). This indicates that 826827 stabilizes the Domain 2 loop and allows for the refinement shown. Residues between T296 and F335 are absent in the electron density map of PDB 5NQG and 1W8K but residues 304–327 are well defined in our structure. Overlays were generated using Pymol, and refined root mean square deviation values are indicated. E) Top view of the PvAMA1 overlays. F) Overlay of PvAMA1 (blue, PDB ID: 9DX6) with PkAMA1 (green, PDB ID: 4UV6). The mobile Domain 2 loops adopt a similar conformation in both structures. G) Top view of PvAMA1 and PkAMA1 overlay. (see Table 7 for data collection and refinement statistics).

[0063] Figs.6(A-E) illustrate the interaction residues of PvAMA1 with RON2 and 826827. A) Sequence alignment of published AMA1 amino acid sequences with thecorresponding binding residues for RON2 (magenta) and humAb heavy chain (826; orange), light chain (827; yellow), or both chains (blue) arrows. The first line of the alignment represents the deposited structure 9DX6 corresponding to the PvAMA1 Palo Alto strain (ACB42438). Pv is the Sal1 variant (PVX_092275). The red box outlines the Domain 2 loop (304–327), highlighting 826827’s interactions with this region of PvAMA1. B) Competition assay using 50 µg / mL of PvRON2 (Asp 2050 – Thr 2088) that competed with varying concentrations of humAb 826827 (40–0.039 µg / mL) to bind recombinant PvAMA1.043038 was used as a negative control. Error bars indicate + / − SEM. C) PvAMA1 residues contacting humAb heavy chain 826 (orange circles), light chain 827 (yellow squares), or both humAb chains (dark blue hexagons) and their mutations are displayed on the X-axis while the Y-axis shows the conservation of that position within 390 clinical isolate sequences. Highlighted amino acids represent those depicted in Fig.6D. D) Structural analysis of the interaction between 826 (orange) 827 (yellow) and PvAMA1 (blue) shows three observed polymorphisms in the binding epitope (G117R, N132D, and N130K; pink). E) The pvama1 gene was sequenced in the seven Pv clinical isolates (Table 6). Amino acids 117, 130, and 132 represent polymorphic contact residues of humAb 826827 to PvAMA1 Sal1 reference strain (PVX_092275). Bars represent the mean (+ SEM) of humAb invasion inhibition of WT compared to an isolate expressing a SNP at each of the three polymorphic residues (n = 7).

[0064] Fig.7 illustrates blocking activity of serum from Cambodian individuals. Inhibition of PvRON2 binding to PvAMA1 by plasma samples from Cambodian donors. Plasma samples were tested at 1 / 50 dilution for the ability to inhibit binding of the PvRON2 loop to PvAMA1 in a plate-based assay. Samples were tested in duplicate. Data show mean and range; the dotted line shows 50% inhibition. PBMCs from donor C5 were selected for sorting B cells specific for PvAMA1 and subsequent MAb generation. Error bars show + / - SEM.

[0065] Fig.8 illustrates a representative Flow diagram depicting PvAMA1-specific B cells that were isolated. B cells were enriched using immunomagnetic positive selection with anti-CD19 magnetic MACS beads (Miltenyi Biotec, upper left panel). SYTOX Green Dead Cell Stain (Invitrogen) was used to gate out dead cells (lower left panel). Doublet discrimination was performed to exclude aggregated cells (upper middle panel) and stained with mouse anti-human CD20 (PE-Cy5.5; Invitrogen) and anti-human IgG Abs (PE-Cy7 clone G18-145; Becton Dickinson) to identify IgG expressing B cell (lower middle panel). PvAMA1-specific B cells were identified using biotinylated PvAMA1 using Streptavidin coupled with Fluorescein isothiocyanate (FITC) or Brilliant Violet 421 (right panel showing sort gate). PvAMA1-cells were sorted on a BD FACSAria II.

[0066] Fig.9 illustrates HumAb Single Cycle Kinetics Curves and Results. A) Binding response curves to various concentrations of PvAMA1 for each humAb. B) kon and koff rates for each humAb determined using SPR.

[0067] Fig.10 illustrates replicates of Pf-PvAMA1 cell line inhibition. Dose response inhibition curves of PvAMA1-specific humAbs against Pf-PvAMA1 transgenic parasites. Each humAb was tested in triplicate as indicated in different colored lines, and IC50 was calculated using R.043038, an anti-tetanus toxoid humAb was used as a negative control.

[0068] Figs.11(A-B) illustrate sporozoite HC04 invasion separated CSP210 / 247. Dose response inhibition curves of Pv sporozoites blocking by PvAMA1 specific humAbs. Murine anti-CSP monoclonal antibodies served as positive control of blocking inhibition. Three different sporozoite isolates were used for this assay. Based on the blocking activity with the anti-CSP monoclonal one can separate two CSP210 and one CSP247 experiments. Shown below in A) are the dose-responses obtained with CSP210 strains and in B) with strain CSP247. The calculated IC 50 for CSP210 is 0.08 µg / mL and for CSP247 IC 50 ~8 µg / mL HumAbs against PvAMA1 were randomly screened with these isolates. Of note, humAb 826827 was screened with both sporozoite strains and shows potent inhibition in both in contrast to the CSP monoclonal.

[0069] Fig.12 illustrates microscopic assessment of P. vivax liver in FRGN huHep mice after administration of anti-AMA1 human monoclonal antibody 826827. The experimental design was identical to that described in the legend of Fig.4. On day 9, liver sections were analyzed microscopically for the presence of parasites described by (Mikolajczak et al. CHM, 2015). Both hypnozoites and schizonts were observed. This analysis shows total parasite forms in the liver (hypnozoites plus schizonts). Of note, one of the control livers could not be adequately evaluated and was not included in the analysis. Each dot represents one mouse. Shown in mean ± SD. Statistics: unpaired t-test.

[0070] Figs.13(A-C) illustrate surface properties of PvAMA1 and its interaction partners RON2 and humAb 826827 A) Schematic overview indicating the Domain 1 of PvAMA1 in gray with the Domain 2 loop in golden and the bound CDR3 loop of 826 in red. B) Hydrophobic surface potential of the PvAMA1 binding site. 826 was removed and rotatedby 180º compared to the orientation in A to show the corresponding bottom interface of the interaction as well as the corresponding PvRON2 peptide. Darker areas represent higher hydrophobicity. Blue areas show hydrophilicity. C) Surface potential of the binding site. Red indicates negatively charged areas. Blue indicates positively charged areas. Figures were generated with Vida 4.4 from OpenEye.

[0071] Figs.14(A-F) illustrate RON2 binding groove conservation and Sequence differences between Pv, PvPNG16, Pf, Pc, Pk, Tg. For all images, a residue-colored orange indicates an amino acid change between PvAMA1_PaloAlto (PDB: 9DX6) and another species’ version of AMA1 A) Sequence conservation of residues surrounding the RON2 binding groove across 390 published clinical isolates B) PvAMA1_PaloAlto versus PvAMA1_PNG16 C) PvAMA1_PaloAlto versus P. cynomolgi D) PvAMA1_PaloAlto versus P. knowlesi E) PvAMA1_PaloAlto versus P. falciparum F) PvAMA1_PaloAlto versus Toxoplasma gondii.

[0072] Figs.15(A-B) illustrate published PvAMA1 clinical isolates sequence conservation A) Percent conservation of each amino acid of PvAMA1 across 390 published clinical isolates. Yellow circles represent Domain 1 residues. Orange triangles represent Domain 2 residues. Blue squares represent Domain 3 residues. B) Percent conservation of PvAMA1 residues that contact the RON2 extracellular loop.

[0073] Figs.16(A-B) illustrate epitope and paratope of the PvAMA1-826827 complex. (A) Surface representation of PvAMA1 with the epitope recognized by 826827 colored in yellow (LC contribution) and orange (HC contribution). (B) Surface representation of humAb 826827 with the paratope of PvAMA1 colored light blue (domain 1 contribution) and blue (domain 2 loop contribution). Residues within a distance cut-off of 5 Å are highlighted.

[0074] Figs.17(A-B) illustrate representative 2Fo-Fc electron density map of the PvAMA1-826827 crystal structure. The final 2Fo-Fc electron density map (blue, contoured at 1.0 σ) of CDR-H3 residues 105-121 (A), and PvAMA1 residues 308-317 of the D2-loop (B).

[0075] Fig.18 illustrates a ribbon representation of a PvAMA1-mAB 864865 complex.

[0076] Fig 19 illustrates a plot showing a competition assay using 50 µg / ml of PvRON2 (Asp 2050 – Thr 2088) that competed with varying concentrations of mAb 826827 or 864865 (40–0.039 µg / mL) to bind recombinant PvAMA1.

[0077] Fig.20 illustrates a graph showing A) mAbs inhibit blood-stage infection. The mean percentage (± SEM) of reticulocytes infected with Pv clinical isolates was calculated in short-term invasion inhibition studies with different mAbs at 100 µg / ml was calculated. Each dot represents a biological replicate from a different clinical isolate. The flow cytometry background of target cells (reticulocytes) without parasites (mean 9%, range 5-15%) was subtracted. Mouse mAb 2C3 (100 µg / ml) binds to Duffy Ag on reticulocytes, thus blocking Pv invasion (positive control). Anti-tetanus C-term mAb 043038 was used as a negative control. B) PvAMA1-specific mAbs inhibit sporozoite invasion of human hepatocyte HCO4 cell line. IC50 were calculated at five concentrations (0.1–1000 μg / ml). Values represent the mean (SEM) of three biological replicates, with each replicate performed in duplicate. 043038 was used as a negative control. Murine anti-CSP 2F2 was used as a positive control. The biological replicate for CSP 2F2 with IC50 ~9 ug / ml was CSP247 variant; the other two isolates were CSP210. DETAILED DESCRIPTION

[0078] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0079] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references,including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol.185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R. I. Freshney.1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp.109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0080] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0081] As used herein, the terms "immunogenic protein, polypeptide, or peptide" or “antigen” refer to polypeptides or other molecules (or combinations of polypeptides and other molecules) that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and / or cellular type directed against the protein. In embodiments, the protein fragment has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the disclosure can comprise or consist essentially of or consists of at least one epitope or antigenic determinant. An "immunogenic" protein or polypeptide, as used herein, may include the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. "Immunogenic fragment" refers to a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above.

[0082] Synthetic antigens are also included within the definition, for example, poly- epitopes, flanking epitopes, and other recombinant or synthetically derived antigens. Immunogenic fragments for purposes of the disclosure may feature at least about 1 amino acid, at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

[0083] As used herein, the term "epitope" refers to the site on an antigen or hapten to which specific B cells and / or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0084] As used herein, the term "immunological response" to a composition or vaccine refers to the development in the host of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. The host may display either a therapeutic or protective immunological response so resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host.

[0085] As used herein, the term "variant" refers to a substantially similar sequence. For polynucleotides, a variant comprises a deletion and / or addition and / or change of one or more nucleotides at one or more sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or an amino acid sequence, respectively. Variants of a particular polynucleotide of the disclosure (e.g., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. "Variant" protein is intended tomean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and / or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they have the ability to elicit an immune response.

[0086] As used herein, the term "acceptor human framework" refers to a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework, or from a human consensus framework.

[0087] As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, primatized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including, e.g., Fab′, F(ab′)2, Fab, Fv, IgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab′)2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non- specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med.24:316, 1983; incorporated herein by reference). Similarly, each heavy chain contains a variable region ("VH-region") and three constant domains ("CH1-," "CH2-," and "CH3-regions") and a hinge region.

[0088] As used herein, an “antibody fragment” or “antigen binding fragment” (i.e. characteristic portion of an antibody) refers to any derivative of an antibody which is less than full-length. In some embodiments, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of such antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, VHH, and Fd fragments. Antibody fragments also include, but are not limited, to Fc fragments.

[0089] Antigen binding fragments can be prepared from full-length antibody by protease digestion. Antigen binding fragments may be produced using standard recombinant DNA methodology by those skilled in the art.

[0090] As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The antibodies described herein may comprise modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a 6-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, 5 Bethesda, Md.1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated.

[0091] CDRs, as antigen binding fragments, can also be incorporated into single domain antibodies, maxi bodies, mini bodies, intrabodies, diabodies, triabodies, tetra bodies, and bis-scFv. Antigen binding fragments of antibodies can be grafted into scaffolds based on polypeptides. Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.

[0092] As used herein, a "single chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0093] As used herein “effector functions” refer to those biological activities attributable to the native Fc region of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation. In order to minimize or eliminate side effects of a therapeutic antibody, it may be preferable to minimize or eliminate effector functions.

[0094] An “engineered antibody” is an antibody that is not naturally produced, and which has been altered or created to achieve a specific purpose or to have a specific characteristic. For example, antibodies which have undergone deliberate modifications to their wild type forms, to have reduced effector functions, are engineered antibodies.

[0095] As used herein, the term “Fc region” refers to the region of the antibody that provides defense to a given antigen.

[0096] As used herein, the term “first portion of the antibody” refers to a portion of a whole antibody, a portion less than the whole, which contains the antigen binding regions of the antibody. “Second portion of the antibody” refers to a portion of a whole antibody, a portion less than the whole, which consists of the portion of the antibody which is not included in the first portion.

[0097] The terms "Fc receptor" or "FcγR" describe a receptor that binds to the Fc region of an IgG. “FcγRI,” “FcγRII,” and “FcγRIII” are subclasses of FcγRs.

[0098] A “modification” to an antibody, antibody fragment, antigen binding fragment and / or Fc region of an antibody, refers to a substitution, insertion, or deletion of one or more amino acids in the protein’s wild type polypeptide sequence. A modified antibody, antibody fragment, antigen binding fragment and / or Fc region is one in which a modification has been artificially made.

[0099] As used herein, “competitively inhibits” refers to competitive inhibition of binding of an isolated antibody, antibody fragment, or antigen binding portion thereof to PvAMA1, by any other molecule.

[0100] As used herein, “Fab fragment” refers to the variable (VL) and constant (CL) domains of the light chain and the variable (VH) and first constant (CH1) domains of the heavy chain. Fab' fragments differ from Fab fragments by the few extra residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab' fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product.

[0101] As used herein, the term "human consensus framework" refers to a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.

[0102] As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos.4,444,887 and 4,716,111; and PCT publications WO 1998 / 46645; WO 1998 / 50433; WO 1998 / 24893; WO 1998 / 16654; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; incorporated by reference herein.

[0103] As used herein, the term “humanized” antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FRregions may also be those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No.6,180,370 to Queen et al.; EP239400; PCT publication WO 91 / 09967; U.S. Pat. No.5,225,539; EP592106; and EP519596; incorporated herein by reference.

[0104] As used herein, the term "monovalent antibody or antigen binding fragment thereof" refers to an antibody or antigen binding fragment thereof comprising a single binding domain, e.g., VH or VHH, for an antigen.

[0105] As used herein, the term "single domain antibody" defines molecules where the antigen binding site is present on, and formed by, a single immunoglobulin domain. Generally, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. The single variable domain may, for example, include a light chain variable domain sequence (a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially is the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

[0106] As used herein, the term "camelid antibody" refers to an antibody derived from a camelid species, for example, in a camel, dromedary, llama, alpaca or guanaco. Camelid antibodies differ from those of most other mammals in that they lack a light chain, and thus include only heavy chains with complete and diverse antigen binding capabilities (Hamers- Casterman, C. et al., Nature, 363:446-8, 1993).

[0107] As used herein, the term "VHH" refers to a single heavy chain variable domain antibody devoid of light chains. VHH chains, for example, can be of the type that can be found in Camelidae that are naturally devoid of light chains or to a synthetic and non- immunized VHH that can be constructed accordingly. Each heavy chain includes a variable region encoded by V-, D- and J-exons. A VHH may be a natural VHH antibody, e.g., a camelid antibody, or a recombinant protein including a heavy chain variable domain.

[0108] As used herein, the terms “single domain antibody (VHH)” and “nanobodies” have the same meaning referring to a variable region of a heavy chain of an antibody, and construct a single domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Generally, the antibodies with a natural deficiency of the light chain and the heavy chain constant region 1 (CH1) are first obtained, the variable regions of the heavy chain of the antibody are therefore cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0109] As used herein, the term an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to a Plasmodium antigen, such as PvAMA1, is substantially free of contaminants, e.g., antibodies that do not bind to a Plasmodium antigen, such as PvAMA1). In addition, an "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that could interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0110] As used herein, the term "specific binding" of an antibody or fragment thereof, polypeptide, or peptidomimetic is binding to a target molecule that is measurably different from binding to molecules that are not target molecules. As used herein, specific binding refers to a greater than 95% preference for binding a particular antigen versus background ("non-specific") binding. "Substantially specific" binding refers to a greater than about 80% preference for binding a particular antigen versus background. Binding can be measured using a variety of methods including, but not limited to, Western blot, immunoblot, enzyme- linked immunosorbent assay ("ELISA"), radioimmunoassay ("RIA"), immunoprecipitation, surface plasmon resonance, bio-layer interferometry, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption / ionization time-of-flight ("MALDI-TOF") mass spectrometry, micro cytometry, microarray, microscopy, fluorescence activated cell sorting ("FACS") and flow cytometry.

[0111] As used herein, the term "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain (e.g., polypeptide portion) foreign to and not substantially homologouswith any domain of the first polypeptide. A chimeric protein may present a foreign domain, which is found (albeit in a different protein) in an organism, which also expresses the first protein, or it may be an "interspecies", "intergenic", etc. fusion of protein structures expressed by different kinds of organisms.

[0112] As used herein, the term "identical" or “substantially identical" with respect to an antibody chain polypeptide sequence may be construed as an antibody chain exhibiting at least 65%, 70%, 80%, 90% or 95% sequence identity to the reference polypeptide sequence present in the variable region of the antigen binding fragment. The term with respect to a nucleic acid sequence may be construed as a sequence of nucleotides exhibiting at least about 65%, 75%, 85%, 90%, 95% or 97% sequence identity to the reference nucleic acid sequence.

[0113] As used herein, the term "individual" refers to a vertebrate, preferably a mammal and more preferably a human. Individuals amenable to treatment include those who are presently asymptomatic, but who are at risk of developing a symptomatic disorder in which the alternative complement pathway plays a role, or in which activation of the alternative complement pathway plays a role.

[0114] As used herein, the term "mammal" refers to any animal classified as a mammal includes humans, higher primates, domestic and farm animals, horses, pigs, cattle, dogs, cats and ferrets, etc. In one embodiment of the invention, the mammal is a human.

[0115] As used herein, “monoclonal antibody" refers to a homogeneous population of antibodies. Such antibodies are highly specific and are directed against a single target antigen. These monoclonal antibodies are homogeneously produced by the hybridoma culture, uncontaminated by other immunoglobulins. Monoclonal antibodies can also be produced by other procedures such as phase display by well known methods.

[0116] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures.

[0117] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.

[0118] As used herein, the term "homology" and "identity" are used synonymously throughout and refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. A degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0119] As used herein, the term "mutant" refers to any change in the genetic material of an organism, in particular a change (i.e., deletion, substitution, addition, or alteration) in a wild type polynucleotide sequence or any change in a wild type protein. The term "variant" is used interchangeably with "mutant". Although it is often assumed that a change in the genetic material results in a change of the function of the protein, the terms "mutant" and "variant" refer to a change in the sequence of a wild type protein regardless of whether that change alters the function of the protein (e.g., increases, decreases, imparts a new function), or whether that change has no effect on the function of the protein (e.g., the mutation or variation is silent).

[0120] As used herein, the term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

[0121] As used herein, the phrases "parenteral administration" and "administered parenterally" are art- recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra- articular, subcapsular, subarachnoid, and intraspinal injection and infusion.

[0122] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, agent or other material other than directly into a specific tissue, organ, or region of the subject being treated (e.g., brain), such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0123] As used herein, the terms "patient", “subject”, "mammalian host," and the like are used interchangeably herein, and refer to mammals, including human and veterinary subjects.

[0124] As used herein, the terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein. As used herein, “polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isomers). “Polypeptide(s)” refers to both short chains, commonly referred as peptides, oligopeptides or oligomers, and to longer chains generally referred to as proteins.

[0125] As used herein, the terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0126] "Recombinant," as used herein, means that a protein is derived from a prokaryotic or eukaryotic expression system.

[0127] As used herein, the terms "therapeutic agent", "drug", "medicament" and "bioactive substance" are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include without limitation pharmaceutically acceptable salts thereof and prodrugs. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may be prodrugs in the form of ethers, esters, amides and the like that are biologically activated when administered into a patient or subject.

[0128] As used herein, the phrase "therapeutically effective amount" or “pharmaceutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on anumber of factors, including: the rate of release of an agent from a polymer matrix, which will depend in part on the chemical and physical characteristics of the polymer; the identity of the agent; the mode and method of administration; and any other materials incorporated in the polymer matrix in addition to the agent.

[0129] As used herein, the term "wild type" refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.

[0130] As used herein, the term “carrier” or “pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” refers to any appropriate or useful carrier or vehicle for introducing a composition to a subject. Pharmaceutically acceptable carriers or vehicles may be conventional but are not limited to conventional vehicles. For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21stEdition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules. Carriers (e.g., pharmaceutical carriers, pharmaceutical vehicles, pharmaceutical compositions, pharmaceutical molecules, etc.) are materials generally known to deliver molecules, proteins, cells and / or drugs and / or other appropriate material into the body. In general, the nature of the carrier will depend on the nature of the composition being delivered as well as the particular mode of administration being employed. In addition to biologically-neutral carriers, pharmaceutical compositions administered may contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like. Patents that describe pharmaceutical carriers include, but are not limited to: U.S. Patent No.6,667,371; U.S. Patent No.6,613,355; U.S. Patent No.6,596,296; U.S. Patent No.6,413,536; U.S. Patent No. 5,968,543; U.S. Patent No.4,079, 038; U.S. Patent No.4,093,709; U.S. Patent No.4,131,648; U.S. Patent No.4,138,344; U.S. Patent No.4,180,646; U.S. Patent No.4,304,767; U.S. Patent No.4,946,931, the disclosures of which are incorporated in their entirety by reference herein. The carrier may, for example, be solid, liquid (e.g., a solution), foam, a gel, the like, or a combination thereof. In some embodiments, the carrier comprises a biological matrix (e.g., biological fibers, etc.). In some embodiments, the carrier comprises a synthetic matrix(e.g., synthetic fibers, etc.). In certain embodiments, a portion of the carrier may comprise a biological matrix and a portion may comprise synthetic matrix.

[0131] Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0132] Embodiments described herein relate to anti-Plasmodium vivax Apical Membrane Antigen 1 (anti-PvAMA1) antibodies, antibody fragments, or antigen binding fragments thereof, vaccines including Plasmodium antigens, and their use in treating and / or preventing Plasmodium infections and / or malaria in a subject in need thereof.

[0133] We identified highly potent neutralizing human derived anti-PvAMA1 monoclonal antibodies that inhibit Plasmodium vivax sporozoite invasion of hepatocytes and merozoite invasion of reticulocytes thereby preventing and / or treating malaria caused by Pv infection or disease. The dual activity against sporozoites and merozoites is valuable because new infections and relapses from dormant liver hypnozoites drive Pv disease. Reducing blood-stage infection also attenuates gametocyte production and, thus, Pv transmission.

[0134] We further determined the crystal structure of PvAMA1 bound to an anti- PVAMA1 antibody described herein shows and found that the anti-PVAMA1 antibody partially occupies a highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. The CDR3 of the heavy chain of the anti-PVAMA1 antibody described herein recognizes a conformational epitope that overlaps with the RON2-loop binding site in Domain 1 and displays a higher affinity for PvAMA1 than PvRON2. Previous structural studies suggested that a mobile loop of PvAMA1 Domain 2 partially obstructs the RON2 binding grove, and this mobile loop must be displaced for a successful PvA-MA1:RON2 interaction to occur. The anti-PVAMA1 antibodies described herein can bind to the Domain 2 loop, thus preventing displacement and further interfering with PvRON2 engagement of PvAMA1. The PvAMA1 contact residues that directly interact with anti-PVAMA1antibodies described herein are conserved or possess single nucleotide polymorphisms (SNP) that do not affect the anti-PVAMA1 antibodies’ potency.

[0135] The discovery of this highly conserved epitope of PvAMA1, which can be targeted by the anti-PVAMA1 antibodies described herein and prevent AMA1-dependent sporozoite invasion into hepatocytes in vivo and merozoite invasion into reticulocytes can provide new treatments that combat Pv infection, disease, and transmission. Moreover, identifying a conserved inhibitory epitope of PvAMA1 effectively targeted by the anti- PVAMA1 antibodies described herein can guide the design of structure-based vaccines for Pv. These insights can also be applied to the PfAMA1 variant for the design of a novel bivalent Pv / Pf vaccine for treating a Pv / Pf infection or disease. The bivalent Pv / Pf vaccine can include a minimalistic antigen, focus the immune response towards the critical epitope of the RON2 binding pocket, and thereby provide a novel malaria vaccine.

[0136] In some embodiments, an anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof described herein specifically binds a conformational epitope of PvAMA1 that overlaps with the RON2-loop binding site in Domain 1 and the mobile loop of Domain 2, thus preventing displacement and competitively inhibiting binding of PvRON2 with PvAMA1. As discussed in the Example, five of the six complementarity-determining regions (CDR, namely: L1, L2, H1, H2, and H3) of an anti-PvAMA1 antibody form direct contacts with PvAMA1 with a buried interaction surface, with the CDR-H3 loop of the anti- PvAMA1 antibody contributing 70% of the buried surface area. The CDR-H3 loop of anti- PvAMA1 antibody forms a disulfide bridged β-hairpin that binds to the hydrophobic groove on PvAMA1 Domain 1, which constitutes part of the RON2-loop receptor binding site. CDR-H3 binding to the PvAMA1-hydrophobic groove involves 53 interatomic contacts. The mobile Domain 2 loop of PvAMA1 is contacted by residues of CDR-H3 and CDR-H1, forming one salt bridge and six hydrogen bonds that stabilize its position on Domain 1. CDR- L2 and H2 interact with PvAMA1 residues of Domain 1 loops that surround the mobile Domain 2 loop, and CDR-L1 forms contacts with a Domain 1 loop next to the hydrophobic groove. Two residues located in the Domain 2 loop of AMA1, Arg317 and Lys321, provide a positively charged patch at the bottom of the RON2-loop binding groove. Otherwise, this binding pocket is largely hydrophobic. One salt bridge between PvAMA1 Lys321 and the anti-PvAMA1 antibody CDR-H3 Glu103 provides an anchoring and orientation point for the observed interaction between the two proteins.

[0137] In some embodiments, the conformational epitope of PvAMA1 that overlaps with the RON2-loop binding site in Domain 1 and the mobile loop of Domain 2 can include residues Thr164 to Gln175 and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), preferably an epitope corresponding to residues Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), and more preferably an epitope corresponding to residues Thr116 to Gln119, Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65).

[0138] In some embodiments, an anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof can specifically bind an epitope corresponding to or defined by residues Thr164 to Gln175 and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), preferably specifically bind an epitope corresponding to or defined by residues Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), and more preferably specifically bind an epitope corresponding to or defined by residues Thr116 to Gln119, Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65).

[0139] In some embodiments, an anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof can include at least one of: a) a CDR-H1 comprising the amino acid sequence of GGSVSSPGY (SEQ ID NO: 3), a CDR-H2 comprising the amino acid sequence of IYYRGSSNQN (SEQ ID NO: 4), and a CDR-H3 comprising the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5); b) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 8), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 9), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 10); c)a CDR-H1 comprising the amino acid sequence of GFTASGFSFSKAWMG (SEQ ID NO: 24), a CDR-H2 comprising the amino acid sequence of GRIKRKIEGGT (SEQ ID NO: 25), and a CDR-H3 comprising the amino acid sequence of DHPGHYDYIWGSYDLTVDS (SEQ ID NO: 26); d) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 28), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 29), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 30); or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment thereof comprising at least one of a), b), c), or d) to PvAMA1.

[0140] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof includes a heavy chain with CDR-H1, CDR-H2, and CDR- H3 amino acid sequences of respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and / or a light chain with CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and / or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof to PvAMA1.

[0141] In other embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof includes a heavy chain with CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 and / or a light chain with CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 and / or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof to PvAMA1.

[0142] In general, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof, typically comprises (at least) three complementarity determining regions (CDRs) on a heavy chain and (at least) three CDRs on a light chain. In general, complementarity determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. Typically, the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor. Usually, the three CDRs (CDR1, CDR2, and CDR3) are arranged non-consecutively in the variable domain. Since antigen receptors are typically composed of two variable domains (on two different polypeptide chains, i.e., heavy and light chain: heavy chain variable region (VH) and light chain variable region (VL)), there are typically six CDRs for each antigen receptor (heavy chain: CDR-H1, CDR-H2, and CDR-H3; light chain: CDR-L1, CDR-L2, and CDR-L3). A classical single antibody molecule usually has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy and / or light chain may be separated by framework regions, whereby a framework region (FR) is a region in the variable domain which is less “variable” than the CDR. For example, a chain (or each chain, respectively) may be composed of four framework regions, separated by three CDR's.

[0143] In some embodiments, the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 to PvAMA1.

[0144] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of one of SEQ ID NO: 2 wherein the CDR sequences as defined above (heavy chainCDR-H1, CDR-H2, and CDR-H3 sequences as set forth in respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5) are maintained.

[0145] In some embodiments, the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 to PvAMA1.

[0146] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of one of SEQ ID NO: 23 wherein the CDR sequences as defined above (heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences as set forth in respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26) are maintained.

[0147] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 to PvAMA1.

[0148] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a light chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 wherein the CDR sequences as defined above (light chain CDR- L1, CDR-L2, and CDR-L3 sequences as set forth in respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10) are maintained.

[0149] In other embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a light chain variable region that includes the 3 CDRs of SEQ ID NO: 27 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a light chain variable region that includes the 3 CDRs of SEQ ID NO: 27 to PvAMA1.

[0150] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a light chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27 wherein the CDR sequences as defined above (light chain CDR- L1, CDR-L2, and CDR-L3 sequences as set forth in respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30) are maintained.

[0151] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 to PvAMA1.

[0152] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 wherein the CDR sequences as defined above (heavy chain CDR-H1, CDR- H2, and CDR-H3 sequences as set forth in respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5) are maintained and a light chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 wherein the CDR sequences as defined above (light chain CDR-L1, CDR-L2, and CDR-L3 sequences as set forth in respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10) are maintained.

[0153] In other embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 27 or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of the anti-PvAMA1antibody, antibody fragment, or antigen binding fragment thereof that includes a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 27 to PvAMA1.

[0154] In other embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes a heavy chain that includes an amino acidsequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 wherein the CDR sequences as defined above (heavy chain CDR-H1, CDR- H2, and CDR-H3 sequences as set forth in respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26) are maintained and a light chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27 wherein the CDR sequences as defined above (light chain CDR-L1, CDR-L2, and CDR- L3 sequences as set forth in respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30) are maintained.

[0155] Sequence identity is usually calculated with regard to the full length of the reference sequence (i.e. the sequence recited in the application). Percentage identity, as referred to herein, can be determined, for example, using BLAST using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1].

[0156] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include a sequence variant. A “sequence variant” has an altered sequence in which one or more of the amino acids in the reference sequence is / are deleted or substituted, and / or one or more amino acids is / are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence which is at least 70% identical to the reference sequence. Variantsequences which are at least 70% identical have no more than 30 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.

[0157] In general, while it is possible to have non-conservative amino acid substitutions, the substitutions are usually conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydoxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.

[0158] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molecule or an enzyme.

[0159] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof binds (specifically) to Plasmodium vivax sporozoites and / or Plasmodium vivax merozoites. The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may provide protection against Plasmodium vivax, in particular the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may inhibit or reduce (symptoms of) Plasmodium vivax infection. Accordingly, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may prevent, reduce, inhibit and / or neutralize infection with Plasmodium vivax. More specifically, the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may (specifically) bind to PvAMA1 according to SEQ ID NO: 65.

[0160] Standard methods to assess binding of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, are known to those skilled in the art and caninclude, for example, an ELISA (enzyme-linked immunosorbent assay). An exemplary standard ELISA may be performed as follows: ELISA plates may be coated with a sufficient amount (e.g., 1 μg / ml of the protein / complex / particle to which binding of the antibody is to be tested. For example, for testing binding to PvAMA1 or an epitope thereof, a protein (e.g., SEQ ID NO: 65) and / or fragments / epitopes thereof (e.g., peptides of SEQ ID NO: 65) may be used. ELISA plates may be coated directly or indirectly (e.g., by coating plates first with avidin and incubating them later with biotinylated protein / complex / particle to which binding of the antibody is to be tested). After the first coating step (avidin or, directly, with the protein / complex / particle to which binding of the antibody is to be tested) plates may be blocked, e.g. with a 1% w / v solution of Bovine Serum Albumin (BSA) in PBS. Before the coated plates are incubated with the antibody to be tested, they may be washed.

[0161] In other embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can have KDto PvAMA1 less than the KDof PvRON2L (e.g., 50 nM) or less about 50 nM, less than about 49 nM, less than about 48 nM, less than about 47 nM, less than about 46 nM, less than about 45 nM, less than about 44 nM, or less than about 43 nM, less than about 42 nM, less than about 41 nM, less than about 40 nM, less than about 39 nM, less than about 38 nM, less than about 37 nM, less than about 36 nM, less than about 35 nM, less than about 34 nM, or less than about 33 nM, less than about 32 nM, less than about 31 nM, less than about 30 nM, less than about 29 nM, less than about 28 nM, less than about 27 nM, less than about 26 nM, less than about 25 nM, less than about 24 nM, or less than about 23 nM, less than about 22 nM, less than about 21 nM, less than about 20 nM, less than about 19 nM, less than about 18 nM, less than about 17 nM, less than about 16 nM, less than about 15 nM, less than about 14 nM, or less than about 13 nM, less than about 12 nM, less than about 11 nM, or less than about 10 nM.

[0162] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof inhibits invasion of sporozoites into hepatocytes at a EC50 of less about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, or less than about 3 nM, e.g., about 3.5 nM.

[0163] To study and quantitate virus infectivity (or “neutralization”) in the laboratory the person skilled in the art knows various standard “neutralization assays”. For a neutralization assay animal viruses are typically propagated in cells and / or cell lines. Forexample, in a neutralization assay cultured cells may be incubated with a fixed amount of Plasmodium vivax sporozoites in the presence (or absence) of the antibody to be tested. As a readout for example flow cytometry may be used. Alternatively, also other readouts are conceivable.

[0164] In some instances, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof may reduce gliding motility of Plasmodium sporozoites. Plasmodium sporozoites are transmitted by mosquito bites into the skin of their vertebrate host. Before sporozoites enter the blood stream, they move rapidly through the dermis, powered by an actomyosin system, using a form of locomotion referred to as “gliding motility”. Accordingly, sporozoite motility is a key prerequisite for parasite transmission and successful infection of the vertebrate host.

[0165] Gliding motility of sporozoites can be assessed by in vitro assays, wherein the sporozoite is allowed to glide on a flat surface, e.g. on a glass surface. F or testing the effect of compounds on sporozoite gliding, sporozoites may be pre-incubated with test compounds before they are allowed to glide. Detailed protocols for gliding assays are known in the art and described, for example, in Example 4 or in Prinz, H. L., Sattler, J. M. & Frischknecht, F. (2017) Plasmodium Sporozoite Motility on Flat Substrates. Bio-protocol7, e2395. Moreover, ex vivo imaging technologies making use of human tissue may be employed to determine the gliding motility of sporozoites,

[0166] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may reduce cell traversal of Plasmodium sporozoites. As sporozoites move towards the liver, they can enter and exit host cells within transient vacuoles, a process known as cell traversal. Traversal allows the sporozoites to cross cellular barriers and evade the host immune response, thereby representing a key prerequisite for successful infection of the vertebrate host.

[0167] Cell traversal of sporozoites can be assessed by in vitro assays, wherein sporozoites are incubated with host cells in a co-culture. For visualization, various (e.g., fluorescent) labels may be used, for example, in the co-culture or sporozoites may be pre-incubated with a label (e.g., as described in Example 5). Alternatively, genetically modified Plasmodium strains may be used, which express, e.g., fluorescent labels. For testing the effect of compounds on traversal, sporozoites may be pre-incubated with test compounds before they are co-cultured with host cells. Detailed protocols for sporozoitetraversal assays are known in the art and described, for example, in Example 5; in Schleicher, T. R., Yang, J., Freudzon, M. et al. (2018).

[0168] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may reduce invasion and / or maturation of Plasmodium sporozoites. Sporozoite invasion of hepatocytes and subsequent maturation into exoerythrocytic forms is an essential step in the establishment of malaria infection.

[0169] Invasion and / or maturation of sporozoites can be assessed by in vitro assays, wherein sporozoites are incubated with host cells (e.g., hepatocytes). For visualization, various (e.g., fluorescent) labels may be used. Alternatively, genetically modified Plasmodium strains may be used, which express, e.g., fluorescent labels. For testing the effect of compounds on invasion and / or maturation, sporozoites may be pre-incubated with test compounds before they are co-incubated with host cells. Detailed protocols for sporozoite invasion / maturation assays are known in the art. Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry.

[0170] In some embodiments, the antibody of the invention is a human antibody. In some embodiments, the antibody of the invention is a monoclonal antibody. For example, the antibody of the invention is a human monoclonal antibody.

[0171] Anti-PvAMA1 antibodies described herein can be of any isotype (e.g., IgA, IgG, IgM i.e. an α, γ or μ heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, anti-PvAMA1 antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass, for example IgG1. Anti-PvAMA1 antibodies described herein may have a κ or a λ light chain. In some embodiments, the anti-PvAMA1 antibody has a lambda or kappa light chain. In some embodiments, the anti-PvAMA1 antibody is of IgG1 type and has a lambda or kappa light chain.

[0172] In some embodiments, the anti-PvAMA1 antibody is of the human IgG1 type. The anti-PvAMA1 antibody may be of any allotype. The term “allotype” refers to the allelic variation found among the IgG subclasses. For example, the anti-PvAMA1 antibody may be of the G1 m1 (or G1m(a)) allotype, of the G1 m2 (or G1m(x)) allotype, of the G1m3 (or G1m(f)) allotype, and / or of the G1m17 (or Gm(z)) allotype. The G1m3 and G1m17 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering). G1 m3 corresponds to R214 (EU), while G1m17 corresponds to K214 (EU). The G1 m1 allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refersto the replacements E356D and M358L. The G1 m2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine. The G1 m1 allotype may be combined, for example, with the G1 m3 or the G1 m17 allotype. In some embodiments, the anti-PvAMA1 antibody is of the allotype G1 m3 with no G1 m1 (G1m3,-1). In some embodiments, the anti- PvAMA1 antibody is of the G1 m17,1 allotype. In some embodiments, the anti-PvAMA1 antibody is of the G1 m3,1 allotype. In some embodiments, the anti-PvAMA1 antibody is of the allotype G1m17 with no G1 m1 (G1 m17,-1). Optionally, these allotypes may be combined (or not combined) with the G1 m2, G1 m27 or G1 m28 allotype. For example, the anti-PvAMA1 antibody may be of the G1 m17,1,2 allotype.

[0173] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, comprises an Fc moiety. The Fc moiety may be derived from human origin, e.g. from human IgG1, IgG2, IgG3, and / or IgG4, such as human IgG1.

[0174] The term “Fc moiety” refers to a sequence derived from the portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof.

[0175] A complete Fc moiety comprises at least a hinge domain, a CH2 domain, and a CH3 domain. An additional lysine residue (K) is sometimes present at the extreme C- terminus of the Fc moiety, but is often cleaved from a mature antibody.

[0176] Each of the amino acid positions within an Fc moiety have been numbered herein according to the art-recognized EU numbering system of Kabat, see e.g., by Kabat et al., in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 1983 and 1987. The EU index or EU index as in Kabat or EU numbering refers to the numbering of the EU antibody (Edelman G M, Cunningham B A, Gall W E, Gottlieb P D, Rutishauser U, Waxdal M J. The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.1969; 63(1):78-85; Kabat E.A., National Institutes of Health (U.S.) Office of the Director, “Sequences of Proteins of Immunological Interest”, 5.sup.th edition, Bethesda, Md.: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, hereby entirely incorporated by reference).

[0177] In some embodiments, an Fc moiety can include at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or avariant, portion, or fragment thereof. An Fc moiety may include at least a hinge domain, a CH2 domain or a CH3 domain. The Fc moiety may be a complete Fc moiety. The Fc moiety may also comprises one or more amino acid insertions, deletions, or substitutions relative to a naturally-occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain or CH3 domain (or portion thereof) may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), (ii) a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iv) a hinge domain (or portion thereof), (v) a CH2 domain (or portion thereof), or (vi) a CH3 domain or portion thereof.

[0178] It will be understood by one of ordinary skill in the art that the Fc moiety may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining at least one desirable function conferred by the naturally-occurring Fc moiety. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding. The portions of naturally occurring Fc moieties, which are responsible and / or essential for such functions are well known by those skilled in the art.

[0179] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, comprises an Fc region. As used herein, the term “Fc region” refers to the portion of an immunoglobulin formed by two or more Fc moieties of antibody heavy chains. For example, the Fc region may be monomeric or “single-chain” Fc region (i.e., a scFc region). Single chain Fc regions are comprised of Fc moieties linked within a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008 / 143954 A2. The Fc region may be dimeric. A “dimeric Fc region” or “dcFc” refers to the dimer formed by the Fc moieties of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc moieties (e.g., an Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties.

[0180] The Fc moieties of the Fc region may be of the same or different class and / or subclass. For example, the Fc moieties may be derived from an immunoglobulin (e.g., a human immunoglobulin) of an IgG1, IgG2, IgG3 or IgG4 subclass. The Fc moieties of the Fc region may be of the same class and subclass. However, the Fc region (or one or more Fcmoieties of an Fc region) may also be chimeric, whereby a chimeric Fc region may comprise Fc moieties derived from different immunoglobulin classes and / or subclasses. For example, at least two of the Fc moieties of a dimeric or single-chain Fc region may be from different immunoglobulin classes and / or subclasses. Additionally or alternatively, the chimeric Fc regions may comprise one or more chimeric Fc moieties. For example, the chimeric Fc region or moiety may comprise one or more portions derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2, or IgG3 subclass) while the remainder of the Fc region or moiety is of a different subclass. For example, an Fc region or moiety of an Fc polypeptide may comprise a CH2 and / or CH3 domain derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2 or IgG4 subclass) and a hinge region from an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, the Fc region or moiety may comprise a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, the chimeric Fc region may comprise an Fc moiety (e.g., a complete Fc moiety) from an immunoglobulin for a first subclass (e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2 or IgG3 subclass). For example, the Fc region or moiety may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or moiety may comprise a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc region or moiety may comprise a portion of a CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of a CH2 domain. For example, an Fc region or moiety may comprise amino acids a positions 292-340 of CH2 derived from an IgG4 moiety and the remainder of CH2 derived from an IgG1 moiety (alternatively, 292-340 of CH2 may be derived from an IgG1 moiety and the remainder of CH2 derived from an IgG4 moiety).

[0181] Moreover, an Fc region or moiety may (additionally or alternatively) for example comprise a chimeric hinge region. For example, the chimeric hinge may be derived, e.g. in part, from an IgG1, IgG2, or IgG4 molecule (e.g., an upper and lower middle hinge sequence) and, in part, from an IgG3 molecule (e.g., a middle hinge sequence). In another example, an Fc region or moiety may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molecule and a middle hingedomain from an IgG1 molecule.

[0182] In some embodiments, the Fc moiety, or the Fc region, comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc region or Fc moiety from a human IgG molecule). However, polypeptides may comprise one or more amino acids from another mammalian species. For example, a primate Fc moiety or a primate binding site may be included in the subject polypeptides. Alternatively, one or more murine amino acids may be present in the Fc moiety or in the Fc region.

[0183] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes in particular in addition to an Fc moiety as described above, other parts derived from a constant region, in particular from a constant region of IgG, such as a constant region of (human) IgG1. The anti-PvAMA1 antibody according describe herein may comprise, in particular in addition to an Fc moiety as described above, all other parts of the constant regions, in particular all other parts of the constant regions of IgG (such as (human) IgG1).

[0184] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may include a (complete) Fc region derived from human IgG1. In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof includes, in particular in addition to a (complete) Fc region derived from human IgG1 also all other parts of the constant regions of IgG, such as all other parts of the constant regions of (human) IgG1.

[0185] In general, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof may be glycosylated. N-linked glycans attached to the CH2 domain of a heavy chain, for instance, can influence C1q and FcR binding, with glycosylated antibodies having lower affinity for these receptors. Accordingly, the CH2 domain of the Fc moiety of the anti-PvAMA1 antibody described herein may comprise one or more mutations, in which a glycosylated residue is substituted by a non-glycosylated residue. For example, the antibody's glycans do not lead to a human immunogenic response after administration.

[0186] Furthermore, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can be modified by introducing (random) amino acid mutations into particular region of the CH2 or CH3 domain of the heavy chain in order to alter their binding affinity for FcR and / or their serum half-life in comparison to unmodified antibodies. Examples of such modifications include, but are not limited to, substitutions of at least oneamino acid from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428. Further examples of such Fc modifications are described in Saxena A, Wu D. Advances in Therapeutic Fc Engineering-Modulation of IgG-Associated Effector Functions and Serum Half-life. Front Immunol.2016; 7:580, which is incorporated herein by reference. In some embodiments, the antibody may comprise the “YTE” mutations (M252Y / S254T / T256E; EU numbering). In some embodiments, the antibody may comprise the mutations M428L and / or N434S in the heavy chain constant region (EU numbering).

[0187] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof may be a single-domain antibody, such as a VHH or V- NAR. Such antibodies exist naturally in camelids and sharks (Saerens, D. et al., Curr. Opin. Pharmacol., 8:600-8, 2008). Camelid antibodies are described in, for example. U.S. Pat. Nos. 5,759,808; 5,800,988; 5,840,526; 5,874,541; 6,005,079; and 6,015,695, the entire contents of each of which are incorporated herein by reference. The cloned and isolated VHH domain is a stable polypeptide that features the full antigen-binding capacity of the original heavy-chain antibody. VHH domains, with their unique structural and functional properties, combine the advantages of conventional antibodies (high target specificity, high target affinity and low inherent toxicity) with important features of small molecule drugs (the ability to inhibit enzymes and access receptor clefts). Furthermore, they are stable, have the potential to be administered by means other than injection, are easier to manufacture.

[0188] In some embodiments, humanized and chimeric anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof thereof can bind to the same epitope on PvAMA1 as the antibodies recited in this application. Such humanized and chimeric antibodies, antibody fragments, and antigen binding fragments thereof can be identified based on their ability to cross-compete with or competitively inhibit the antibodies and antigen binding fragments thereof in standard PvAMA1 binding assays. Thus, all humanized and chimeric antibodies, antibody fragments, and antigen binding fragments thereof that competitively inhibit the binding of antibodies and the antigen binding fragments thereof are encompassed by this disclosure.

[0189] The antigen binding fragments of the anti-PvAMA1 antibodies can be identified following protease digestion. These include, for example, the "Fab fragment", "Fab' fragment" (a Fab with the heavy chain hinge region), and "F(ab')2 fragment" (a dimer of Fab' fragments joined at the heavy chain hinge region). Recombinant methods have been used togenerate such fragments and to generate even smaller antibody fragments, e.g., those referred to as "single chain Fv" (variable fragment) or "scFv," consisting of VL and VH joined by a synthetic peptide linker (VL-linker-VH or VH-linker-VL). Fab fragments, Fab' fragments and scFv fragments are monovalent or monospecific for antigen binding, as they each include only one antigen binding domain including one VH / VL dimer.

[0190] Even smaller monovalent antibody fragments are the dAbs, which include only a single immunoglobulin variable domain, e.g., VH or VL, that alone specifically binds antigen, i.e., without the need for a complementary VL or VH domain, respectively. A dAb binds antigen independently of other V domains; however, a dAb can be present in a homo- or hetero-multimer with other VH or VL domains where the other domains are not required for antigen binding by the dAb, i.e., where the dAb binds antigen independently of the additional VH or VL domains.

[0191] In some embodiments, the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof is a single domain antibody that is a heavy chain variable domain (VH, e.g., VHH) or a light chain domain (VL). One means of generating single domain antibodies specific for PvAMA1 is to amplify and express the VH and VL regions of the heavy chain and light chain gene sequences isolated, for example, from a cell that expresses the anti-PvAMA1 antibody. The boundaries of VH and VL domains are set out, for example, by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1991). The information regarding the boundaries of the VH and VL domains of heavy and light chain genes is used to design PCR primers that amplify the V domain from a heavy or light chain coding sequence encoding an antibody known to bind a PvAMA1.

[0192] The amplified V domains are inserted into a suitable expression vector, e.g., pHEN-1 (Hoogenboom, H. et al., Nucleic Acids Res., 19:4133-7, 1991) and expressed, for example, as a fusion of the VH and VL in a scFv or other suitable monovalent format. The resulting polypeptide can then be screened for high affinity monovalent binding to a PvAMA1. Screening for binding can be performed by methods known in the art. Single domain antibodies can be generated using methods known in the art (WO2005118642; Ward, E. et al., Nature, 341:544-6, 1989; Holt, L. et al., Trends Biotechnol., 21:484-90, 2003). Each light chain domain may be either of the kappa or lambda subgroup. Methods for isolating VH and VL domains have been described in the art (EP0368684).

[0193] In one embodiment, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include a single domain antibody that is obtained from a human, humanized rodent, camelid or shark. Any such single domain antibody can be optionally humanized. Humanization of camelid single domain antibodies requires the introduction and mutagenesis of a limited number of amino acids in a single polypeptide chain. This is in contrast to humanization of scFv, Fab, (Fab')2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

[0194] In some embodiments, the single domain antibody includes VHH domains. In some embodiments, the VHH domains correspond to the VHH domains of naturally occurring heavy chain antibodies directed against a PvAMA1. Such VHH sequences can be generated, for example, by suitably immunizing a species of camelid with a PvAMA1 antigen, (i.e., so as to raise an immune response and / or heavy chain antibodies directed against PvAMA1) by obtaining a suitable biological sample from said camelid (such as a blood sample, serum sample or sample of B-cells), and by generating VHH sequences directed against a PvAMA1, starting from said sample, using any suitable technique known in the art (e.g., the gene encoding the single domain antibody may be cloned by PCR, or the B-cell(s) encoding the single domain antibody may be immortalized by EBV transformation, or by fusion to an immortal cell line).

[0195] Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries may be used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling (WO 00 / 43507). In a certain embodiment, a VHH library is constructed, transformed into a host cell, and expressed on phages after infection of the host cell with a helper phage. After several rounds of bio- panning, single domain antibodies can be isolated and efficiently expressed.

[0196] A library of fusion proteins including VHH or VHH fragments can be displayed on a phage, or suitable microorganism (such as yeast), to facilitate screening.

[0197] Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) fusion proteins including VHH or VHH fragments are known in the art (WO 03 / 054016; Hoogenboom, H., Nat. Biotechnol., 23:1105-16, 2005).

[0198] Other suitable methods and techniques for obtaining the single domain antibodies and / or nucleic acids encoding the same, starting from naturally occurring VHsequences or VHH sequences may, for example, include combining one or more parts of one or more naturally occurring VH sequences (such as one or more framework region (FR) sequences and / or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more framework region sequences or CDR sequences), and / or one or more synthetic or semi- synthetic sequences, in a suitable manner, so as to provide a monovalent single domain antibody or a nucleotide sequence or nucleic acid encoding the same. Nucleotide sequences encoding framework sequences of VHH or single domain antibodies are known in the art and may alternatively be obtained by polymerase chain reaction (PCR) starting from the nucleotide sequences obtained using the methods described herein. Such compositions can be suitably combined with nucleotide sequences that encode the desired CDRs (for example, by PCR assembly using overlapping primers), to provide a single domain antibody, or antibody fragment fused with a regulator of the alternative complement pathway or fragment thereof.

[0199] Anti-PvAMA1 antibodies or antigen binding fragments that recognize the same epitope as a parent antibody can be generated by known techniques. For example, anti-and PvAMA1 antibody or antigen binding fragments can be prepared by proteolytic hydrolysis of an anti-PvAMA1 antibody or by expression in E. coli of the DNA coding for the fragment. The anti-PvAMA1 antibody or antigen binding fragments are antigen binding portions of an antibody, such as Fab, F(ab')2, and scFV, and can be obtained by pepsin or papain digestion of whole anti-PvAMA1 antibodies by conventional methods or by genetic engineering techniques.

[0200] An anti-PvAMA1 antibody or antigen binding fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide a 100 kDa fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 50 kDa Fab' monovalent fragments. Alternatively, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly (U.S. Pat. Nos.4,036,945 and 4,331,647; Nisonoff, A. et al., Arch. Biochem. Biophys., 89:230-44, 1960; Porter, R., Biochem. J., 73:119-26, 1959; Edelman et al., in Methods in Enzymology Vol. I, page 422 (Academic Press 1967), and Coligan et al., Current Protocols in Immunology, Vol.1, pages 2.8.1-2.8.10 and 2.10.-2.10.4 (John Wiley & Sons 1991).

[0201] Other methods of cleaving antibodies, such as separation of heavy chains toform monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0202] Another form of an anti-PvAMA1 antibody or antigen binding fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using reverse transcriptase followed by the polymerase chain reaction to synthesize the variable region from RNA of antibody producing cells (Larrick, J & Fry, K. METHODS--a companion to Methods in Enzymology Volume: New Techniques in Antibody Generation, 2:106-110, 1991); Courtenay- Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles And Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc.1995)).

[0203] Other antibody or antigen binding fragments, for example single domain antibody fragments, are known in the art and may be used in the claimed constructs (Muyldermans, S. et al., Trends Biochem. Sci., 26:230-5, 2001; Yau, K. et al., J. Immunol. Methods, 281:161-75, 2003; Maass, D. et al., J. Immunol. Methods, 324:13-25, 2007).

[0204] In some embodiments, an anti-PvAMA1 single domain antibody or nanobody can include a single CDR that includes the amino acid sequence of X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10(SEQ ID NO: 11); wherein: X2is R or S; X3is S, A, or G; X4is R, H, N, or Y; X5is G or A; X6is Y or H; X7 is F or G; X8is T, S, or N; X9 is T or S; X10is D or E; and wherein SEQ ID NO: 11 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%identical to the amino acid sequence of RSRGEGYCSFGTCYTLFYYFD (SEQ ID NO: 64).

[0205] In other embodiments, the anti-PvAMA1 nanobody can include a CDR that includes the amino acid sequence of CX1X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10X11W, (SEQ ID NO: 12); wherein: X1is A or V; X2 is R or S; X3is S, A, or G; X4 is R, H, N, or Y; X5is G or A; X6 is Y or H; X7is F or G; X8 is T, S, or N; X9is T or S; X10 is D or E; X11is Y or N; and wherein SEQ ID NO: 12 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5).

[0206] For example, the anti-PvAMA1 nanobody can include a CDR selected from CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5), CARAHGEGHCSGGSCYSLFYYFDYW (SEQ ID NO: 13), CARARGEAYCSGGTCYTLFYYFDYW (SEQ ID NO: 14), CARAHGEGYCSGGSCYSLFYYFDYW (SEQ ID NO: 15), CARAYGEGYCSFGTCYTLFYYFDNW (SEQ ID NO: 16), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 17), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 18), CARGRGEGYCSGGNCYTLFYYFDYW (SEQ ID NO: 19), CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 20), CASGNGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 21), CVSANGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 22), and truncated sequences thereof.

[0207] By way of example, a nanobody that include a CDR having the amino acid sequence of SEQ ID NO: 11 or 12 can include a polypeptide that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 32 or 33, wherein the CDR as defined above (CDR sequence as set forth in respectively SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NOs: 13-22) is maintained.

[0208] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof also include hybrid antibody molecules that comprise the six CDRs from an antibody described herein as defined above and one or more CDRs from another antibody to an antigen. For example, the antibody may be bispecific.

[0209] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof may be provided in purified form. Typically, the anti-PvAMA1 antibody will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

[0210] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein may be immunogenic in non-human (or heterologous) hosts e.g., in mice. In particular, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof may have an idiotope that is immunogenic in non-human hosts, but not in a human host. In particular, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.

[0211] Other embodiments described herein relate to a composition that includes a nucleic acid or nucleic acid molecule encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein.

[0212] In some embodiments, the nucleic acid encodes a heavy chain variable region atleast about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 are maintained.

[0213] In some embodiments, the nucleic acid that encodes a heavy chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 are maintained includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 66.

[0214] In other embodiments, the nucleic acid that encodes a heavy chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are maintained.

[0215] In other embodiments, the nucleic acid, which encodes a heavy chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are maintained, includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 68.

[0216] In some embodiments, the nucleic acid encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 wherein the light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 are maintained.

[0217] In other embodiments, the nucleic acid, which encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 wherein the light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 are maintained, includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 67.

[0218] In other embodiments, the nucleic acid encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27 wherein the light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 are maintained.

[0219] In other embodiments, the nucleic acid, which encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27 wherein the light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 are maintained, includes a nucleotide sequence at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 69.

[0220] In some embodiments, the composition includes a nucleic acid that encodes a heavy chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 are maintained, and a nucleic acid that encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 wherein the light chain CDR-L1, CDR-L2, and CDR- L3 amino acid sequences as set forth in respectively SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 are maintained.

[0221] In other embodiments, the composition includes a nucleic acid that encodes a heavy chain variable region with heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of respectively SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and that includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 66, and a nucleic acid that encodes a light chain variable region with light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of respectively SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 and that includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 67.

[0222] In some embodiments, the composition includes a nucleic acid that encodes a heavy chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at leastabout 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 wherein the heavy chain CDR-H1, CDR-H2, and CDR-H3 sequences as set forth in respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are maintained, and a nucleic acid encodes a light chain variable region at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO: 27 wherein the light chain CDR-L1, CDR-L2, and CDR-L3 sequences as set forth in respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 are maintained.

[0223] In other embodiments, the composition includes a nucleic acid that encodes a heavy chain variable region with heavy chain CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of respectively SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 and that includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 68, and a nucleic acid that encodes a light chain variable region with light chain CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of respectively SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 and that includes a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 69.

[0224] In other embodiments, the composition includes a nucleic acid that encodes a nanobody that includes a CDR having the amino acid sequence of SEQ ID NO: 11 or 12. For example, the nucleic acid can encode a polypeptide that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 32.

[0225] In some embodiments, a nucleic acid that encodes amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 32 can include a nucleotide sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a nucleotide sequence of SEQ ID NO: 70.

[0226] In other embodiments, the composition includes a nucleic acid that encodes a nanobody that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 33.

[0227] Examples of nucleic acid molecules and / or nucleotides include, e.g., a recombinant nucleotide, a vector, an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA, an siRNA, or a tRNA, or a DNA molecule such as a cDNA. Nucleic acids may encode the light chain and / or the heavy chain of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein. In other words, thelight chain and the heavy chain of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein may be encoded by the same nucleic acid molecule (e.g., in bicistronic manner). Alternatively, the light chain and the heavy chain of the antibody may be encoded by distinct nucleic acid molecules.

[0228] Due to the redundancy of the genetic code, this disclosure also includes sequence variants of nucleotide sequences, which encode the same amino acid sequences. The nucleic acids encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein (or the complete nucleic acid molecule) may be optimized for expression of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein. For example, codon optimization of the nucleotide sequence may be used to improve the efficiency of translation in expression systems for the production of the antibody. Moreover, the nucleic acid molecule may comprise heterologous elements (i.e., elements, which in nature do not occur on the same nucleic acid molecule as the coding sequence for the (heavy or light chain of) the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein. For example, a nucleic acid may comprise a heterologous promotor, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous Poly-A-tail, and the like.

[0229] In some embodiments, the nucleic acid may also comprise further elements in addition to the nucleotide encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are covalently linked to each other by phosphodiester-bonds of a sugar / phosphate-backbone. The term “nucleic acid” also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.

[0230] In general, the nucleic acid molecule may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and / or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and / or insertions into the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragmentthereof described herein amino acid sequence. Such point mutations can modify effector functions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes). Alternatively, a mutation in a nucleotide sequence may be “silent”, i.e. not reflected in the amino acid sequence due to the redundancy of the genetic code. In general, mutations can be introduced in specific sites or can be introduced at random, followed by selection (e.g., molecular evolution). For instance, one or more nucleic acids encoding any of the light or heavy chains of an (exemplary) the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes can be the result of an iterative process wherein initial changes are retained and new changes at other nucleotide positions are introduced. Further, changes achieved in independent steps may be combined.

[0231] In some embodiments, the nucleic acid encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein, (or the (complete) nucleic acid molecule) may be codon-optimized. The skilled artisan is aware of various tools for codon optimization, such as those described in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 Aug. 2014, Pages 2210-2212; or in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel D C, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res.2005 Jul.1; 33(Web Server issue):W526-31; or, for example, Genscript's OptimumGene™ algorithm (as described in US 2011 / 0081708 A1).

[0232] For example, the nucleic acid molecule may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs 67-69; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0233] In some embodiments, a composition described herein can include a combination of first and second nucleic acid molecules, wherein the first nucleic acid molecule encodes the heavy chain of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein; and the second nucleic acid molecule encodes the corresponding light chain of the same the anti-PvAMA1 antibody, antibodyfragment, or antigen binding fragment thereof described herein. The above description regarding the (general) features of the nucleic acid molecule applies accordingly to the first and second nucleic acid molecules of the combination. Accordingly, one or both of the nucleic acid molecules encoding the heavy and / or light chain(s) of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein, is / are codon-optimized. For example, the combination may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs 67-69; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0234] Other embodiments described herein relate to vectors, for example, expression vectors, that include the nucleic acid described herein. Usually, a vector comprises a nucleic acid molecule as described above.

[0235] In some embodiments, a first vector can be provided in combination with a second vector, wherein the first vector includes a first nucleic acid molecule as described above (for the combination of nucleic acid molecules) and the second vector comprises a second nucleic acid molecule as described above (for the combination of nucleic acid molecules), in particular wherein the first and the second nucleic acid molecules are selected from the same (embodiment of) combination of nucleic acid molecules as described above. More specifically, the first and the second nucleic acid molecules may be selected such that they encode together (i) the six CDRs, (ii) the variable regions VH and VL; or the light and heavy chain of any one of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein.

[0236] A vector is usually a recombinant nucleic acid molecule, i.e., a nucleic acid molecule which does not occur in nature. Accordingly, the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature). For example, the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector) and the like. A vector can be suitable for incorporating or harboring a nucleic acid having a desired nucleotide sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a (heavy and / or light chain of a) desired anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein. An expression vector may be used for production of expression products such as RNA, e.g., mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector may be, e.g., an RNA vector or a DNA vector. For example, a vector in the sense can include a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. A vector may be a plasmid vector.

[0237] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein can be made by any method known in the art. For example, the general methodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C, 1975; Kozbar et al.1983). In some embodiments, the alternative EBV immortalization method described in WO2004 / 076677 is used.

[0238] In some embodiments, the method as described in WO 2004 / 076677, which is incorporated herein by reference, is used. In this method B cells producing the antibody of the invention are transformed with a B cell activator. Additional stimulants of cellular growth and differentiation may optionally be added during the transformation step to further enhance the efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. The immortalized B cells produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.

[0239] Another method is described in WO 2010 / 046775. In this method plasma cells are cultured in limited numbers, or as single plasma cells in microwell culture plates. Antibodies can be isolated from the plasma cell cultures. Further, from the plasma cell cultures, RNA can be extracted and PCR can be performed using methods known in the art.

[0240] The VH and VL regions of the anti-PvAMA1 antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector that isthen transfected into HEK293T cells or other host cells. The cloning of nucleic acid in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.

[0241] The anti-PvAMA1 antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.

[0242] Standard techniques of molecular biology may be used to prepare DNA sequences encoding the anti-PvAMA1 antibodies described herein. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site- directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

[0243] Any suitable host cell / vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention. Eukaryotic, e.g., mammalian, host cell expression systems may be used for production of antibody molecules, such as complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Also, prokaryotic, e.g. bacterial host cell expression systems may be used for the production of antibody molecules, such as complete antibody molecules. Suitable bacterial host cells include, but are not limited to, E. coli cells.

[0244] In some embodiments, a process for the production of the anti-PvAMA1 antibody described herein can include culturing a (heterologous) host cell comprising a vector encoding a nucleic acid described herein under conditions suitable for expression of protein from DNA encoding the antibody molecule herein, and isolating the antibody molecule.

[0245] For production of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein that includes both heavy and light chains, a cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.

[0246] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragmentthereof may be produced by (i) expressing a nucleotide sequence in a host cell, e.g., by use of a vector described herein, and (ii) isolating the expressed antibody product. Additionally, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of the desired specificity or function.

[0247] The screening step may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies.

[0248] Individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art.

[0249] Nucleic acids from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.

[0250] The immortalized B cell clones or the transfected host-cells can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.

[0251] Other embodiments described herein relate to a composition that includes immortalized B memory cells or transfected host cells that produce anti-PvAMA1 antibodies described herein.

[0252] The immortalized B cell clone or the cultured plasma cells may also be used as a source of nucleic acid for the cloning of antibody genes for subsequent recombinant expression. Expression from recombinant sources may be more common for pharmaceutical purposes than expression from B cells or hybridomas e.g., for reasons of stability, reproducibility, culture ease, etc.

[0253] In some embodiments, a method for preparing a recombinant cell can include the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and / or light chain mRNAs) from the B cell clone or the cultured plasma cells that encodes the anti-PvAMA1 antibody of interest; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting thevector into a (heterologous) host cell in order to permit expression of the antibody of interest in that host cell.

[0254] In other embodiments, a method for preparing a recombinant cell can include the steps of: (i) sequencing nucleic acid(s) from the B cell clone or the cultured plasma cells that encodes the anti-PvAMA1 antibody of interest; and (ii) using the sequence information from step (i) to prepare nucleic acid(s) for insertion into a host cell in order to permit expression of the anti-PvAMA1 antibody of interest in that host cell. The nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, to change codon usage, and / or to optimize transcription and / or translation regulatory sequences.

[0255] Other embodiments described herein relate to a method of preparing a transfected host cell that includes the step of transfecting a host cell with one or more nucleic acids that encode an anti-PvAMA1 antibody of interest, wherein the nucleic acids are nucleic acids that were derived from an immortalized B cell clone or a cultured plasma cell of the invention. Thus, the procedures for first preparing the nucleic acid(s) and then using it to transfect a host cell can be performed at different times by different people in different places (e.g., in different countries).

[0256] These recombinant cells can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production. They can also be used as the active ingredient of a pharmaceutical composition. Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites fluid, hollow-fiber type bioreactor cartridge, modular minifermenter, stirred tank, microcarrier culture, ceramic core perfusion, etc.

[0257] The transfected host cell can be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a human liver cell), as well as plant cells. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, expression hosts can glycosylate the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, particularly with carbohydrate structures that are not themselves immunogenic in humans. In some embodiments the transfected host cell may be able to grow in serum-free media. In further embodiments the transfected host cell may be able to grow in culture without the presence of animal-derived products. The transfected host cell may also be cultured to give a cell line.

[0258] In other embodiments, a method for preparing one or more nucleic acid molecules (e.g., heavy and light chain genes) that encode the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells; (ii) obtaining from the B cell clone or the cultured plasma cells nucleic acid that encodes the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof.

[0259] In other embodiments, a method for obtaining a nucleic acid sequence that encodes the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, can include the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (ii) sequencing nucleic acid from the B cell clone or the cultured plasma cells that encodes the antibody of interest.

[0260] In further embodiments, a method of preparing nucleic acid molecule(s) that encode the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can include the step of obtaining the nucleic acid that was obtained from a transformed B cell clone or cultured plasma cells. Thus, the procedures for first obtaining the B cell clone or the cultured plasma cell, and then obtaining nucleic acid(s) from the B cell clone or the cultured plasma cells can be performed at different times by different people in different places (e.g., in different countries).

[0261] Other embodiments described herein relate to a method for preparing an the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, (e.g., for pharmaceutical use). The method includes the steps of: (i) obtaining and / or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from the selected B cell clone or the cultured plasma cells expressing the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof; (ii) inserting the nucleic acid(s) into or using the nucleic acid(s) sequence(s) to prepare an expression vector; (iii) transfecting a host cell that can express the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof; (iv) culturing or sub-culturing the transfected host cells under conditions where the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, is expressed; and, optionally, (v) purifying the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, of interest.

[0262] Still other embodiments relate to a method of preparing the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof by culturing or sub-culturing a transfected host cell population, e.g., a stably transfected host cell population, under conditions where the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, is expressed and, optionally, purifying the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, wherein the transfected host cell population has been prepared by (i) providing nucleic acid(s) encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof that is produced by a B cell clone or cultured plasma cells, (ii) inserting the nucleic acid(s) into an expression vector, (iii) transfecting the vector in a host cell that can express the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, and (iv) culturing or sub-culturing the transfected host cell comprising the inserted nucleic acids to produce the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof. Thus, the procedures for first preparing the recombinant host cell and then culturing it to express the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof can be performed at very different times by different people in different places (e.g., in different countries).

[0263] Other embodiments described herein relate to a pharmaceutical composition that includes one or more of: (i) the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof; (ii) the nucleic acid or a combination of nucleic acids encoding the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof; (iii) the vector or a combination of vectors as described herein; and / or (iv) a cell expressing the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof or comprising the vector described herein and, optionally, a pharmaceutically acceptable diluent or carrier. In other words, the pharmaceutical composition can include the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, the nucleic acid encoding the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, the vector as described herein and / or the cell described herein.

[0264] The pharmaceutical composition may optionally also contain a pharmaceutically acceptable carrier, diluent and / or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. In some embodiments, the pharmaceuticallyacceptable carrier, diluent and / or excipient in the pharmaceutical composition according to the present invention is not an active component in respect to P. vivax infection and / or malaria.

[0265] Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

[0266] Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.

[0267] Pharmaceutical compositions described herein may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis™ and Herceptin®, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

[0268] In some embodiments, the (only) active ingredient in the composition is the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from degradation but which release the anti-PvAMA1 antibody once it has been absorbed from the gastrointestinal tract.

[0269] A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0270] Pharmaceutical compositions generally have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and / or pyrogen free. The composition may be isotonic with respect to humans. In some embodiments pharmaceutical compositions of the invention are supplied in hermetically-sealed containers.

[0271] The compositions can present in several forms of administration; the forms include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and / or dispersing agents. Alternatively, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, may be in dry form, for reconstitution before use with an appropriate sterile liquid.

[0272] A vehicle is typically understood to be a material that is suitable for storing, transporting, and / or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and / or administering a pharmaceutically active compound, in particular the antibodies according to the present invention. Once formulated, the compositions can be administered directly to the subject. In some embodiments the compositions are adapted for administration to mammalian, e.g., human subjects.

[0273] The pharmaceutical compositions may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions. Optionally, the pharmaceutical composition may be prepared for oral administration, e.g., as tablets, capsules and the like, for topical administration, or as injectable, e.g., as liquid solutions or suspensions. In some embodiments, the pharmaceutical composition is an injectable. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the pharmaceutical composition may be in lyophilized form.

[0274] For injection, e.g., intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required. Whether it is an antibody, a peptide, a nucleic acid molecule, or another pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is usually in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present invention may be provided for example in a pre-filled syringe

[0275] The pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0276] The pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g., including accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the pharmaceutical composition may be formulated in a suitable ointment, containing the pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.

[0277] Dosage treatment may be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition may be provided as single-dose product. In some embodiments, the amount of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, in the pharmaceutical composition—in particular if provided as single-dose product—does not exceed 200 mg, for example it does not exceed 100 mg or 50 mg.

[0278] For a single dose, e.g. a daily, weekly or monthly dose, the amount of the anti- PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof in the pharmaceutical composition according to the present invention, may not exceed 1 g or 500 mg. In some embodiments, for a single dose, the amount of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof in the pharmaceutical composition according to the present invention, may not exceed 200 mg, or 100 mg. For example, for a single dose, the amount of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof in the pharmaceutical composition according to the present invention, may not exceed 50 mg.

[0279] Pharmaceutical compositions typically include an “effective” amount of one or more the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, i.e., an amount that is sufficient to treat, ameliorate, attenuate, reduce or prevent a desired Pv disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction or attenuation in pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend upon their size, weight, and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of a clinician. An effective dose may generally be from about 0.005 to about 100 mg / kg, for example from about 0.0075 to about 50 mg / kg or from about 0.01 to about 10 mg / kg. In some embodiments, the effective dose will be from about 0.02 to about 5 mg / kg, of the anti-PvAMA1 antibody, antibody fragment,or antigen binding fragment thereof (e.g., amount of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof in the pharmaceutical composition) in relation to the bodyweight (e.g., in kg) of the individual to which it is administered.

[0280] Moreover, the pharmaceutical composition n may also comprise an additional active component, which may be a further antibody or a component, which is not an antibody. Accordingly, the pharmaceutical composition may comprise one or more of the additional active components.

[0281] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, can be present either in the same pharmaceutical composition as the additional active component or, alternatively, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof is comprised by a first pharmaceutical composition and the additional active component is comprised by a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active component is envisaged, each additional active component and the antibody according to the present invention may be comprised in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered either combined / simultaneously or at separate times or at separate locations (e.g., separate parts of the body).

[0282] The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof and the additional active component may provide an additive therapeutic effect, such as a synergistic therapeutic effect. The term “synergy” is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in “synergistic inhibition” of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term “synergistic therapeutic effect” refers to a therapeutic effect observed with a combination of two or more therapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.

[0283] In some embodiments, a composition described herein may include the anti- PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein, wherein the anti-PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein, may make up at least 50% by weight (e.g., 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition. In the composition, the anti-PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein may be in purified form.

[0284] Also described herein is a method of preparing a pharmaceutical composition that includes the steps of: (i) preparing the anti-PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein; and (ii) admixing the anti-PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein with one or more pharmaceutically-acceptable carriers.

[0285] In other embodiments, a method of preparing a pharmaceutical composition includes admixing the anti-PvAMA1 antibodies, antibody fragments, or antigen binding fragments thereof described herein with one or more pharmaceutically-acceptable carriers, wherein the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragments thereof described herein is a monoclonal antibody that was obtained from a transformed B cell or a cultured plasma cell.

[0286] As an alternative to delivering the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein or B cells for therapeutic purposes, it is possible to deliver nucleic acid (typically DNA) that encodes the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof described herein derived from the B cell or the cultured plasma cells to a subject, such that the nucleic acid can be expressed in the subject in situ to provide a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

[0287] Pharmaceutical compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g., less than 0.01%. Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity. For example, a concentration of 10±2 mg / ml NaCl is typical. Further, pharmaceutical compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg / ml (e.g., 25 mg / ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material. The pH of a composition for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilization.

[0288] The compositions may also comprise one or more immunoregulatory agents. Insome embodiments, one or more of the immunoregulatory agents include(s) an adjuvant.

[0289] In some embodiments, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragments thereof, the nucleic acid molecule (or the combination of nucleic acid molecules), the vector (or the combination of vectors) the cell or the pharmaceutical composition described herein can be used in the prophylaxis and / or treatment of subject having or at risk of malaria.

[0290] The term “at risk” of malaria (such as erythrocytic malaria infection), refers to a subject (e.g., a human) that is predisposed to contracting the disease and / or expressing one or more symptoms of the disease. Such subjects include those at risk for failing to elicit an immunogenic response to a vaccine against the disease. This predisposition may be genetic (e.g., a particular genetic tendency to expressing one or more symptoms of the disease, such as heritable disorders, the presence of bacterial species blocking antibodies, the presence of reduced levels of bactericidal antibodies, etc.), or due to other factors (e.g., immune suppressive conditions, environmental conditions, exposures to detrimental compounds, including immunogens, present in the environment, etc.). The term subject “at risk” includes subjects “suffering from disease,” i.e., a subject that is experiencing the disease. It is not intended that subject at risk of malaria be limited to any particular signs or symptoms. Thus, it is intended that subjects to be treated are those experiencing any range of malaria, from sub-clinical infection to full-blown disease, wherein the subject exhibits at least one of the indicia (e.g., signs and symptoms) associated with the disease.

[0291] Initial manifestations of malaria, common to all malaria species, are similar to flu-like symptoms and can resemble other conditions such as septicemia, gastroenteritis, and viral diseases. The presentation may include headache, fever, shivering, arthralgia (joint pain), vomiting, hemolytic anemia, jaundice, hemoglobinuria, retinal damage, and convulsions. Owing to the non-specific nature of disease presentation, diagnosis of malaria in non-endemic countries requires a high degree of suspicion, which might be elicited by any of the following: recent travel history, splenomegaly (enlarged spleen), fever without localizing signs, thrombocytopenia, and hyperbilirubinemia combined with a normal peripheral blood leukocyte count.

[0292] The classic symptom of malaria is paroxysm—a cyclical occurrence of sudden coldness followed by rigor and then fever and sweating, occurring every two days (tertian fever) in P. vivax infections, and every three days (quartan fever).

[0293] In some embodiments, a method of reducing malaria, or lowering the risk of P. vivax infection includes administering to a subject in need thereof, a therapeutically effective amount of the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragments thereof, the nucleic acid molecule (or the combination of nucleic acid molecules), the vector (or the combination of vectors), the cell or the pharmaceutical composition as described herein.

[0294] Moreover, the anti-PvAMA1 antibody, antibody fragment, or antigen binding fragments thereof, the nucleic acid molecule (or the combination of nucleic acid molecules), the vector (or the combination of vectors), the cell or the pharmaceutical composition as described herein can be used in the manufacture of a medicament for prophylaxis, treatment or attenuation of malaria.

[0295] Other embodiments described herein relate to the treatment and / or prophylaxis of Plasmodium infection / malaria (used interchangeably herein) by administration of vaccines disclosed herein. In some embodiments, such vaccines are administered to a subject suffering from malaria or susceptible to Plasmodium infection. In some embodiments, a subject is considered to be suffering from malaria, if the subject is displaying one or more symptoms commonly associated with therewith. In some embodiments, the subject is known or believed to have been exposed to at least one Plasmodium species. In some embodiments, a subject is considered to be susceptible to Plasmodium infection, if the subject is known or believed to have been exposed to a Plasmodium species. In some embodiments, a subject is known or believed to have been exposed to a Plasmodium species, if the subject has been in contact with other individuals known or suspected to have been infected with the same, and / or if the subject is or has been present in a location in which malaria is known or thought to be prevalent. Vaccines disclosed herein may be administered prior to or after development of one or more symptoms of Plasmodium infection / malaria.

[0296] In some embodiments, the Plasmodium vaccine composition includes a PvAMA1 antigen, PfAMA1 antigen, and optionally at least one adjuvant and / or at least one physiologically acceptable carrier.

[0297] In one embodiment, the Plasmodium vaccine composition elicits an immune response to a Plasmodium species, e.g., Plasmodium vivax and / or Plasmodium falciparum, in a subject upon administration to the subject. In another embodiment, the immune response is sufficient to impede or prevent infection by a Plasmodium species, e.g., Plasmodium vivaxand / or Plasmodium falciparum. In another embodiment, the Plasmodium vaccine is for the treatment of or protection from erythrocytic (blood stage) and / or pre-erythrocytic (liver stage) malaria infection in a subject. In another embodiment, the Plasmodium vaccine is for the treatment of or protection from erythrocytic malaria infection in a subject. In another embodiment, the Plasmodium vaccine is for the treatment of or protection from pre- erythrocytic malaria infection in a subject. In still another embodiment, the treatment manifests itself in the subject as the parasitemia being under control and / or the infection being cleared.

[0298] In some embodiments, the PvAMA1 antigen includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 36.

[0299] In other embodiments, the PvAMA1 antigen includes a nucleic acid, such as mRNA, encoding an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 36.

[0300] In some embodiments, a nucleic acid encoding an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 36 can include a nucleotide sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 72.

[0301] In some embodiments, the PvAMA1 antigen includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 75.

[0302] In other embodiments, the PvAMA1 antigen includes a nucleic acid, such as mRNA, encoding an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 75.

[0303] In some embodiments, a nucleic acid encoding an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at leastabout 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 36 can include a nucleotide sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 74.

[0304] In some embodiments, the PfAMA1 antigen includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 38.

[0305] In other embodiments, the PfAMA1 antigen includes a nucleic acid, such as mRNA, encoding an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 38.

[0306] In some embodiments, a shorter fragment within such PvAMA1 antigen amino acid is contemplated and can potentially elicit a protective immune response. For example,PvAMA1 antigen amino acid can be a polypeptide that can include or be composed entirely of a polypeptide having a sequence that is about 90% to 100% identical to SEQ ID NO: 36 or SEQ ID NO: 75 or a fragment of at least 10 amino acids thereof.

[0307] In some embodiments, a shorter fragment within such PfAMA1 antigen amino acid is contemplated and can potentially elicit a protective immune response. For example, PvAMA1 antigen amino acid can be a polypeptide that can include or be composed entirely of a polypeptide having a sequence that is about 90% to 100% identical to SEQ ID NO: 38 or a fragment of at least 10 amino acids thereof.

[0308] In some embodiments, the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid can include amino acid variants. Variants of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid can include polypeptide analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced, or wherein one or more non-specified amino acids are added without loss of one or more of the biological activities or immunological characteristics (activity) specific for the PvAMA1 antigen amino acid and / or the PfAMA1 antigen amino acid; or with specific disablement of a particular biological activity of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid. Deletion variants contemplated also include fragments lacking portions of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid not essential for biological activity, and insertion variants include fusion polypeptides in which the wild-type polypeptide or fragment thereof have been fused to another polypeptide.

[0309] Variants of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid can include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides. Conservative substitutions are recognized in the art to classify amino acids according to their related physical properties and are known in the art (see, for example, Lehninger (Biochemistry, Second Edition (1975) W. H. Freeman & Co., pp.71-77).

[0310] Variant of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid include mature PvAMA1 antigen amino acids and PfAMA1 antigen amino acids, i.e., wherein leader or signal sequences are removed, having additional amino terminal residues. Variants contemplated herein also include gene products wherein amino-terminal sequences derived from other proteins have been introduced, as well as variants comprising amino-terminal sequences that are not found in naturally occurring proteins.

[0311] Variants of the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid can also polypeptides having additional amino acid residues resulting from the use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as a fusion protein with glutathione-S-transferase (GST) provide the desired polypeptide having an additional glycine residue at position −1 following cleavage of the GST component from the desired polypeptide. Variants that result from expression using other vector systems are also contemplated.

[0312] In an additional embodiment, at least one of the PvAMA1 antigen amino acid or PfAMA1 antigen amino acid can be coupled to a carrier protein. Suitable carrier proteins may include, without limitation, albumin, ovalbumin, a toxin, a growth factor, poly-L-lysine, poly-L-glutamine, or mannose-6-phosphate.

[0313] In some embodiments, the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid can each be linked respectively of a linker segment which self assembles into a nanoparticle, such as described in U.S. Patent Publication No.2007 / 0014804 and U.S. Patent Publication No.2012 / 0015000, both of which are incorporated by reference in their entirety. The PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid each linked respectively to a linker segment of self-assembling PvAMA1 antigen amino acid and the self- assembling PfAMA1 antigen amino acid can be combined or mixed at, for example, a 1:1 mixture to form a self-assembled peptide nanoparticle (SAPN) that displays both the PvAMA1 antigen amino acid and the PfAMA1 antigen amino acid to elicit a protective immune response against P. vivax and P. falciparum.

[0314] In some embodiments, the self-assembled peptide nanoparticle can include a self-assembling PvAMA1 antigen amino acid sequence that include a PvAMA1 amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 75 and / or a self- assembling PfAMA1 antigen amino acid sequence comprising an amino acid sequence atleast about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence to the amino acid sequence of SEQ ID NO: 38.

[0315] For example, the self-assembling PvAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 76.

[0316] In some embodiments, the self-assembling PfAMA1 antigen amino acid sequence can include an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 39.

[0317] The PvAMA1 antigen amino acid, PfAMA1 antigen amino acid, and self- assembling sequences thereof described herein can be encoded by a nucleic acid. The nucleic acid can be expressed and translated in a suitable in vitro or in vivo expression system. Such systems are generally known in the art. In addition to the nucleotides that encode the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid, the nucleic acid can include additional nucleotides that can be regulatory and / or encode additional transcribed proteins,such as selectable markers and / or reporter proteins. Examples of selectable markers include, but are not limited to, DNA and / or RNA segments that contain restriction enzyme sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g., FLAG- and His-tags), and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0318] Additional nucleotides can be operatively linked to the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid encoding nucleotides at the 5′ and / or 3′ end of the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid. In some embodiments, the nucleic acids encoding the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid can include a polyadenylation region at the 3′ end of the coding region of the nucleic acid encoding the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid. In addition to the nucleic acids encoding the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid, nucleotides for linkers and / or nucleotides that improves or otherwise regulates synthesis, purification, expression, and / or identification of the translated peptide can be operatively linked to the nucleic acids encoding the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid.

[0319] In some embodiments, the nucleic acids described herein can be incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector can contain one or more regulatory sequences or one or more other sequences used to facilitate the expression of the nucleic acids encoding the PvAMA1 antigen amino acid and PfAMA1 antigen amino acid. The expression vector can contain one or more regulatory sequences or one or more other sequences used to facilitate the replication of the expressionvector. The expression vector can be suitable for expressing the nucleic acids in a cell, such as a bacteria cell, yeast cell, or mammalian cell.

[0320] All or part of the vectors can be capable of being transcribed in vitro without a host cell or in a host cell. The vectors can be capable of being replicated by a host cell. All or part of the vector or an RNA molecule produced from the vector template can be capable of being integrated directly or indirectly into a host cell genome. The vectors can be viral vectors, i.e., vectors that are virus based or incorporate viral proteins or nucleic acids corresponding to a viral protein. Suitable viral vectors can include adenoviral, lentiviral, retroviral, and alpha viral vectors.

[0321] In some embodiments, the Plasmodium vaccine composition can be in the form of an antigen delivery system. Any appropriate antigen delivery system may be considered for delivery of the PvAMA1 antigen and PfAMA1 antigen described herein.

[0322] In certain embodiments, the antigen delivery system comprises an adeno- associated virus vector-based antigen delivery system, such as but not limited to the adeno- associated virus vector type 9 (AAV9 serotype), AAV type 8 (AAV8 serotype), etc. Examples of other antigen delivery systems include: adenoviruses such as but not limited to Ad5, Ad26, Ad35, etc., as well as carriers such as lipid nanoparticles, polymers, peptides, etc. In other embodiments, the antigen delivery system comprises a vesicular stomatitis virus (VSV) vector.

[0323] In certain embodiments, nucleic acids encoding the PvAMA1 antigen and PfAMA1 antigen can operatively linked to a generic promoter. For example, in certain embodiments, nucleic acids encoding the PvAMA1 antigen and PfAMA1 antigen are operatively linked to a CMV promoter. In certain embodiments, the antigen or antigens (e.g., epitopes) are operatively linked to a CAG, EFIA, EFS, CBh, SFFV, MSCV, mPGK, hPGK, SV40, UBC, or other appropriate promoter.

[0324] In some embodiments, the Plasmodium vaccine composition comprises a molecular adjuvant and / or one or more T Cell enhancement compositions. The adjuvant and / or enhancement compositions may help improve the immunogenicity and / or long-term memory of the Plasmodium vaccine composition. Non-limiting examples of molecular adjuvants include CpG, such as a CpG polymer, and flagellin. In some embodiments, the Plasmodium vaccine composition can include a T cell attracting chemokine. Non-limiting examples of T cell attracting chemokines include CCL5, CXCL9, CXCL10, CXCL11,CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.

[0325] In some embodiments, the Plasmodium vaccine composition can include a composition that promotes T cell proliferation. Non-limiting examples of compositions that promote T cell proliferation include IL-7, IL-15, IL-2, or a combination thereof.

[0326] In some embodiments, the T-cell enhancement compositions described herein (e.g. CXCL9, CXCL10, IL-7, IL-2) may be integrated into a separate delivery system from the Plasmodium vaccine composition. In other embodiments, the T-cell enhancement compositions described herein (e.g., CXCL9, CXCL10, IL-7, IL-2) may be integrated into the same delivery system as the vaccine composition.

[0327] In some embodiments, mRNA sequences encoding any of the vaccine compositions or portions thereof herein can be used. Modified mRNA sequences encoding any of the Plasmodium vaccine compositions or portions thereof can be used as well as DNA sequences encoding any of the Plasmodium vaccine compositions or portions thereof herein.

[0328] In certain embodiments, nucleic acids of a Plasmodium vaccine composition herein are chemically modified. In some embodiments, the nucleic acids of a vaccine composition therein are unmodified. In some embodiments, all or a portion of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. In some embodiments, all or a portion of the uracil in the open reading frame has an N1-methyl pseudouridine in the 5-position of the uracil.

[0329] In certain embodiments, an open reading frame of a Plasmodium vaccine composition herein encodes one antigen. In some embodiments, an open reading frame of a vaccine composition herein encodes two or more antigens. In some embodiments, an open reading frame of a vaccine composition herein encodes five or more antigens. In some embodiments, an open reading frame of a vaccine composition herein encodes ten or more antigens.

[0330] In some embodiments, the Plasmodium vaccine composition can be used in a method treat (e.g., alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of one or more symptoms or features of) and / or prevent Plasmodium infection.

[0331] In some embodiments, methods of vaccination and / or treatment (such as thosedescribed in the sections below) involve stratification of a patient population based on prior exposure to Plasmodium strains. Such methods involve steps of determining whether a patient has been previously exposed to one or more of the strains. In some embodiments, if it is determined that a patient has been previously been exposed to one or more of the strains, that patient may receive less concentrated, less potent, and / or less frequent doses of the inventive vaccine or composition. If it is determined that a patient has not been previously been exposed to one or more of the Plasmodium strains, that patient may receive more concentrated, more potent, and / or more frequent doses of the inventive vaccine.

[0332] In one embodiment, the vaccine treats more than one Plasmodium infection, i.e., infection with more than one Plasmodium strain / species. In another embodiment, the vaccine according to the invention is administered in combination with a distinct therapy.

[0333] A number of factors can be taken into account when determining the distinct therapy: the infecting species of Plasmodium parasite, the clinical situation of the patient (for example, adult, child, or pregnant female, with either mild or severe malaria), and the drug susceptibility of the infecting parasites. Drug susceptibility is determined by the geographic area where the infection was acquired. Different areas of the world have malaria types that are resistant to certain medications.

[0334] Furthermore, while mild malaria can be treated with oral medication; severe malaria (one or more symptoms of either impaired consciousness / coma, severe anemia, renal failure, pulmonary edema, acute respiratory distress syndrome, shock, disseminated intravascular coagulation, spontaneous bleeding, acidosis, hemoglobinuria [hemoglobin in the urine], jaundice, repeated generalized convulsions, and / or parasitemia ([parasites in the blood]>5%) requires intravenous (IV) drug treatment and fluids in the hospital.

[0335] Known drug treatments of malaria include, but are not limited to, chloroquine, quinine sulfate plus doxycycline, tetracycline, clindamycin, atovaquone-proguanil, artemisinin-derived combination therapy (ACTs)—drug combinations like artesunate- amodiaquine, artesunate-mefloquine, artesunate-pyronaridine, dihydroartemisinin- piperaquine, and chlorproguanil-dapsoneartesunate, and spiroindolones.

[0336] In some embodiments, the Plasmodium vaccine composition can induce an immune response in the recipient or host of the vaccine. The vaccine can induce protection against infection upon subsequent challenge with a Plasmodium species. Protection refers to resistance (e.g., partial resistance) to persistent infection of a host animal with at leastone Plasmodium species. Neutralizing antibodies generated in the vaccinated host can provide this protection.

[0337] In some embodiments, the Plasmodium vaccine composition can be used to prevent or reduce infection or disease by inducing immune responses, to PvAMA1 antigen and / or PfAMA1 antigen in an individual. For example, vaccines can be used prophylactically in naive individuals or therapeutically in individuals already infected with at least one Plasmodium species.

[0338] Protective responses can be evaluated by a variety of methods. For example, either the generation of neutralizing antibodies against Plasmodium proteins, specifically, PvAMA1 or PfAMA1, and / or the generation of a cell-mediated immune response against such proteins can indicate a protective response. Protective responses also include those responses that result in lower number of bacteria colonized in a vaccinated host animal exposed to a given inoculum (of the bacteria) as compared to a host animal exposed to the same inoculum, but that has not been administered the vaccine.

[0339] The Plasmodium vaccine composition can be administered to an individual in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of who / what is being treated. Prescription of treatment, e.g., final decisions on acceptable dosage etc., will be dictated by Vaccine Regulatory Authorities, after review of safety and efficacy data following human immunizations. Thus, a vaccine according to the invention comprises an immunoprotective or immunotherapeutic and non-toxic amount of the vaccine strain. Suitable dosage amounts can be determined by the person skilled in the art.

[0340] In some embodiments, the Plasmodium vaccine composition can include in addition to the PvAMA1 antigen and PfAMA1 antigen one or more inactive, agents such as a sterile, biocompatible pharmaceutical carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution. Alternatively or additionally, the Plasmodium vaccine composition may comprise a pharmaceutically acceptable excipient.

[0341] In some embodiments, the mode of administration of the Plasmodium vaccine composition is any suitable route that delivers an immunoprotective or immunotherapeutic amount of the Plasmodium vaccine composition to the subject and is described below.

[0342] The Plasmodium vaccine composition can be administered using any amount and any route of administration effective for treatment and / or vaccination. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like. Vaccines are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the vaccines will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the severity of the malaria infection; the activity of the specific vaccine composition employed; the half-life of the composition after administration; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific components employed; the duration of the treatment; drugs used in combination or coincidental with the specific components employed; and like factors, well known in the medical arts.

[0343] The Plasmodium vaccine composition can be administered by any route. In some embodiments, the vaccines are administered by a variety of routes, including oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical or transcutaneous (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, intranasal, buccal, enteral, vitreal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray, nasal spray, and / or aerosol, and / or through a portal vein catheter.

[0344] In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the Plasmodium vaccine composition being administered (e.g., its stability upon administration), the condition of the subject (e.g., whether the subject is able to tolerate a particular mode of administration), etc. In specific embodiments, vaccines may be administered intranasally. In specific embodiments, vaccines may be administered by intratracheal instillation. In specific embodiments, Plasmodium vaccine compositions may be administered by bronchial instillation. In specific embodiments, Plasmodium vaccine compositions may be administered by inhalation. In specific embodiments, Plasmodium vaccine compositions may be administered as a nasalspray. In specific embodiments, Plasmodium vaccine compositions may be administered mucosally. In specific embodiments, Plasmodium vaccine compositions may be administered orally. In specific embodiments, Plasmodium vaccine compositions may be administered by intravenous injection. In specific embodiments, Plasmodium vaccine compositions may be administered by intramuscular injection. In specific embodiments, Plasmodium vaccine compositions may be administered by subcutaneous injection. The oral or nasal spray or aerosol route (e.g., by inhalation) is most commonly used to deliver therapeutic agents (e.g., an immunogenic composition, comprising the Plasmodium vaccine composition) directly to the lungs and respiratory system. However, the invention encompasses the delivery of such a composition by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

[0345] For oral administration, a vaccine according to the invention may be presented as capsules, tablets, dissolvable membranes, powders, granules, or as a suspension. The Plasmodium vaccine composition may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The Plasmodium vaccine composition may also be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins. Additionally, the Plasmodium vaccine composition may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The Plasmodium vaccine composition may be further presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the Plasmodium vaccine composition may be presented with lubricants, such as talc or magnesium stearate.

[0346] For parenteral administration, the Plasmodium vaccine composition may be prepared with a sterile aqueous solution, which is preferably isotonic with the blood of the subject. Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The Plasmodium vaccine composition may be presented in unit or multi-dose containers, such as sealed ampoules or vials. The Plasmodium vaccine composition also may be delivered by any mode of injection, including any of those described herein.

[0347] Solid dosage forms for oral administration include capsules, tablets, pills, powders, dissolvable membranes, and granules. In such solid dosage forms, theimmunogenic composition is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), taste / olfactory components, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0348] Dosage forms for topical and / or transdermal administration of a vaccine in accordance may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and / or patches. Generally, the Plasmodium vaccine composition is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required. Additionally, the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active therapeutic agent (e.g., the Plasmodium vaccine composition) to the body is contemplated. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the immunogenic composition in the proper medium. Alternatively or additionally, the rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0349] For intranasal administration (e.g., nasal sprays) and / or pulmonary administration (administration by inhalation), a Plasmodium vaccine composition, including an aerosol formulation, may be prepared in accordance with procedures well known to persons of skill in the art. Aerosol formulations may comprise either solid particles or solutions (aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.) and atomizers may be used to produce aerosols from solutions (e.g., using a solvent such as ethanol); metered-dose inhalers and dry-powder inhalers may be used to generate small- particle aerosols. The desired aerosol particle size can be obtained by employing any one of a number of methods known in the art, including, without limitation, jet-milling, spraydrying, and critical-point condensation.

[0350] Plasmodium vaccine compositions can be used with a metered-dose inhaler device may include a finely-divided powder containing the immunogenic composition as a suspension in a non-aqueous medium. For example, the c Plasmodium vaccine composition may be suspended in a propellant with the aid of a surfactant (e.g., sorbitan trioleate, soya lecithin, or oleic acid). Metered-dose inhalers typically use a propellant gas (e.g., a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon) stored in a container (e.g., a canister) as a mixture (e.g., as a liquefied, compressed gas). Inhalers require actuation during inspiration. For example, actuation of a metering valve may release the mixture as an aerosol. Dry-powder inhalers use breath-actuation of a mixed powder.

[0351] The Plasmodium vaccine composition also may be released or delivered from an osmotic mini-pump or other timed-release device. The release rate from an elementary osmotic mini-pump may be modulated with a microporous, fast-response gel disposed in the release orifice. An osmotic mini-pump would be useful for controlling release, or targeting delivery, of the Plasmodium vaccine composition.

[0352] The Plasmodium vaccine composition can be administered to a subject, either alone or in combination with one or more drugs used to treat the infection or a symptom of the same. A Plasmodium vaccine composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0353] In some embodiments, the dosage of Plasmodium vaccine composition (or other composition) can be determined by, for example, first identifying doses effective to elicit a prophylactic and / or therapeutic immune response. This may be accomplished by measuring the serum titer of Plasmodium-specific immunoglobulins (anti-PvAMA1 or anti-PvAMA1 antibodies) and / or by measuring the inhibitory ratio of antibodies in serum samples. The dosages can be determined from animal studies, including animals that are not natural hosts to the parasite species in question. For example, the animals can be dosed with a vaccine candidate, e.g., a Plasmodium vaccine composition, to partially characterize the immune response induced and / or to determine if any neutralizing antibodies have been produced. In addition, routine human clinical studies can be performed to determine the effective dose for humans.

[0354] In one embodiment, a Plasmodium vaccine composition dose consists of a range of about 1 μg to about 1 mg total protein. In another embodiment, the range is about 0.1 mg to about 1.0 mg total protein. Such a dosage could be adjusted based on the amount of polypeptide delivered. More precise dosages can further be determined by assessing the immunogenicity of the Plasmodium vaccine composition.

[0355] Effective doses may be extrapolated from dose-response curves derived from in vitro and / or in vivo animal models. For example, various immunization schedules could be evaluated for optimum ensuing protection (and therapy).

[0356] An immunologically effective amount, based upon human studies, would, in one embodiment, be sufficient to stimulate an acceptable level of protective immunity in a population. For some vaccines (in certain embodiments), this immunologically effective level would provide an 80% efficacy against a malaria. For other vaccines (in other embodiments), an immunologically effective amount would be one that protects against severe malaria but may not protect against all symptoms of the infection.

[0357] In some embodiments, the Plasmodium vaccine composition may be administered to a subject at risk of developing malaria, in an amount effective to prevent the disorder in the subject. As used herein, the phrase “effective to prevent the disorder” includes effective to hinder or prevent the development or manifestation of clinical impairment or symptoms resulting from the disorder, or to reduce in intensity, severity, and / or frequency, and / or delay of onset of one or more symptoms of the disorder.

[0358] The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto. Example

[0359] This example discloses 12 human-derived monoclonal antibodies (humAbs) that target PvAMA1. These antibodies were taken from the blood cells of a person from Cambodia who had a history of Pv infection. They have been shown to stop PvAMA1 from binding to PvRON2. We have thoroughly studied the physical properties of these 12 humAbs. We tested their ability to stop the growth and invasion of merozoites and sporozoites in vitro. Additionally, we looked at how well our most effective humAb, referred to as 826827, could stop Pv infection in a chimeric FRG-humHep mouse model. In short, we have found a powerful humAb that can stop RON2-loop binding and consistently preventblood stage and sporozoite infection in both in vitro with multiple clinical isolates and in vivo. Our structural data shows that this humAb attaches to the RON2 hydrophobic binding groove and targets conserved amino acids in its epitope. Methods Blood samples

[0360] Peripheral blood mononuclear cells (PBMC) were obtained from Cambodians with documented Pv malaria residing in Pursat Province, as described previously. Samples were screened for blocking antibodies to PvAMA1. Institutional review boards from the National Institutes of Health (National Institute of Allergy and Infectious Diseases protocol 08-N094, clinicaltrials.gov identifier NCT00663546), Cambodian Ministry of Health, and University Hospitals of Cleveland Medical Center Institutional Review Board (no.04-14-19), approved the protocols. Written informed consent was obtained from all study participants or their parents / guardians. Protein expression and purification

[0361] The protein sequences for each of the antigens were selected: PfAMA1 (XP_001348015.1; 3D7 genotype), PvAMA1 (ACB42433.1; Palo Alto genotype), PvAMA1 (PNG 20 genotype) and PkAMA1 (XP_002259339.1; Strain H). At the N-terminus of the sequences, the native signal peptide was removed and replaced with a signal peptide for tissue plasminogen activator, followed by a 6-histidine tag. At the C-terminus of all sequences, the sequence was truncated to remove the transmembrane domain and cytoplasmic tail. All sequences were assessed for potential glycosylation sites (https: / / services.healthtech.dtu.dk / services / NetNGlyc-1.0 / ). The AMA1 sequences for each species were then modified to prevent potential glycosylation (PfAMA1, six changes; PvAMA1, three changes; PkAMA1, seven changes). Preparation of the PvAMA1 probe and cell sorting

[0362] The addition of biotin to PvAMA1_Palo Alto was done using EZ-Link NHS- PEG12-Biotin based on the manufacturers’ instructions. Using a Zeba spin 7 kDa cutoff buffer exchange spin column eliminated free biotin and changed to Bicine buffer, 50 mM pH 8.3. PvAMA1_Palo Alto biotin was quantified by BCA Protein assay (Pierce) andbiotin / molecule was calculated as 2 biotin per molecule using FluoReporter® Biotin Quantitation Assay, Invitrogen.

[0363] CD19+ human B cells were isolated from 30 × 106cryopreserved PBMCs using Miltenyi CD19 microbeads and then stained at 4°C for LIVE / DEAD, CD20, IgG, PvAMA1_Palo Alto_biotin tetramers prepared with Streptavidin-FITC and Streptavidin- Brilliant Violet 421. Single cells were sorted as a 96 well sort to a chilled wells on a FACS_ARIA_SORP. 252 single cells were isolated into 4 μL of catch buffer. Stained CD19 cells were stored at 4°C overnight and 252 more cells were collected as a dry catch. Cell staining and sorting of PvAMA1-specific Memory B cells

[0364] Single cells were identified and sorted from cryopreserved PBMC without activation. Briefly, staining and single-cell sorting of PvAMA1-specific IgG+ MBCs were performed as follows: B cells were enriched using immunomagnetic positive selection with anti-CD19 magnetic MACS beads (Miltenyi Biotec) and stained with mouse anti-human CD20 (PE-Cy5.5; Invitro- gen) and anti-human IgG Abs (PE-Cy7 clone G18-145; Becton Dickinson) along with biotinylated PvAMA1 using Streptavidin coupled with allophycocyanin (Becton Dickinson) and SYTOX Green Dead Cell Stain (Invitrogen) to gate out dead cells. Stained CD19+cells were sorted on a BD FACSAria II equipped with chilled stage sorting based on size and complexity. Doublet discrimination was performed to exclude aggregated cells. Individual AMA1-specific CD20+, IgG+ MBCs were single cell sorted directly into 4 µL of mRNA extraction buffer on a cooled 96- well metal block. After cells were collected, plates were frozen immediately on dry ice and stored at −80°C until further processing. cDNA synthesis

[0365] The 96−well plates with single cells were thawed on ice; a cold volume of 7 µL containing 300 ng of random hexamers (Qiagen Operon), 12 U Rnasin (Promega), and 0.9% NP−40 (Thermo Scientific Pierce) was added to each well. After thorough pipetting and rinsing, wells were capped, centrifuged at 4°C, heated to 68°C in a thermal cycler for 5 min, and placed on ice for at least 1 min. Reverse transcription was performed with the addition of 7 µL containing 3.6 µL of 53 reverse transcriptase buffer, 10 U RNAsin (Promega), 62 U Superscript III reverse transcriptase (Invitrogen), 0.62 µL dNTPs 25 mM each (V Bio−Tek), and 1.25 µL of 0.1 M DTT (Sigma− Aldrich). All wells were capped, and the plate placed in a cold rack and vortexed for 10 sec before centrifugation at 300 x g. Thermal cycler conditions for reverse transcription were as follows: 42°C for 5 min, 25°C for 10 min, 50°C for 60 min, 94°C for 5 min, and 4°C hold. When completed, 10 µL of nuclease−free PCR water was added to each well. Ig gene amplification

[0366] Immediately following cDNA synthesis, IgG genes (Igg) were amplified in a total of 20 µL per well for thefirst round of nested PCR for IgG H chain (Iggh), IgG k (Iggk) and IgG l (Iggl), using master mix (Table 1) and primers (Table 2). cDNA from individual sorted B cells were added and Igg amplified under the following conditions: thermal cycle PCR at 94°C for 15 min; 50 cycles at 94°C for 30 sec, then 58°C (Iggh and Iggk) or 60°C (Iggl) for 30 sec, then 72°C for 55 sec; then one cycle at 72°C for 10 min. Second round of nested PCR for Iggh, Iggk, and Iggl used master mix (Table 1), 2 µL offirst−round PCR product with second−round primers (Table 3) and the same master mix protocol, with the following conditions; thermal cycle PCR at 94°C for 15 min; 50 cycles at 94°C for 30 sec, then 58°C (Iggh and Iggk) or 60°C (Iggl) for 30 sec, then 72°C for 45 sec; then one cycle at 72°C for 10 min. The PCR product generated was purified and sequenced, with V(D)J genes determined using IMGT / V−Quest. Table 1 – PCR Round 1 Master Mix Specifics Component Volume for 1 well (μL)DNA Template 2.00 Total Volume (w / out DNA) 18.005’ 3’ Gamma LVH Group Mix CGammaCH15’ 3’ G A VH G Mi I GI t l ho Table SpecificsComponent Volume for 1 well (μL) Specific V(D)J regi

[0367] Primers specific with restriction enzyme sites for V and J regions were used to amplify thefirst−round PCR product to generate a fragment for cloning based on previouslydescribed primers. PCR product was purified, and restriction enzyme digested, cloned into Iggh, Iggk, or Iggl expression vectors, and chemically transformed into 5 μL of cDNA of aliquots of TOP10 E. coli cells (Thermo Fisher Scientific). Successful transformants were screened by PCR amplification (Table4) using a vector−specific primer paired with an insert−specific primer with the following conditions; thermal cycle PCR at 94°C for 15 min; 50 cycles at 94°C for 30 sec, then 57°C (Mu / Gamma / Kappa) or 60°C (Lambda) for 30 sec, then 72°C for 45 sec; then one cycle at 72°C for 10 min. Products were sequenced and compared with the second−round PCR product sequence. Definition of clonal groups

[0368] Clonal groups were based on H−chain nucleotide sequences. Any PCR product with 0.8% nucleotide sequences with a Phred score of <20 was excluded. We determined H chain alleles from PCR−amplified sequences using IMGT / V−QUEST (http: / / www.imgt.org, international ImMunoGeneTic information system). Because of primer mixture ambiguities, thefirst 20–22 nt of IgGH variable regions were designated germline; thus, this region was not evaluated for somatic hypermutations. IMGT / V−QUEST was used to assign V(D)J organization and shared IgHV genes and CDR3 length−grouped sequences. Clonal grouping was determined using Sequence Manipulation Suite: Ident and Sim, and the AB model in the open−source software CodonPhyML described by A. Mirsky, et al. This software utilizes specific clusters TIVLMFA, YHWGCPQ, SNKR, and DE to calculate the similarity between CDR3 sequences. A clonal group is defined by the same VDJ gene usage, CDR3 length, 84% similarity CDR3 aa sequence. HumAb expression and purification

[0369] Briefly, following transformation and sequencing to confirm that the plasmid contains the desired nucleotide sequence, glycerol stocks of E. coli containing the proper plasmid were stored at −80°C. Plasmids were harvested from ampicillin grown E. coli cultures (50 − 250 mL) using the ZymoPURE Midiprep Purification Kit. Purified DNA was quantified using a NanoDrop and stored at 4 °C until use. Transfection sizes were then dependent on DNA yield. Cells were transfected at a density of 3 − 5× 106viable cells per mL. On the day before transfection (Day −1), Expi293F™ culture was split to afinal density of 2.5 – 3.0 × 106viable cells / mL and allowed to grow overnight. The cells reached a densityof approximately 4.5 – 5.5 × 106viable cells / mL at the day of transfection. Cells were diluted with Expi293™ Expression Medium, pre-warmed to 37°C to afinal density of 3 × 106viable cells / mL. Plasmid DNA was diluted in Opti-MEM™ I Reduced Serum Medium at 1 μg / mL of transfected cells. ExpiFectamine™ 293 Reagent was diluted with Opti- MEM™ I Reduced SerumMedium and incubated at room temperature for 5 minutes prior to initiating the plasmid DNA complexation reaction. Diluted ExpiFectamine™ 293 Reagent was added to diluted plasmid DNA and mixed gently by swirling. ExpiFectamine™ 293 / plasmid DNA complexes were incubated at RT for 10–20 minutes and slowly transferred to the cell culture with continuous mixing. Cells were incubated at 37°C with a humidified atmosphere of 8% CO2 in the air with continuous orbital shaking.18–22 hours post-transfection, Expi- Fectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 were added to theflask per company instruction and gently mixed. Theflask was returned to the 37°C incubator with a humidified atmosphere of 8% CO2with shaking. Six to seven days post transfections cells are centrifuged at 4000 x g for 20 minutes at RT. The supernatant is decanted andfiltered through a 0.22 µm sterilefilter and stored at 4°C until purification. The volume of the cleared cell supernatant was supplemented with 1 M Tris pH 8.0 and 5 M NaCl solutions to afinal concentration of 0.1 M Tris pH 8.0 and 0.5 M NaCl and loaded onto a 5 mL HiTrap MabSelect PrismA column. Once the complete sample was loaded onto the column, the unbound sample was washed out with 10 column volumes (CV) 1x PBS. Elution was carried out with 10 CV using a commercial IgG Elution buffer pH 2.5 (Thermo Fisher) into a collection tube containing 1 mL of 1 M Tris pH 8.0 for immediate neutralization. Following the elution, the column was washed with 10 CV of 50 mM NaOH and re-equilibrated to 1 x PBS pH 7.4. Eluted humAbs were dialyzed overnight in 1 x PBS at 4°C. Protein concentrations were determined by Nanodrop (Thermo Fisher Scientific) using OD 1.3 for a 1 mg / mL concentration. Antibody titration curve (MagPix) methods

[0370] A total of 3.6 µl of 1 mg / mL PvAMA1_PaloAlto was conjugated to 300 µL of MagPix bead 19 following the manufacturer’s protocol (Luminex, Corp, Austin, TX). 1 µl of 4 mg / mL PfAMA1_3D7 was conjugated to 200 µL of MagPix bead 20, 4 µL of 1 mg / mL PvAMA1_PNG16 was con- jugated to 200 µL of MagPix bead 18, 4 µL of 1 mg / mL PkAMA1 was conjugated to 200 µL of MagPix bead 56, 4 µL of 1 mg / mL TgAMA1 wasconjugated to 200 µL of MagPix bead 12.50 µL of humAb at the following concentrations (1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125 µg / mL) were mixed with 50 µL of bead master mix (diluted 1:1000 in 1% BSA-TBST). Samples were incubated for 10 min at RT while shaking, and an additional 20 min while sitting. Plate was placed on a magnet for 5 min and excess liquid was removed. Plate was washed two times with 1x TBST for 2 min with shaking, followed by an additional 5 min on a magnet. Donkey anti-Human IgG (PE conjugated, Jackson ImmunoResearch) secondary antibody was diluted 1:400.25 µL of secondary antibody was added to the plate and samples were incubated for 10 min at RT while shaking, and an additional 20 min while sitting. Plate was placed on a magnet for 5 min and excess liquid was removed. Plate was washed two times with 1x TBST for 2 min with shaking, followed by an additional 5 min on a magnet.150 µL of 1% BSA-TBST was added to the plate, shaken for 2 min, and read using the MagPix machine. Antibody avidity (MagPix) methods

[0371] The beads used in the humAb titration were also used to measure antibody avidity.50 µL of 0.2 µg / mL of humAb was mixed with 50 µL of bead master mix (diluted 1:1000 in 1% BSA-TBST). Samples were incubated for 10 min at RT while shaking, and an additional 20 min while sitting. Plate was placed on a magnet for 5 min and excess liquid was removed. P late was washed two times with 1x TBST for 2 min with shaking, followed by an additional 5 min on a magnet.50 µL of Ammonium Thiocyanate at the followingfinal concentrations of 0, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 3.9 M was added to the plate. Samples were incubated for 5 min at RT while shaking, and an additional 5 min while sitting. The plate was placed on a magnet for 5 min and excess liquid was removed. The plate was washed two times with 1x TBST for 2 min with shaking, followed by an additional 5 min on a magnet. Donkey anti-Human IgG (PE conjugated, Jackson ImmunoResearch) secondary antibody was diluted 1:400.25 µL of secondary antibody was added to the plate and samples were incubated for 10 min at RT while shaking, and an additional 20 min while sitting. Plate was placed on a magnet for 5 min and excess liquid was removed. Plate was washed two times with 1x TBST for 2 min with shaking, followed by an additional 5 min on a magnet. 150 µL of 1% BSA-TBST was added to the plate, shaken for 2 min, and read using the MagPix machine. GraphPad Prism was used to calculate the IC50 of each humAb.Measurement of antibody affinity

[0372] To determine the binding affinity of each humAb to PvAMA1, Surface Plasmon Resonance (SPR) was utilized to perform single cycle kinetics. One CM5 chip (GE Healthcare)flow cell was coated with 275 RUs of anti-Human IgG antibody (Abcam). An untreated cell was used as a blank reference. Data was collected at 25°C and 10 Hz on a Biacore T200 system. Pilot studies of the interaction and follow-up regeneration protocols were established before data collection. Three start-up cycles were run with 1x HBSS Running Buffer (GE Healthcare). A humAb, at a concentration of 80 nM, was passed over all theflow cells at a rate of 10 µL / min for a contact time of 30 sec and allowed to stabilize for 120 sec. Increasing concentrations (5 nM, 10 nM, 20 nM, 40 nM, 80 nM) of rPvAMA1 were passed over allflow cells at a rate of 30 µL / min. Each injection was allowed to stabilize for 60 sec and was given 120 sec for dissociation. Anti-Human Fc chip was regenerated to baseline using IgG Elution Buffer (Thermo Scientific) and the process was repeated for all humAbs in the panel. Data was processed using the double referencing method using Scrubber 2.0 (BioLogic Software). Transgenic Pf-PvAMA1 growth inhibition assays

[0373] Invasion assays were performed as previously described, with minor changes. Parasites were synchronized with sorbitol, then inoculated at late trophozoite and schizont stages at 0.5% parasitemia in 4% hematocrit in 45 µL RPMI media, with5 µL antibodies in 1x PBS. Cultures were left to invade, then harvested at the ring stage byfixation with 0.25% glutaraldehyde. Parasites were stored in 1x PBS at 4°C. Cultures were later stained with 1× SYBR Green I (S7563, Invitrogen), and parasitemia assayed byflow cytometry in a BD FACSCanto™ II system, with further analysis in FlowJo™ v10.9.0 (BD Life Sciences). Parasites were gated from uninfected erythrocytes using the FITC-A and PE-A channels. Net growth rates were calculated by subtracting the parasitemia of parasites inoculated with 1 mg / mL heparin, then dividing by the parasitemia of parasites inoculated with 5 µL PBS. Growth percentages were subtracted from 100% to give invasion inhibition.

[0374] IC50 calculations were performed in R [R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https: / / www.R-project.org / ] using the packages drc and ggplot2. Assays wereperformed in biological triplicate, and IC50s calculated independently for each replicate. The parasite lines were based on W2mef, with the AMA1 locus replaced with the P. vivax locus. Parasite culturing and transgenic PfPvAMA1 inhibition experiments

[0375] P. falciparum asexual stage parasites were maintained in culture in human erythrocytes (blood group type O +) at a hematocrit of 4% in RPMI-HEPES supplemented with 0.25% (w / v) Albumax™ (Invitrogen) and 5% (v / v) heat-inactivated human serum. P. falciparum was synchronized using sorbitol and heparin treatments as described previously. Transgenic W2mef P. falciparum strains expressing PfAMA1 W2mef allele (W2-W2), PfAMA13D7 allele (W2- 3D7) and PvAMA1 Palo Alto allele (W2-PvAMA1) were generated in previous studies. In vitro invasion inhibition assay using Pv clinical isolates, DNA extraction amplification and sequencing of PvAMA1

[0376] Clinical isolates of P. vivax were either used fresh after blood collection or cryopreserved and infected RBCs were thawed and cultured in IMDM medium (Gibco) supplemented with 0.5% Albumax II (Gibco), 2.5% heat-inactivated human serum, 25 mM HEPES (Gibco), 20 μg / mL gentamicin (Sigma) and 0.2 mM hypoxanthine (C-C Pro) for ~24 or ~48 hr until a majority of schizont stage parasites were observed as previously described. The schizont-infected erythrocytes were enriched using KCl-Percoll density gradient, then mixed at a ratio of 1 erythrocyte to 1 reticulocyte enriched from cord blood and previously labeled with CellTrace Far Red dye following manufacturer’s instructions. The cultures were incubated for ~10 hr in afinal volume of 50 μL in 384 well plates, in presence of the humAbs. The threshold for P. vivax invasion of reticulocytes in controls is ≥0.2% parasitemia. This separates new invasion events from uninfected reticulocytes on FACS analysis. For the experiments performed with AMA1 humAbs, invasion rates in controls varied from 0.24 to 0.7%. The mouse monoclonal anti-Duffy 2C3 at 100 μg / mL was used as positive invasion inhibition control. Cells were stained with DNA stain Höchst 33342 post- invasion and examined byflow cytometry. Reticulocytes, which were Höchst 33342 and Far Red positive, were scored as new invasion events. For quantification, data were normalized against parasites mock-treated with 1x PBS. Invasion of reticulocytes in absence of antibodies ranged from 0.2% to 12.2% (mean 3.37%). The work presented here wasapproved by the National Ethics Committee for Health Research of Cambodia (192NECHR). All patients and / or their parents / guardians provided informed written consent.

[0377] Genomic DNA was extracted from an aliquot of the parasitized blood samples using the QIAamp DNA Blood Minit Kit (QUIAGEN), according to manufacturer’s instructions. PvAMA1 sequences were determined by PCR and Sanger sequencing (Macrogen, Seoul, South Korea) using the following conditions. The PCR was conducted in 20 µL reaction consisting of 2 µL of DNA, 0.25 µM of primers (Forward 5’- AAGCTGCTCACCCGTTAGTG-3’ (SEQ ID NO: 62); Reverse 5’-GGGTGGGAAGGTG- CATTCTG-3’) (SEQ ID NO: 63) and 1X HOT FirePol Blend MasterMix (Solis BioDyne, Tartu, Estonia) under the following conditions: 95°C for 1 min, followed by 34 cycles 95°C for 20 sec, 63.8°C for 30 sec, 72°C for 2 min and afinal extension at 72°C for 5 min. Nucleotides and corresponding amino acids were analyzed using MEGA 11 software. The sequences generated were compared with the reference strain Sal1 (PVX_092275). RON2 peptide competition

[0378] We conducted a RON2 binding assay. 96−well plates were coated with PvAMA1. HumAb was serially diluted in 0.1% casein in 1x PBS by a factor of two, with concentrations ranging from 40–0.039 µg / mL. 50 μL of humAb was added to each well.50 μL 0.1% casein in 1x PBS was added to additional wells as blank controls. The optical density (OD) of these were later subtracted from each of the other wells to correct for background. Each well, except for the blank control wells, was incubated with 50 µL of 1 µg / mL biotinylated PvRON2 peptide (Asp 2050 – Thr 2088)29in 1x PBS. Streptavidin– horseradish peroxidase conjugated protein (Thermo Fisher Scientific, 21130) was added at 1 / 500. Plates were incubated with ABTS for 20 min before reading. In vitro invasion into HC04 hepatocytes using Pv Sporozoite in Thailand and primary human hepatocytes in Cambodia

[0379] Anopheles dirus mosquitoes were fed on blood collected from symptomatic patients attending malaria clinics in Tak, Songkla, and Ubon- Ratchathani Provinces in Thailand and in Kampong Speu province in Cambodia. Samples were confirmed positive for only P. vivax via microscopy and RT-PCR. P. vivax-infected blood samples were collected from patients under the approved protocol by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2018-016). Sample collection in Cambodia wasapproved by the National Ethics Committee for Health Research of Cambodia (192NECHR). Written informed consent was obtained prior to blood collection.

[0380] Briefly, P. vivax infected blood was drawn into heparinized tubes and kept at 37°C until processing. Infected blood was washed once with RPMI 1640 incomplete medium. Packed infected blood was resuspended in warm, non-heat inactivated naive human AB serum (Thailand) or in heat-inactivated naive human AB serum (Cambodia) for afinal hematocrit of 50%. Resuspended blood was fed to laboratory-reared female Anopheles dirus mosquitoes for 30 min via an artificial membrane attached to a water-jacketed glass feeder kept at 37°C. Engorged mosquitoes were kept on 10% sugar at 26°C under 80% humidity at the designated insectary at the Mahidol Vivax Research Unit or the Malaria Research Unit of Institut Pasteur du Cambodge. Sporozoites were dissected from the salivary glands of infected mosquitoes 14–21 days after blood feeding and pooled in DMEM supplemented with 200 mg / mL penicillin-streptomycin (Thai- land) or in RPMI without NaHCO3(Cambodia).

[0381] In assays performed in Thailand, a 96-well plate was seeded with HC04 hepatocytes (50,000 cells / well). Isolated sporozoites (100,000 per well) were pre-incubated for 30 min with the humAbs at various concentrations (0.1, 1, 10, 100, and 1000 µg / mL). Sporozoite / humAb mixture was added to hepatocyte culture and incubated for 4 days at 37°C in duplicate. Murine anti-PvCSP polyclonal antibodies CSP 210 (clone 2F2) and 247 were used as positive controls. Anti-tetanus toxoid humAb 043038 was used as a negative control.

[0382] In the assay performed in Cambodia a 384-well plate was seeded with primary human hepatocytes (18,000 cells / well from BioIVT M00995). Isolated sporozoites (16,000 per well) were pre-incubated for 20 min with the humAb 826827 at various concentrations (0.1, 1, 10, 100, and 1000 µg / mL), with the humAb 043038 at 1000 µg / mL and with non- treated cell culture medium (latter two as negative controls). Each condition was tested in five technical replicates. Cell culture medium was changed to non-treated medium after 24 h of infection and the plate was incubated for 6 days at 37°C. Greater than 60 liver stage parasites / well in controls are used as cut-offs for a valid experiment. The mean of invasion controls varied from 72 to 904 for experiments using humAbs to PvAMA1. Cells werefixed and stained using sporozoite specific polyclonal antibodies UIS4 and DAPI. Wells were then analyzed usingfluorescent microscopy and invaded hepatocytes were quantified.In vivo infection of mouse human liver chimera mice

[0383] All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Oregon Health and Science University (IACUC protocol number: [IP00002518]). The FRG-humHep mouse studies were conducted similarly to studies previously published with modifications. FRG-humHep mice (n = 12) on the C57Bl / 6 background were purchased from Yecuris, Inc (Beaverton, OR, USA). Mice were pre- screened to have a serum human albumin level indicative of >90% humanization of hepatocytes. Mice were injected intravenously with the indicated quantity of antibody, diluted in PBS, approximately 3 hours before the challenge. Anopheles dirus mosquitoes were fed on blood collected from symptomatic patients of Plasmodium vivax in Thailand, parasites were allowed to mature to the sporozoite stage and migrate to salivary glands. Infected mosquitoes were shipped to the US. The salivary glands are dissected and diluted in Schneider’s insect media to allow sporozoites released from salivary glands. Sporozoites were centrifuged at 400 x g supernatant to remove salivary gland debris, re-washed, and counted. Mice were immediately injected with 400,000 freshly dissected sporozoites, diluted in Schneider’s insect media.

[0384] Blood was collected on day -1 (pre-infection) on days 2 and 9 post- infection using heparinized capillary tubes and 100 μL of whole blood was transferred to 1.9 mL of nucliSENS Lysis Buffer (Biomerieux Inc. Cat# 200292). Blood was allowed to lyse at room temperature for at least 30 minutes before storage at −80°C until qRT-PCR analysis. The remaining whole blood was centrifuged at 600 x g for 10 minutes, and plasma was frozen to measure human mAbs levels. Mice were then humanely euthanized, and liver samples were collected. Liver pieces were weighed and placed in 4 mL of nucliSENS Lysis Buffer and subsequently homogenized using a Bead Ruptor Elite (OMNI International, model #: NE48611 / A) with ceramic beads on the Liver-7mL tube setting 1 disruptor cycle at 5 m / s for 30 seconds. Following homogenization, the supernatant was collected for qRT-PCR quantification of parasite burden. The weight of the liver sample processed was used to standardize the resultant qRT-PCR values. Protein expression and purification of PvAMA1 and humAb 826827 for crystallization

[0385] For crystallization purposes, PvAMA1 (ACB42433.1; Palo Alto genotype) and humAb 826827 were expressed using transient transfection of Expi293 cells at a density of2.5 × 106cells / mL following the manufacturer’s protocol. Five days after transfection, supernatants were harvested by centrifugation at 7000 x g and dialyzed overnight into 30 mM Tris pH 7.5, 300 mM NaCl. Dialyzed PvAMA1-supernatant was incubated with Ni-NTA resin (Qiagen) for 1 hr at 4°C followed by stepwise elution using gravityflow chromatography. Size exclusion chromatography (SD200 increase in 20 mM HEPES pH 7.5, 150 mM NaCl) was used as thefinal purification step. Supernatant of humAb 826827 was loaded onto a 5 mL Mabselect PrismA column (Cytiva) equilibrated in 1x PBS. After washing the column with 1x PBS, humAb 826827 was eluted using 0.1 M citric acid pH 3.0 and neutralized with 1 M Tris pH 9.0 and buffer exchanged into 1x PBS. Complex formation, crystallization, and structure determination

[0386] HumAb 826827 was incubated with enzyme FabALACTICA (IgdE) at a ratio of 50 μg enzyme / mg humAb for 24–48 hr at 37°C. Fab fragments were purified by affinity chromatography using a 1 mL HiTrap Kappa-Select column (Cytiva), eluted with 0.1 M glycine pH 1.5 and immediately neutralized with 1 M Tris pH 9.0.

[0387] For crystallization, PvAMA1- Fab 826827 complexes were formed by incubating the PvAMA1 and Fab 826827 at a molar ratio of 1.2:1 for 1 hr on ice. This complex was separated from excess Fab using size exclusion chromatography in 20 mM HEPES pH 7.5, 150 mM NaCl. Crystallization screens were set up at the Monash Macromolecular Crystallization Platform (MMCP, Clayton, VIC, Australia) with 3 − 6 mg / mL at 20°C. Initial PvAMA1-Fab 826827 crystals grew in 22% PEG Smear Broad, 0.1 M Tris pH 8.5 (BCS screen, Molecular Dimensions) and were optimized by additive screening in a sitting drop vapor diffusion experiment (Additive Screen HT, Hampton Research). A PvAMA1-Fab 826827 crystal obtained in 22% PEG Smear Broad, 0.1 M Tris pH 8.5, 0.3 M Glycyl-glycyl-glycine was harvested with 30% glycerol in mother liquor as cryo-protectant and used for data collection. X-ray diffraction data was collected at the MXII beamline of the Australian Synchrotron at 100 K. The XDS package was used for data processing. Molecular replacement with Phaser was used to solve the phase problem using structural coordinates of PvAMA1 (PDB ID 5NQG) and Fab coordinates of PBD ID 6FG1. Iterative cycles of structure building and refinement was carried out using Coot and Phenix. Figures of the structure were prepared with PyMOL (Version 2.5.2 Schrödinger, LLC) orVIDA 4.4 (OpenEye, Cadence Molecular Science). The atomic coordinates and structure factorfiles have been deposited in the Protein Data Bank under PDB ID 9DX6. Results Isolation and expression of human monoclonal antibodies

[0388] We tested plasma from seven malaria-exposed Cambodian adults for antibodies that could inhibit the binding of PvRON2 to PvAMA1. Using the donor with the highest RON2 binding inhibition activity (Fig.7), we isolated 157 PvAMA1-specific B cells (Fig.8) that were PCR-amplified and sequenced for the immunoglobulin heavy chain (IGH) V-D-J region. B cells were placed into 67 clonal groups based on the same VDJ segment, CDR3 length, and 85% or greater amino acid similarity for CDR3. We generated 12 human PvAMA1-specific monoclonal antibodies that represent 12 clonal groups. The clones selected for the generation of humAbs were based on whether there was a corresponding immunoglobulin light chain (IGL), the quality of sequence, the degree of somatic hypermutations (SHM), and the selection of one B cell IGH + IGL pair from a clonal group (Fig.1A, B). The humAb are named with thefirst 3 numbers referring to IGH and the last three IGL. For example, humAb 826827 comes from the 826-heavy chain and 827- light chain.

[0389] To determine the specificity of 12 PvAMA1 humAbs, we tested the binding capabilities to recombinant AMA1 in a multiplex immunoassay format by coating magnetic microbeads with recombinant proteins corresponding to two strains of PvAMA1 (Palo Alto, the variant used to sort B cells and PvAMA1_PNG16, a sequence with significant poly- morphisms compared to Palo Alto), PkAMA1, PfAMA1_3D7, and TgAMA1 (Fig.1C). The sequence identity of the AMA1 constructs with respect to PvAMA1_Palo Alto was 97.2%, 85.6%, 60.3%, and 30.3% for PvAMA1_PNG16, PkAMA1, PfAMA1_3D7, and TgAMA1 respectively. All humAbs recognized PvAMA1_Palo Alto, ten recognized PvA- MA1_PNG16, seven recognized PkAMA1, two recognized PfAMA1_3D7, and none recognized TgAMA1 in a multiplex immunoassay. The isotype control humAb 043038, specific to tetanus toxoid C-terminal fragment, did not bind to any AMA1 recombinant proteins (Fig.1C). The humAb titers recognizing the different recombinant AMA1 con- structs varied among the different humAbs and were associated with differences in humAb avidity (Fig.1C, D). We measured avidity using the chaotropic reagent NH4SCN. Thecalculated Avidity Index 50 (AI50) represents the molar concentration of the chaotropic reagent where 50% of the binding of the humAb to AMA1 is lost (Table 5). Higher AI50 represents stronger binding. The humAbs 806807, 826827, 832833, and 838839 had the highest avidity. Of note, humAb 826827 has a higher avidity for PkAMA1 than PvAMA1. To further characterize the biophysical properties of the humAbs, we measured the affinity to PvAMA1_Palo Alto using Surface Plasmon Resonance (SPR). Affinities of humAbs to PvAMA1 ranged from 10.7 × 10−9to 47.7 × 10−9M (Fig.1E, Fig.9). We could not accurately determine the KD for three humAbs, 800801, 804805 and 808809. The PvAMA1 humAbs had various biophysical characteristics that may have further therapeutic potential. Table 5 –Avidity Index (AI50) of PvAMA1 specific humAbs to different AMA1 constructsTable 6 – List of SNP and haplotypes observes in AMA1 sequences in isolates tested in response to 826827 Haplot es 1 H2 H3 H4 H5 H6 H7.Table 7 - Crystallography data collection and refinement statistics PvAMA1-Fab 826827R.m.s. deviations Bond lengths (Å) 0004Table 8 – Interaction sof PvAMA1 – Fab 826827 based on PISA PvAMA1 Group 826827 Location Group Distance .6Gln 314 O Tyr 34 CDR-H1 OH 3.6 Asn 315 ND2 Tyr 117 CDR-H3 OH 2.8 73Ser 331 Pro 32 Gly 33 Ser 28 Ser 50 Thr 53

[0390] Because long-term in vitro culture of P. vivax is not currently possible, we used a modified Pf parasite line that can express PvAMA1 as a model to assess the ability of the humAbs to inhibit merozoite invasion. Fig.2A shows the IC50 curves of the four humAbs, 808809, 826827, 828829, and 830831. These humAbs demonstrated the lowest IC50s (2.6– 11.5 µg / mL). Other humAbs targeting PvAMA1 showed higher IC50 values or did not show blocking activity in this assay, e.g., 814815 and 816817 (Fig.10). HumAb 043038 wasemployed as a negative control and did not inhibit invasion at the tested concentrations. This assay shows humAb 826827 was the most potent, with an IC50 of 2.6 µg / ml. HumAb inhibition of Pv clinical isolates from Cambodia

[0391] To assess the ability of humAbs to PvAMA1 to inhibit Pv clinical isolates, we used in vitro reticulocyte invasion assays. This assay com- bines schizont-enriched red cells from P. vivax infected subjects with enriched reticulocytes from human cord blood donors and cultured for ~10 hours to allow schizonts to rupture and release merozoites to invade reticulocytes in the presence of various humAbs. The results of this assay are shown in (Fig.2B). Only humAb 826827 significantly inhibited Pv invasion of reticulocytes compared to the control humAb 043038 (67.4% (± 8.6 SEM) vs 4.7% (± 3.8 SEM), one-way ANOVA, P = < 0.0001). We conducted dose-response invasion assays using humAb 826827 ranging from 7.8 to 1000 µg / mL against four additional Pv clinical isolates to determine the IC50. The average IC50obtained from four clinical isolates was 48 µg / mL (± 6.6 SEM) (Fig.2C). These results and the in vitro Pf-PvAMA1 transgenic parasite experiments suggest that human 826827 is highly effective in inhibiting AMA1-dependent erythrocyte invasion. HumAb inhibition of Pv sporozoites in human hepatocytes

[0392] We examined humAb ability to inhibit Pv sporozoite invasion of hepatocytes in vitro (Fig.3). Sporozoites were isolated from the salivary glands of Anopheles dirus mosquitoes fed on blood collected from Pv-infected subjects from Thailand, and humAbs were tested for their ability to inhibit sporozoite invasion of HC04 hepatocytes. Five humAbs showed IC50s (0.38–2.6 μg / mL) that were comparable to the positive control anti- CSP murine monoclonal 2F2 (IC50CSP210 = 0.2 µg / mL, Fig.3A, Fig.11). For three biological replicates of humAb 826827 (Fig.3A), IC50s ranged from less than 0.07 to 1.8 μg / mL. We also performed the Pv sporozoite invasion assay on a Cambodian isolate using primary human hepatocytes and the humAb 826827 (Fig.3B). In this experiment, the IC50was 3.7 μg / mL. Thus, humAb 826827 can inhibit AMA1-dependent sporozoite invasion from multiple clinical isolates.

[0393] To assess whether humAb 826827 inhibited liver stage infection in vivo, we used liver chimeric mice (FRG-humHep), which have been transplanted with human hepatocytes and support P. vivax sporozoites infection and liver stage development. FRG-humHep mice were inoculated intravenously with 30 μg or 300 μg of humAb 826827 or 300 µg of negative control anti-tetanus toxoid humAb 043038 three hours before challenge with 400,000 freshly dissected P. vivax sporozoites. The average serum mAb concentration in the 300 μg 826827- treated group was 27.9 μg / mL 2 days after the sporozoite challenge and 4.1 μg / mL in the 30 μg-treated group (Fig.4A). To determine the effect of passive immunization on liver infection, animals were sacrificed on 9 days post-infection. This represents the peak of liver-stage growth and the beginning of schizont egress from the liver. Mice that received 300 μg humAb 826827 showed a significant reduction in liver-stage parasites assessed by 18S quantitative RT qPCR compared to control humAb (Fig.4B). However, 18S copies detected in the liver exhibited more variability in the 30 μg humAb 826827 treatment group, with one mouse showing comparable 18S copies to that observed in animals treated with 300 μg humAb 826827. The variability in 18S copies was not associated with the serum concentration of humAb 826827 in this treatment group. It is possible that some dead sporozoites remain in the liver by day 9 and their residual DNA is being detected due to the sensitivity of 18 s RT qPCR, accounting for the signal shown in the 300 μg humAb 826827 treatment group as previously seen in this model. There was also a significant reduction in microscopically identified merozoite or hypnozoites in the liver (Fig.12). Therefore, 300 μg humAb 826827 administration significantly reduced sporozoite invasion of hepatocytes in vivo. Structural studies defining humAb 826827 interaction with PvAMA1

[0394] We obtained a 2.4 Å resolution structure of humAb 826827 bound to the PvAMA1 ectodomain with an Rwork / Rfree of 19.2% and 23.5% (Table 7). A 1:1 complex is present in the asymmetric unit (Fig.5A). The resulting electron density map allowed the tracing of PvAMA1 from residues 46 to 474 with main chain gaps at residues 211–215, 296– 303, 328–334, and 402–415. Heavy and light chains of the antigen-binding fragment of 826827 were fully traced from 1–231 and 1–214, respectively. Our structure shows that 826827 interacts with PvAMA1 residues of Domain 1 and the mobile loop of Domain 2 (Fig.5A, panel i and ii). Five of the six complementarity-determining regions (CDR, namely: L1, L2, H1, H2, and H3) form direct contacts with PvAMA1 with a buried interaction surface of ~1392 Å2, with the CDR-H3 loop of 826827 contributing 70% of the buried surface area (Fig.5B, Fig.16). The CDR-H3 loop of 826827 forms a disulfidebridged β-hairpin that binds to the hydrophobic groove on PvAMA1 Domain 1, which constitutes part of the RON2-loop receptor binding site (Fig.5A–C). CDR-H3 binding to the PvAMA1-hydrophobic groove involves 53 interatomic contacts with distances <3.8 Å of which six are hydrogen bonds (Table 8). The mobile Domain 2 loop of PvAMA1 is contacted by residues of CDR-H3 and CDR-H1, forming one salt bridge and six hydrogen bonds that stabilize its position on Domain 1 (Fig.5, Fig.17). CDR-L2 and H2 interact with PvAMA1 residues of Domain 1 loops that surround the mobile Domain 2 loop, and CDR-L1 forms contacts with a Domain 1 loop next to the hydrophobic groove (Fig.5). Two residues located in the Domain 2 loop of AMA1, Arg317 and Lys321, provide a positively charged patch at the bottom of the RON2-loop binding groove. Otherwise, this binding pocket is largely hydrophobic (Fig.13). One salt bridge between PvAMA1 Lys321 and humAb 826827 CDR-H3 Glu103 provides an anchoring and orientation point for the observed interaction between the two proteins. The remainder of the interactions are hydrophobic and hydrogen bonding interactions in nature (Fig.5I).

[0395] Compared to structures of unbound PvAMA1 (PDB ID: 1W8K) and the PvAMA1 bound to a peptide representing the ß-hairpin loop of RON2 (PDB ID 5NQG), our PvAMA1 structure overlays with low root mean square (r.m.s.) deviation values of 0.463 Å (over 2137 atoms) and 0.417 Å (over 2121 atoms) respectively (Figs.5D, E). A major difference exists in our structure, where the mobile Domain 2 loop with residues 304–327 is visible while it is unstructured in the other PvAMA1 structures. Structural analysis with Plasmodium AMA1 homologs showed that the Domain 2 loop of our structure adopts a similar ‘closed’ conformation as in unbound PkAMA1 (PDB ID: 4UV6) which shares 90% sequence identity in this loop (Figs.5F, G). Our crystal structure clearly shows that in the presence of 826827, the Domain 2 loop of PvAMA1 is stabilized and remains bound to Domain 1, effectively blocking the RON2 binding site. The displacement of the Domain 2 loop is required to expose the complete RON2 binding site (Figs.5F, G). Our proposed model aligns with molecular dynamics simulations performed on PfAMA1-PfRON2 and TgAMA1-TgRON2 complexes. Our co-crystallization structure confirms that humAb 826827 binds to an important region of PvAMA1, providing a mechanistic explanation for its high potency.HumAb 826827 binds in a highly conserved binding pocket of PvAMA1

[0396] Previous co-crystal structures of Pf- and PvAMA1 reveal a hydrophobic binding pocket on AMA1 that interacts with the extracellular RON2-loop. Visually, our co- crystallization structure of humAb 826827 with PvAMA1 indicates that this antibody may interact with the same PvAMA1 residues as the RON2-loop. Fig.6A shows that the contact residues between PvAMA1 and RON2-loop overlap with those of PvAMA1 that contact humAb 826827 CDR-H3 residues. Sequence and structural analysis of the contact residues indicate that humAb 826827’s epitope is highly conserved for PkAMA1 and PcAMA1. However, little epitope conservation is observed in PfAMA1, accounting for the lack of humAb recognition of the recombinant protein (Figs.1C, 6A; Fig.12). Comparing the RON2 and CDR-H3 binding site of 826827 to other available Plasmodium species and model systems reveals that P. cynomolgi AMA1 is 100% conserved and would therefore serve as a predictive non-human primate model for Pv challenge infections to evaluate humAb 826827 (Fig.6A, Fig.14). HumAb 826827 competes for the same epitope on PvAMA1 as PvRON2

[0397] To confirm that the binding of humAb 826827 to PvAMA1 inhibits the extracellular RON2-loop from binding, we performed a dose-response competition assay between the two proteins. We found that humAb 826827 at a concentration as low as 2.5 μg / mL inhibits the binding of the biotinylated PvRON2-loop peptide to PvAMA1 (Fig.6B). This result demonstrates that humAb 826827 competes for the same epitope as the RON2- loop, further confirming humAb 826827-induced invasion inhibition mechanism. Sequence conservation across 390 clinical PvAMA1 isolates

[0398] To evaluate how the sequence conservation and polymorphisms in PvAMA1 may impact humAb efficacy, we examined 390 published PvAMA1 sequences from clinical isolates. We found 98% conservation of PvAMA1’s total amino acid sequence across the 484 residues. Domain 1 is the largest and most polymorphic of the domains of PvAMA1. Domain 1 (1–248) contains 21 polymorphic amino acid residues, Domain 2 (248–385) contains 6, and Domain 3 (386–484) contains 3. All polymorphic residues are 44–80% conserved across 390 PvAMA1 sequences (Fig.15).

[0399] There is 97% conservation of PvAMA1 residues that contact the CDR3 loop of 826827 (Fig.6C). We evaluated whether the PvAMA1 polymorphisms of the Pv clinical isolates used in the invasion assays affected the potency of 826827. We classified the invasion data by assessing the impact of the polymorphisms of AMA1 within the epitope recognized by humAb 826827 (Table 6). None of the 7 isolates tested against 826827 (Fig.2B) shared the same haplotype with the wild type (WT) reference Sal1 strain (PVX_092275) and had unique amino acid sequences. Among the 7 isolates, 4 were WT for the residues recognized by 826827; 2 had two mutations, and 1 had three mutations. All mutations were observed in residues 117, 130, and 132. As predicted, these polymorphisms did not impact the invasion inhibition by 826827 in terms of number of mutations compared to the WT or when considering individually each mutation compared to the WT, supporting our structural interpretation of the interaction (Fig.6D, E; Tables 6, 8).

[0400] Structural analysis of the two residues N130 (N 86.4%, K 13.9%) and N132 (D 52.9%, N 46.7%, G 0.5%) with the highest variability between PvAMA1 sequences are unlikely to disrupt humAb 826827 binding significantly as the interactions are either maintained (backbone interactions) or do not directly lie in the groove of the binding pocket (Fig.6D). An additional mutation within hydrogen bonding distance in the proximity of the CDR3 loop is G117 (G 99.0%, R 1%), which is unlikely to alter the interaction as it is pointing towards the solvent and is not interacting with other residues on the protein (Fig.6D). These data support our observations that humAb 826827 consistently inhibit liver and reticulocyte invasion with multiple clinical isolates.

[0401] We describe thefirst reported humAb that recognizes PvAMA1 and inhibits the invasion of pre-erythrocytic and blood-stage parasites. HumAb 826827 is potent and blocks the invasion of multiple clinical isolates. The dual activity against sporozoites and merozoites is valuable because new infections and relapses from dormant liver hypnozoites drive Pv disease. Reducing blood-stage infection also attenuates gametocyte production and, thus, Pv transmission.

[0402] 826827 was the most potent humAb to inhibit merozoite invasion into erythrocytes using a Pf transgenic parasite line expressing PvAMA1 (IC50 = 3.0 µg / mL) and in short-term invasion experiments into reticulocytes using Pv clinical isolates (IC50= 48 µg / mL). Only humAb 826827 showed significant inhibition of multiple Pv clinical isolates consistent with its recognition of a conserved epitope on PvAMA1. The other growth-inhibiting humAbs may target polymorphic epitopes that differ among the clinical isolates from the PvAMA1_Palo Alto, the variant used in Pf transgenic parasites and to sort B cells. These humAbs are more potent than previously reported PfAMA1-specific murine mAbs IF9 and 4G2, which display IC50s of 292 and 105 µg / mL, respectively, against the Pf WT strain 3D7 in blood stage invasion studies and rat mAb R31C2. They exhibit similar potency to a PfAMA1 humAb produced from an IgG sequence isolated from a Ghanaian with an IC50 of 35 µg / mL against the Pf 3D7 variant in vitro. Of note, a single-component PfAMA1-RON2L immunogen was developed as a vaccine candidate to produce an antibody response to complexed PfAMA1-RON2. Although the elicited polyclonal antibodies displayed Pf strain- transcending properties like our humAb 826827, they had poor potency with IC50s ranging from 1.5 to 4.5 mg / mL against Pf 3D7.

[0403] HumAb 826827 also blocked sporozoite invasion into human hepatocytes. This is consistent with recent studies showing AMA1 is utilized during sporozoite penetration of hepatocytes and entry into mosquito salivary glands. It has been previously demonstrated that mouse polyclonal antibodies generated by PfAMA1 or PvAMA1 inhibit blood sporozoite invasion into human hepatocytes but require a concentration of 0.5 to 1 mg / mL. By contrast, our humAb 826827 has an IC50of 0.3 µg / mL when preventing sporozoite invasion of human hepatocyte cell line HC04 and an IC50of 3.7 µg / mL in primary human hepatocytes. Using different clinical isolates across the different assays and observing slight variation in inhibition suggests that humAb 826827 is strain-transcendent with high potency. Indeed, the potency of humAb 826827 was comparable to the murine anti-circumsporozoite protein mAb 2F2, which was used as a positive control in these experiments. This is notable as passive transfusion of a suboptimal dose of mAb 2F2 in liver-humanized mice and challenged with Pv sporozoites reduced parasite relapse by 62%, associated with a corresponding reduction of hypnozoite numbers. This suggests that humAb 826827, when used in that same model, could achieve a similar or more significant reduction in hypnozoites, though this would require future testing. Of note, we validated humAb 826827 ability to inhibit sporozoite invasion into primary human hepatocytes. This model yields much higher infection rates than the HC04 cell line and allows for the development of both hypnozoites and schizonts. The difference in humAb 826827 concentrations required to inhibit sporozoites (IC50= 0.3– 3.7 µg / mL) in hepatocytes and merozoite invasion (IC50 = 48 µg / mL) may be attributed to variations in levels of antigen availability or the nature of molecular interactions that occurduring invasion. Parasite load, and thus, the number of PvAMA1 molecules expressed, differs significantly between the different in vitro assays. While 1 × 105sporozoites are used in the liver invasion assay, the number of merozoites present during the clinical isolate blood stage assay is a log-fold higher. Furthermore, the structural conformation of different epitopes displayed may vary between pre-erythrocytic and erythrocytic stages of invasion. However, the combination of our pre- and erythrocytic invasion inhibition data confirms that PvAMA1 plays an important role during sporozoite invasion into hepatocytes and merozoite invasion into reticulocytes. This confirms that AMA1 is a viable multi-stage therapeutic target against Plasmodium infection and requires further research.

[0404] The humAb 826827 also reduced liver-parasite burden in vivo using an FRG- humHep human liver chimeric mice model. When the animals received 300 µg of PvAMA1- specific humAb 826827 and achieved mean blood antibody levels of 27.9 µg / mL two days after sporozoite challenge, there was a significant decrease in liver parasite burden. This confirmed the in vitro observations. Administering a lower dose (30 µg) of humAb 826827 resulted in high variability in liver infection. Some animals had low parasite burdens in the liver comparable to those observed with higher antibody doses. The variation in liver burden did not correlate with serum antibody levels at the lower humAb dose. However, the variability may be attributed to the difference in the viability of injected sporozoites or variation in the AMA1 expression on the sporozoites that could affect antibody efficacy at lower doses. This model only tested activity against sporozoite invasion of the liver, not the blood-stage infection. Intravenous injection of parasites circumvents sporozoite migration in the skin, which might also be susceptible to antibody elimination. However, FRG-humHep chimeric mice are a recognized screening tool that allowed us to evaluate the inhibitory potential of humAb 826827 in vivo.

[0405] The crystal structure of recombinant PvAMA1_Palo Alto bound to humAb 826827 reveals why this humAb is potent and strain transcendent. The CDR3 of the heavy chain (826) recognizes a conformational epitope that overlaps with the RON2-loop binding site in Domain 1 and displays a higher affinity for PvAMA1 than PvRON2, 30.5 nM and 50.0 nM respectively. Previous structural studies suggested that a mobile loop of PvAMA1 Domain 2 partially obstructs the RON2 binding grove. This mobile loop must be displaced for a successful PvA-MA1:RON2 interaction to occur. HumAb 826827 binds the Domain 2 loop, thus preventing displacement and further interfering with PvRON2 engagement ofPvAMA1. The PvAMA1 contact residues that directly interact with humAb 826827 are conserved or possess single nucleotide polymorphisms (SNP) that do not affect humAb potency. For example, the SNP (N132D) frequently occurs in Pv clinical isolates. The Pv clinical isolates data indicates this mutation does not impair the hydrogen bonding capabilities between PvAMA1 and humAb 826827 (Fig.6D). Similarly, D133N maintains hydrogen bonding capabilities and does not impact the IC50 of 826827 (Fig.6D). The mutation N130K does change the interaction between PvAMA1 and 826827. However, the sidechains point towards the solvent, and only the backbone of the residue interacts with the light chain of 826827. As expected, there is no variation in humAb activity (Fig.6D). Of note, 826827 displays a much higher avidity to PkAMA1 than to PvAMA1_Palo Alto. Two amino acid changes (M153L & M171I) occur in 826827’s binding epitope from the PvAMA1 to PkAMA1 amino acid sequences. This variation in amino acid sequence creates a more hydrophobic and presumably stronger interaction between 826827 and PkAMA1, likely leading to increased avidity.

[0406] Recent studies suggest that PvAMA1 might interact directly with reticulocytes independent of RON2. Another study shows that some antibodies that do not inhibit PfAMA1-PfRON2 interaction can still be protective. This suggests that there might be antibodies that block AMA1 action by not blocking RON2. We did notfind such antibodies to PvAMA1. The humAbs to PvAMA1 that blocked sporozoite or merozoite invasion also inhibited PvAMA1-RON2 interaction to some extent. We may have biased selection of PvAMA1-RON2 blocking Abs because the individual from whom we isolated humAbs had potent PvAMA1-RON2 blocking activity in serum. The isolation of humAbs from PvAMA1 by other individuals might identify such humAbs.

[0407] In conclusion, we have discovered a highly conserved epitope of PvAMA1 that can be targeted by humAb 826827, preventing AMA1- dependent sporozoite invasion into hepatocytes in vitro and in vivo and merozoite invasion into reticulocytes. HumAb 826827 may lead to developing a new treatment to combat Pv infection, disease, and transmission. Moreover, identifying a conserved inhibitory epitope of PvAMA1 effectively targeted by 826827 could guide the design of structure-based vaccines for Pv.Example 2

[0408] This example discloses another human-derived monoclonal antibody (humAb) that targets PvAMA1. The humAb referred to as 864865 has been shown to stop PvAMA1 from binding to PvRON2.

[0409] Variable region 864865 CDRs are underlined in each sequence (hCDR3 bold): >864 EVQLVESGGGLVKPGGSLRLSCAASGFTASGFSFSKAWMGWVRQAPGKGLEWVGR IKRKIEGGTTDYTAPVKGRFTISRDDSRDTMYLQMDSLKTEDTAVYYCTTDHPGHY DYIWGSYDLTVDSWGQGTLVTVSS >865 EIVMTQSPATLSVSPGERATLSCRASQSVSRTLAWYQQKPGQAPRLLIFSASTRAAGI PARFSGSGSGTEFTLTINSLQSEDFAVYFCQQYNKWPPRPTFGGGTKVEIK

[0410] Fig.18 illustrates ribbon representation of a PvAMA1-mAB 864865 complex showing where 864865 is predicted to bind to an open domain 2 conformation of PvAMA1. Circled in red is the Domain 2 loop of AMA1 that is predicted to be in an open conformation when 864865 binds while it is in a closed conformation when 826827 binds (our crystal structure). Domain 2 loop is one of our vaccine candidates as it is highly conserved in P. vivax. The same loop with a different sequence is also highly conserved in P. falciparum, but a cross-species vaccine would be unlikely as the sequence identity is only 60%. However, a vaccine with both antigens displayed could be used for Pv and Pf immunization at the same time.

[0411] Fig.19 illustrates a plot showing competition assay indicating that 864865 and 826827 overlap with PvRON2 binding pocket in support of the predicted Competition assay using 50 µg / ml of PvRON2 (Asp 2050 – Thr 2088) that competed with varying concentrations of mAb 826827 or 864865 (40–0.039 µg / mL) to bind recombinant PvAMA1.

[0412] Fig.20 illustrates a graph showing A) MAbs inhibit blood-stage infection. The mean percentage (± SEM) of reticulocytes infected with Pv clinical isolates was calculated in short-term invasion inhibition studies with different mAbs at 100 µg / ml was calculated. Each dot represents a biological replicate from a different clinical isolate. The flow cytometry background of target cells (reticulocytes) without parasites (mean 9%, range 5-15%) was subtracted. Mouse mAb 2C3 (100 µg / ml) binds to Duffy Ag on reticulocytes, thus blocking Pv invasion (positive control). Anti-tetanus C-term mAb 043038 was used as a negative control. B) PvAMA1-specific mAbs inhibit sporozoite invasion of human hepatocyte HCO4cell line. IC50 were calculated at five concentrations (0.1–1000 μg / ml). Values represent the mean (SEM) of three biological replicates, with each replicate performed in duplicate. 043038 was used as a negative control. Murine anti-CSP 2F2 was used as a positive control. The biological replicate for CSP 2F2 with IC50 ~9 ug / ml was CSP247 variant; the other two isolates were CSP210.

[0413] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. An anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof that specifically binds an epitope corresponding to residues Thr164 to Gln175 and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), preferably an epitope corresponding to residues Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65), and more preferably an epitope corresponding to residues Thr116 to Gln119, Phe128 to Ile135, Thr164 to Gln175, and Gln310 to Lys321 of PvAMA1 (SEQ ID NO: 65).

2. An anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof comprising at least one of: a) a CDR-H1 comprising the amino acid sequence of GGSVSSPGY (SEQ ID NO: 3), a CDR-H2 comprising the amino acid sequence of IYYRGSSNQN (SEQ ID NO: 4), and a CDR-H3 comprising the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5); b) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 8), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 9), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 10); c) a CDR-H1 comprising the amino acid sequence of GFTASGFSFSKAWMG (SEQ ID NO: 24), a CDR-H2 comprising the amino acid sequence of GRIKRKIEGGT (SEQ ID NO: 25), and a CDR-H3 comprising the amino acid sequence of DHPGHYDYIWGSYDLTVDS (SEQ ID NO: 26);d) a CDR-L1 comprising the amino acid sequence of ASQSVSRT (SEQ ID NO: 28), a CDR-L2 comprising the amino acid sequence of SGSGTE (SEQ ID NO: 29), and a CDR-L3 comprising the amino acid sequence of YNKWPPRPT (SEQ ID NO: 30); or an antibody, antibody fragment or antigen binding fragment thereof that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment thereof comprising at least one of a), b), c), or d) to PvAMA1.

3. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a heavy chain variable region that includes the 3 CDRs of one of SEQ ID NO: 2 or SEQ ID NO: 23; or a heavy chain variable region that competitively inhibits binding of an antibody, antibody fragment or antigen binding fragment thereof comprising a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 or SEQ ID NO: 23 to PvAMA1.

4. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a heavy chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of one of SEQ ID NO: 2 or SEQ ID NO:

23.

5. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a light chain variable region that includes the 3 CDRs of one of SEQ ID NO: 7 or SEQ ID NO: 27; or a light chain variable region thatcompetitively inhibits binding of an antibody, antibody fragment or antigen binding fragment comprising a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7 or SEQ ID NO: 27 to PvAMA1.

6. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a light chain variable region that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO:

27.

7. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a) a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 2 and a light chain variable region that includes the 3 CDRs of SEQ ID NO: 7; or b) a heavy chain variable region that includes the 3 CDRs of SEQ ID NO: 23 and a light chain variable region that includes the 3 CDRs of SEQ ID NO:

27.

8. An anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof comprising: a) a heavy chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2 and a light chainthat includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 7; or b) a heavy chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 23 and a light chain that includes an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:

27.

9. The anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof of any of claims 1 to 8, wherein the antibody is a monoclonal antibody, antibody fragment or antigen binding fragment thereof.

10. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof of any of claims 1 to 9 being human, humanized, de-immunized, or chimeric.

11. The anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof of any of claims 1 to 10, wherein anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof is recombinant.

12. The anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof of any one of claims 1 to 11, wherein the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof.

13. The anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof of any one of claims 1 to 12, wherein the anti-PvAMA1 antibody, antibody fragment or antigen binding fragment thereof is a Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, nanobody, or a single domain antibody.

14. An anti-PvAMA1 nanobody comprising: a CDR that includes the amino acid sequence of X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10(SEQ ID NO: 11); wherein X2 is R or S; X3is S, A, or G; X4is R, H, N, or Y; X5is G or A; X6is Y or H; X7is F or G; X8is T, S, or N; X9is T or S; and X10is D or E; and wherein SEQ ID NO: 11 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of RSRGEGYCSFGTCYTLFYYFD (SEQ ID NO: 64).

15. An anti-PvAMA1 nanobody comprising: a CDR that includes the amino acid sequence of CX1X2X3X4GEX5X6CSX7GX8CYX9LFYYFX10X11W, (SEQ ID NO: 12); wherein: X1is A or V;X2is R or S; X3 is S, A, or G; X4is R, H, N, or Y; X5 is G or A; X6is Y or H; X7 is F or G; X8is T, S, or N; X9 is T or S; and X10is D or E; X11 is Y or N; and wherein SEQ ID NO: 12 is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5).

16. The anti-PvAMA1 nanobody of claim 14 or claim 15, including a CDR selected from: CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 5), CARAHGEGHCSGGSCYSLFYYFDYW (SEQ ID NO: 13), CARARGEAYCSGGTCYTLFYYFDYW (SEQ ID NO: 14), CARAHGEGYCSGGSCYSLFYYFDYW (SEQ ID NO: 15), CARAYGEGYCSFGTCYTLFYYFDNW (SEQ ID NO: 16), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 17), CARGHGEGYCSGGTCYSLFYYFEYW (SEQ ID NO: 18), CARGRGEGYCSGGNCYTLFYYFDYW (SEQ ID NO: 19), CARSRGEGYCSFGTCYTLFYYFDYW (SEQ ID NO: 20), CASGNGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 21), CVSANGEGYCSGGTCYSLFYYFDYW (SEQ ID NO: 22), or truncated sequence thereof.

17. The anti-PvAMA1 nanobody of any of claims 14 to 16, comprising a polypeptide that includes an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

33.

18. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 17 binding to Domain 1 and / or Domain 2 of PvAMA1.

19. The anti-PvAMA1 antibody, antibody fragment, or antigen binding fragment thereof, or nanobody of any of claims 1 to 18, competitively inhibiting binding of RON2 to PvAMA1.

20. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 19 for use in inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax sporozoite invasion of hepatocytes.

21. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 19 for use in inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax merozoite invasion of erythrocytes.

22. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 19 for use in treating and / or preventing Plasmodium infection, preferably Plasmodium vivax infection, preferably in a subject at risk of becoming infected with Plasmodium vivax.

23. The anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 19 for use in treating and / or preventing malaria in a subject in need thereof.

24. A composition comprising a nucleic acid having nucleotide sequence encoding an anti-PvAMA1 antibody, antibody fragment, antigen binding fragment thereof, or nanobody of any of claims 1 to 19.

25. The composition of claim 24, wherein the nucleic acid encodes a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 23, and / or a light chain variable region at least about 90% identical to the amino acid sequence SEQ ID NO: 7 or SEQ ID NO:

27.

26. The composition of claim 24, wherein the nucleic acid includes a nucleotide sequence at least about 90%, at least about 95%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO:

69.

27. The composition of claim 24, wherein the nucleic acid encodes a nanobody having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

33.

28. The composition of claim 24, wherein the nucleic acid encoding a nanobody includes a nucleotide sequence at least about 90% identical to the nucleotide sequence of SEQ ID NO:

70.

29. A composition of any of claims 24 to 28, including vector or a combination of vectors comprising the nucleic acid.

30. The composition of any of claims 24 to 29 for use inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax sporozoite invasion of hepatocytes.

31. The composition of any of claims 24 to 29 for use inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax merozoite invasion of erythrocytes.

32. The composition of any of claims 24 to 29 for use in treating and / or preventing Plasmodium infection, preferably Plasmodium vivax infection, preferably in a subject at risk of becoming infected with Plasmodium vivax.

33. The composition of any of claims 24 to 19 for use in treating and / or preventing malaria in a subject in need thereof.

34. A Plasmodium vaccine composition comprising a PvAMA1 antigen amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 75 or a nucleic encoding the PvAMA1 antigen amino acid sequence.

35. The Plasmodium vaccine composition of claim 34, wherein the nucleic acid includes a nucleotide sequence at least about at least about 90% identical to the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO:

74.

36. The Plasmodium vaccine composition of claim 34 or 35, further comprising a PfAMA1 antigen amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 38 and / or a nucleic acid encoding the PfAMA1 antigen amino acid sequence.

37. The Plasmodium vaccine composition of claim 36, wherein the nucleic acid encoding the PvAMA1 antigen amino acid sequence and / or the PfAMA1 antigen amino acid sequence includes DNA or RNA.

38. The Plasmodium vaccine composition of claim 34, comprising: a self-assembling PvAMA1 antigen amino acid sequence comprising an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37 and / or SEQ ID NO: 76 and / or a self-assembling PfAMA1 antigen amino acid sequence comprising an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 39.

39. A self-assembled peptide nanoparticle (SAPN) comprising: a self-assembling PvAMA1 antigen amino acid sequence comprising an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 36 and / or SEQ ID NO: 75 and / or a self-assembling PfAMA1 antigen amino acid sequence comprising an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO:

38.

40. The SAPN of claim 39, wherein the self-assembling PvAMA1 antigen amino acid sequence comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO:

76.

41. The SAPN of claims 39 or 40, wherein the self-assembling PfAMA1 antigen amino acid sequence comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO:

39.

42. The vaccine or SAPN of any of claims 34 to 41, for use in inhibiting Plasmodium sporozoite invasion of hepatocytes, preferably, Plasmodium vivax and / or Plasmodium falciparum sporozoite invasion of hepatocytes.

43. The vaccine or SAPN of any of claims 34 to 41 for use in inhibiting Plasmodium merozoite invasion of erythrocytes, preferably, Plasmodium vivax and / or Plasmodium falciparum merozoite invasion of erythrocytes.

44. The vaccine or SAPN of any of claims 34 to 41, for use in treating and / or preventing Plasmodium infection, preferably Plasmodium vivax and / or Plasmodium falciparum infection, preferably in a subject at risk of becoming infected with Plasmodium vivax and / or Plasmodium falciparum.

45. The vaccine or SAPN of any of claims 34 to 41, for use in treating and / or preventing malaria in a subject in need thereof.

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