PFCSP-based immunogens and related compositions and methods

Abstract

Described herein are malaria immunogens or variants thereof comprising at least a portion of a wild-type PfCSP amino acid sequence lacking a KQ motif. In certain aspects, the malaria immunogen lacks a KQP motif. For example, in certain aspects, the immunogens described herein do not comprise the C-terminal domain of the PfCSP. In other aspects, the immunogens described herein are particularly free of KQ or KQP motifs. In certain aspects, the immunogens described herein do not comprise an N-terminal KQ or KQP motif that is part of the N-terminal linking region in the PfCSP.

Description

Technical Field

[0001] The present invention relates to immunogens. In particular, the present invention relates to malaria immunogens based on the PfCSP antigen, and related compositions and methods. Background Art

[0002] Plasmodium falciparum (Pf) is the parasite responsible for most malaria deaths worldwide, but an effective pre-erythrocytic subunit vaccine remains elusive. Previous studies, such as Foquet, L. et al. (J. Clin. Invest. 124, 140–144, 2014), White, MT et al. (PLoS One 8, e61395, 2013), RTSS Clinical Trials Partnership (Lancet 386, 31–45, 2015), and Sumitani, M. et al. (Insect Mol. Biol. 22, 41–51, 2013), have shown the presence of protective antibodies against malaria that recognize the NANP repeats of the circumsporozoite protein (CSP) on the surface of Pf sporozoites.

[0003] The RTS,S / AS01 malaria subunit vaccine (GSK) contains 18.5 CSP NANP repeats and the complete C-terminal domain (C-CSP) displayed on virus-like particles composed of hepatitis B surface antigen building blocks. In a recent phase III trial in Africa, RTS,S / AS01 provided protection to approximately 50% of vaccine recipients, but its efficacy declined rapidly (Agnandji et al., 2011; RTSS Clinical Trials Partnership, 2015).

[0004] U.S. Patent Application Publication Nos. 2013 / 0259890 and 2016 / 0038580 describe nucleotide sequences and other constructs for expressing recombinant Plasmodium falciparum circumsporozoite protein in bacterial cells (e.g., E. coli). A process for producing soluble recombinant Plasmodium falciparum CSP by E. coli is described. A method for producing a highly immunogenic anti-malarial vaccine for human use based on CSP is demonstrated. Recombinant Plasmodium falciparum circumsporozoite protein itself or in combination with other malarial antigens or adjuvants is described as the basis for forming an effective malaria vaccine.

[0005] International Patent Application Publication No. 2018 / 193063 describes a Plasmodium circumsporozoite protein fragment, for example for use in a malaria vaccine, a nucleic acid encoding a Plasmodium CSP fragment, a composition comprising a Plasmodium CSP fragment, and an antibody that binds to a Plasmodium CSP fragment. The antibody specifically binds to Plasmodium falciparum sporozoites and can be used to treat and / or prevent malaria.

[0006] International Patent Application Publication No. 2012 / 154199 describes nucleotide sequences and other constructs for expressing recombinant Plasmodium falciparum circumsporozoite protein in bacterial cells (e.g., E. coli). A process for producing soluble recombinant Plasmodium falciparum CSP by E. coli is described. A method for producing a highly immunogenic anti-malarial vaccine for human use based on CSP is described.

[0007] There is a need to develop effective malaria vaccines as well as alternative vaccine platforms and related vaccines, compositions and methods. Summary of the invention

[0008] According to one aspect, there is provided a malarial immunogen or a variant thereof comprising at least a portion of a wild-type PfCSP amino acid sequence lacking a KQ motif.

[0009] In one aspect, the malarial immunogen lacks the KQP motif.

[0010] In one aspect, the malarial immunogen lacks an N-terminal ETG motif.

[0011] In one aspect, the malarial immunogen comprises the following motif:

[0012] -NPDP a ,PDPN a DPNP a or PNPD a ;

[0013] -NANP b ,ANPN b NPNA b or PNAN b ;

[0014] -NVDP c 、VDPN c DPNV c or PNVD c ;and

[0015] -NANP d ,ANPN d NPNA d or PNAN d ,

[0016] - wherein a, b, c and d are each independently 0 or greater, and wherein a+b+c+d is at least 2.

[0017] In one aspect, a, b, c, d, or any combination thereof, are each independently at least about 1.

[0018] In one aspect, a, b, c, d, or any combination thereof, are each independently from about 1 to about 40.

[0019] In one aspect, a, b, c, and d are each independently from about 1 to about 100, e.g., from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 75, about 80, about 90, or about 100, e.g., from about 1 to about 40, or from about 1 to about 20, or from 1 to about 10.

[0020] In one aspect, a is 1.

[0021] In one aspect, b is 1.

[0022] In one aspect, c is 3.

[0023] In one aspect, d is 5.

[0024] In one aspect, d is 18.5.

[0025] In one aspect, when a, b, c and / or d is greater than 1 such that the corresponding motif is at least partially repeated, each of the repeated motifs is independently contiguous.

[0026] In one aspect, when a, b, c and / or d is greater than 1 such that the corresponding motif is at least partially repeated, each of the repeated motifs is independently discontinuous.

[0027] In one aspect, the order of the motif is NPDP a -NANP b -NVDP c -NANP d .

[0028] In one aspect, the malarial immunogen further comprises an N-terminal ADG or PADG motif, wherein the ADG or PADG motif is optionally repeated alone or in combination with NPDP a , NANP b NVDP c and NANP d At least one combination of the motifs is repeated.

[0029] In one aspect, the variant has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the wild-type PfCSP amino acid sequence lacking the KQ motif.

[0030] In one aspect, the malarial immunogen is fused directly or indirectly to the nanocage monomer peptide.

[0031] In one aspect, the nanocage monomer is ferritin, apoferritin, encapsulin, SOR, dioxotetrahydropterin synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P or MS2 coat protein or a fragment or variant thereof.

[0032] In one aspect, the nanocage monomer is provided as two or more self-assembling subunits.

[0033] In one aspect, the nanocage monomer peptide is from Helicobacter pylori.

[0034] In one aspect, the nanocage monomer peptide is not human.

[0035] In one aspect, the nanocage monomer peptide is modified to reduce the anti-nanocage monomer peptide immune response.

[0036] In one aspect, the nanocage monomer peptides are modified to enhance antigenic immune responses.

[0037] In one aspect, the nanocage monomer peptide is at least partially or completely covered.

[0038] In one aspect, the nanocage monomer peptide is at least partially covered with a glycan.

[0039] In one aspect, the nanocage monomer peptide is completely covered with glycans.

[0040] In one aspect, the nanocage monomer comprises at least one NXT and / or NXS glycosylation motif.

[0041] In one aspect, the nanocage monomer is glycosylated at N79 and / or N99.

[0042] In one aspect, the nanocage monomers are glycosylated.

[0043] In one aspect, the nanocage monomers are glycosylated with high mannose glycans.

[0044] In one aspect, a plurality of nanocage monomer peptides self-assemble into a nanocage.

[0045] In one aspect, the immunogenic peptide decorates the interior and / or exterior surface of the nanocage.

[0046] In one aspect, the malarial immunogen further comprises a peptide that provides exogenous T cell help and / or a peptide that provides autologous T cell help.

[0047] In one aspect, peptides that provide exogenous T cell help include PADRE peptides and / or peptides derived from pathogenic molecules, such as tetanus toxoid peptides.

[0048] In one aspect, the PADRE peptide comprises the amino acid sequence AKFVAAWTLKAAA or a functional variant thereof having at least 70% sequence identity, or a fragment of either thereof.

[0049] In one aspect, the peptide that provides autologous T cell help comprises a PfCSP T cell peptide epitope.

[0050] In one aspect, peptides providing exogenous T cell help and / or peptides providing autologous T cell help independently decorate the interior and / or exterior surfaces of the assembled nanocages.

[0051] In one aspect, the malarial immunogen further comprises a linker between any one or more of the motifs, the nanocage monomer, and any other peptide (eg, a peptide that provides exogenous T cell help and / or a peptide that provides autologous T cell help).

[0052] In one aspect, the linker is a GGS linker.

[0053] In one aspect, the linker comprises the following amino acid sequence:

[0054] GGS;

[0055] GGGGSGGSGGSGGS; and / or

[0056] GGGGGSGGSGGSGGS.

[0057] In one aspect, the malarial immunogen comprises or consists of the following sequence:

[0058] ADG-NPDP-NANPNVDP3-NANP5-Hpferr-PADRE;

[0059] ADG-NPDP-NANPNVDP3-NANP18-Hpferr-PADRE;

[0060] ADG-NPDP-NANPNVDP3-NANP18.5-Hpferr-PADRE;

[0061] ADG-NPDP-NANPNVDP3-NANP5-LS-PADRE;

[0062] ADG-NPDP-NANPNVDP3-NANP18-LS-PADRE; and / or

[0063] ADG-NPDP-NANPNVDP3-NANP18.5-LS-PADRE.

[0064] In one aspect, the malarial immunogen comprises any of the following amino acid sequences: 203:

[0066] 205:

[0068] 206:

[0070] 207:

[0072] 208:

[0074] 209:

[0076] 210:

[0078] 211:

[0080] 212:

[0082] 213:

[0084]

[0085] or a functional variant thereof having at least 70% sequence identity thereto, or a functional fragment of either.

[0086] According to one aspect, a nucleic acid molecule encoding a malaria immunogen as described herein is provided.

[0087] According to one aspect, a vector comprising a nucleic acid molecule described herein is provided.

[0088] According to one aspect, a host cell comprising the vector of claim 40 and producing the malaria immunogen described herein is provided.

[0089] According to one aspect, there is provided a vaccine comprising a malaria immunogen as described herein.

[0090] In one aspect, the vaccine further comprises an adjuvant.

[0091] According to one aspect, there is provided an antibody that binds to a malaria immunogen as described herein.

[0092] According to one aspect, there is provided a method for treating and / or preventing malaria, comprising administering the immunogen, nucleotide, vector, cell or vaccine described herein.

[0093] According to one aspect, there is provided use of the immunogen, nucleotide, vector, cell or vaccine described herein for treating and / or preventing malaria.

[0094] In one aspect, the immunogens, nucleotides, vectors, cells or vaccines described herein are used to treat and / or prevent malaria.

[0095] In one aspect, the prophylactic and / or therapeutic effects are potentiable.

[0096] In one aspect, the prophylactic and / or therapeutic effects persist for at least about 6 months or longer, e.g., about 9 months or longer, about 12 months or longer, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years or longer.

[0097] The novel features of the present invention will be apparent to those skilled in the art after reviewing the following specific embodiments of the present invention. However, it should be understood that the specific embodiments and specific examples of the present invention, while indicating certain aspects of the present invention, are provided for illustrative purposes only, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on the specific embodiments and claims of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] The present invention will be further understood through the following description with reference to the accompanying drawings, in which:

[0099] Figure 1 : Strong recruitment of IGHV3-33 and IGHV3-15 expressing B cells in response to immunization with KQP-containing immunogens. Mice were subcutaneously immunized with immunogens containing N-linked amino acids and NANP units repeated 5 or 18 times in Hp ferritin or dioxotetrahydropteroidine synthase nanoparticles (126, 127 or 128 or 129) and SAS adjuvants on days 1, 28 and 70 and sacrificed on day 77. Cells from lymph nodes and bone marrow were collected. Use of IGHV gene fragments. A) Bone marrow plasma cells (BM PC), lymph node plasma cells (LN PC) and germinal center B cells (LN GCB). B) CSP+ and CSP- germinal center cells (GCB CSP+ and GCB CSP-) identified by flow cytometry and mixed plasma cells from lymph nodes and bone marrow (PC). Points represent individual animals, and only populations with >10 cells per animal were included in the analysis. N = 2 experiments, total number of sequences was 11430 (average 476 sequences per animal).

[0100] Figure 2 : Dominant pairing of IGHV3-15 and IGLV3-10. Figure 1 Usage of IGK / LV gene segments in CSP+ germinal center B cells expressing VH3-15 antibody.

[0101] Figure 3 : VH3-15 / VL3-10 monoclonal antibodies are specific for KQPA peptides with nanomolar affinity. A. ELISA reactivity of VH3-15 / VL3-10 (circles) and VH3-33 / VK1-5 (triangles) monoclonal antibodies to KQPA peptides when the peptides were coated directly onto the plate (y-axis) or when the C-terminally biotinylated KQPA peptides were captured via the streptavidin coating of the plate (x-axis). B. Affinity of VH3-15 / VL3-10 monoclonal antibodies (n=13) to the indicated peptides representing the N-terminal linker (KQPA and NPDP peptides) and the central repeat region (NANP3 peptide) of PfCSP, measured by surface plasmon resonance. The amino acid sequences of the corresponding peptides are shown in the figure below.

[0102] Figure 4 : Compared with high affinity NANP-reactive monoclonal antibodies with known parasite inhibitory activity (2A10 and 317), two representative high affinity VH3-15 / VL3-10 monoclonal antibodies have lower sporozoite binding and inhibition. A. Bar graphs show the binding intensity of VH3-33 / VK1-5 (left) and VH3-15 / VL3-10 (right) monoclonal antibodies to Pb-PfCSP transgenic sporozoites in flow cytometry. B. The percentage of sporozoites bound by selected VH3-15 / VL3-10 (n=7) and VH3-33 / VK1-5 (n=4) monoclonal antibodies. C. The percentage inhibition of Pf sporozoite (spz) in vitro hepatocyte crossing activity by the indicated monoclonal antibodies at the indicated concentrations. Monoclonal antibodies 2A10 and mGO53 were used as positive and negative controls, respectively. The red line represents the mean value. D. Ig gene signatures and KQPA peptide affinity of two representative VH3-15 / VL3-10 monoclonal antibodies tested. Data are representative of at least two independent experiments. B. Mann-Whitney test. **P<0.001.

[0103] Figure 5 : Fine epitope specificity of VH3-15 / VL3-10 monoclonal antibodies. A. Affinity of VH3-15 monoclonal antibody (n=13) for the indicated peptides representing the N-linked (KQPA and NPDP) and central repeat regions (NANP3 peptides) of PfCSP, measured by surface plasmon resonance. B. Affinity of VH3-15 / VL3-10 (circles) and VH3-33 / VK1-5 (triangles) monoclonal antibodies for the indicated peptides. The amino acid sequences of the peptides are shown.

[0104] Figure 6 : Crystal structure of 509893_121 Fab in complex with KQPA peptide. 509893 Fab is shown in the surface, with HC and LC framework regions in dark grey and white, respectively; HCDR1, -2 and -3 and LCDR1, -2 and -3 highlighted in light blue, sky blue, dense blue, light green, lime and green, respectively. Bound KQPA peptide is represented by magenta sticks with labeled residues. The total buried surface area (BSA) per peptide residue is listed in the table on the right. The contribution of KQP residues to the peptide interaction (40%) is shown in red.

[0105] Figure 7 : Removal of KQP residues from 155 immunogens results in loss of binding of the low inhibitory VH3-15 / VL3-10 mAbs. BLI binding curves of 155 and 203 nanoparticles with anti-KQP A specific Fabs 509893_121 and 511205_132. All samples were diluted to 10 μg / mL in kinetic buffer.

[0106] Figure 8 : Immunogens optimized at the N-terminus by removal of the KQP motif expressed well, were of high purity, and had good biophysical properties. SDS-PAGE and negative staining electron micrographs (scale bar, 50 nm). BLI binding curves of 203 immunogens with anti-PfCSP Fabs 4493, 317, 1210, 1710, 5D5, and 509893_121. 1710 and 5D5 served as negative controls because their epitopes were not present in the immunogen, while 4493, 317, and 1210 served as positive controls because their epitopes were present in the immunogen. All samples were diluted to 10 μg / mL in kinetic buffer.

[0107] Fig. 9 : Reactivity of sera from Kymice immunized at the indicated doses with two different classes of immunogens (155-A containing KQP; 203 lacking KQP) to peptides containing KQP residues. Serum IgG reactivity to truncated N-terminal peptides (N-CSPshort) with the indicated amino acid sequences containing KQP residues. The black line represents the arithmetic mean. Data represent at least two independent experiments.

[0108] Fig.10: Immunogenicity of immunogens with (155-A and 155-B) and without (203) KQP residues. Kymice (n=6 mice per group) were immunized with the indicated immunogens and doses and adjuvants or with adjuvant alone. Serum IgG responses to peptides with the indicated amino acid sequences were measured by ELISA at different time points after the first (d0) or second (d29) immunization, representing the N-terminal connection and NANP repeat domain (P126; top) or NANP repeat domain (NANP, bottom). The mean and standard deviation of the response over time for each group (left) and the serum IgG level of each mouse at d36 (right) are shown (circles). The lines in the right figure represent the mean. The data represent at least two independent experiments.

[0109] Fig.11 : Immunization with the 203 immunogen lacking KQP residues provides strong protection against challenge in wild-type mice. Groups of wild-type mice (n=5) were immunized intramuscularly with the 203 immunogen and adjuvant (red) according to a prime-boost schedule (d0, d28) and exposed three weeks later to three bites of Pb-PfCSP-infected mosquitoes. The adjuvant-only control group is shown in black. Parasitemia was examined daily by flow cytometry of blood samples.

[0110] Fig.12 : Immunogenicity of immunogens without KQP residues. Wild-type (WT) mice (n=5 per group) were immunized intramuscularly with the indicated high-mannose glycosylated (203F+Kif) or non-glycosylated (211F) immunogens and doses (0.5 μg or 10 μg / mouse) and adjuvant or adjuvant alone. Serum IgG responses against the indicated peptides were measured by ELISA at different time points after the first (d0) or second (d28) immunization. Data show the mean and standard deviation of three independent experiments averaged from 5 mice per group.

[0111] Fig.13 : Immunogenicity of immunogens without KQP residues. Wild-type (WT) mice (n=5 per group) were immunized intramuscularly with the indicated high-mannose glycosylated (203F+Kif) or non-glycosylated (211F) immunogens and doses (0.5 μg or 10 μg / mouse) and adjuvant or adjuvant alone. Serum IgG responses against the indicated peptides were measured by ELISA at different time points after the first (d0) or second (d28) immunization. Data show the mean and standard deviation of 5 mice per group for three independent experiments.

[0112] Fig.14: Highly protective serum responses induced by immunogens after removal of KQP residues. Serum inhibitory activity induced by immunization (intramuscularly) with glycosylated (203F, green) or non-glycosylated (211F, blue) immunogens and adjuvants compared with NANP 5.5 Figure 2 shows the relationship between serum IgG titers and the serum IgG titers of the immunized mice. (A) Results of the sporozoite crossing inhibition test of five mixed serum dilutions (day 35 (D35) and day 51 (D51)) (N=3). The mean + / -SEM of three repeated experiments is shown. (B) Results of the sporozoite crossing inhibition test of three serum dilutions (D35 and D51) (N=3). The mean + / -SEM of each experiment is shown. (C) In vivo protection data. On day 52 after immunization, immunized mice and controls were bitten by 3 infected mosquitoes. Blood parasitemia was detected for 10 consecutive days after infection. The summary results of two independent experiments are shown (n=5 per group / per experiment). DETAILED DESCRIPTION

[0113] definition

[0114] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The definition of common terms in molecular biology is found in Genes V by Benjamin Lewin, published by Oxford University Press in 1994 (ISBN 0-19-854287-9); The Encyclopedia of Molecular Biology by Kendrew et al. (editor), published by Blackwell Science Co., Ltd. in 1994 (ISBN 0-632-02182-9); and Molecular Biology and Biotechnology: a Comprehensive Desk Reference by Robert A. Meyers (editor), published by VCH Press in 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein may be used in the test practice of the present invention, typical materials and methods are described herein. The following terms will be used in the description and claims of the present invention.

[0115] It should also be understood that the terms used herein are only for describing specific aspects and are not intended to be limiting. Reference is made herein to a number of patent applications, patents, and publications to help understand the various aspects described. These references are incorporated herein in their entirety by reference.

[0116] In understanding the scope of the present application, "a", "an", and "the" are intended to indicate the presence of one or more elements. In addition, the term "comprising" and its derivatives used herein are open terms, indicating the presence of the features, elements, components, groups, integers and / or steps, but not excluding the presence of other unspecified features, elements, components, groups, integers and / or steps. The above also applies to words with similar meanings, such as the terms "including", "having" and their derivatives.

[0117] It should be understood that any aspect described as "comprising" certain components may also be "consisting of" or "consisting essentially of" these components, where "consisting of" has a closed or restrictive meaning, and "consisting essentially of" means including the specified components, but excluding other components except for materials present as impurities, unavoidable materials present due to the process used to provide the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, etc. Typically, a composition consisting essentially of a group of components will contain less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of non-specified components.

[0118] It should be understood that any component defined herein as included can be expressly excluded from the claimed invention by way of proviso or negative limitation. For example, in some aspects, the immunogens described herein do not comprise the C-terminal domain of PfCSP. In other aspects, the immunogens described herein specifically do not comprise a KQ or KQP motif. In some aspects, the immunogens described herein do not comprise an N-terminal KQ or KQP motif, or a KQ or KQP motif as part of the N-terminal connecting region in PfCSP.

[0119] In addition, no matter whether clearly stated, all scopes provided herein include the endpoints of scope and any middle range point.About four amino acid whose motifs as described herein, it should be understood that, for example, the scope of the 0-20 tumor-necrosis factor glycoproteins of these motifs also includes one-fourth integer, for example 0.25, 0.5 and 0.75, and every other one-fourth integer is considered herein.This shows that tumor-necrosis factor glycoproteins are not necessarily repeated completely, and for example NANP can be repeated by NANPN, NANPNA, NANPNAN, NANPNANP, N, NA, NAN, NANP, A, AN, ANP, ANPN, N, NP, NPN, NPNA, P, PN, PNA or PNAN etc.

[0120] The degree terms such as "substantially", "approximately" and "approximately" used herein refer to a reasonable deviation amount of the modified term so that the end result will not be significantly changed. These degree terms should be interpreted as including a deviation of at least ± 5% of the modified term, provided that such deviation does not negate the meaning of the word it modifies.

[0121] It should also be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to represent non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the term "for example". Unless the context clearly indicates otherwise, the term "or" includes "and".

[0122] The terms "protein nanoparticles" and "nanocages" are used interchangeably herein to refer to multi-subunit protein-based polyhedral structures. The subunits or nanocage monomers are composed of proteins or polypeptides (e.g., glycosylated polypeptides), and are optionally composed of the following single or multiple features: nucleic acids, prosthetic groups, organic and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct, and Mol. Biol., 15:939-947, 2008, incorporated herein by reference), sulfoxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, incorporated herein by reference), dioxotetrahydropteridine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 96:1240-1245, 1999, incorporated herein by reference). Ferritin, apoferritin, capsule protein, SOR, dioxotetrahydropteridine synthase and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes, in some cases consisting of 24, 60, 24, 60 and 60 protein subunits, respectively. Ferritin and apoferritin are generally used interchangeably herein, and it is understood that both are suitable for fusion proteins, nanocages and methods described herein. It is contemplated herein to use carboxysomes, vault proteins, GroEL, heat shock proteins, E2P and MS2 coat proteins to produce nanocages. In addition, it is also contemplated herein to use fully or partially synthesized self-assembling monomers.

[0123] It should be understood that each nanocage monomer can be divided into two or more subunits that will self-assemble into a functional nanocage monomer. For example, ferritin or apoferritin can be divided into an N-subunit and a C-subunit, essentially divided into two halves, so that each subunit can be separately bound to a different biologically active moiety and then self-assembled into a nanocage monomer and then self-assembled into a nanocage. "Functional nanocage monomer" refers to a nanocage monomer that can self-assemble with other such monomers into a nanocage as described herein.

[0124] A "vaccine" is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines induce antigen-specific immune responses to antigens of pathogens (e.g., viral pathogens) or cellular components associated with a pathological condition. A vaccine can comprise a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In a specific non-limiting example, the vaccine induces an immune response that reduces and / or prevents malaria compared to a control group. In another non-limiting example, the vaccine induces an immune response that reduces the severity of malaria-related symptoms and / or reduces parasite load compared to a control group.

[0125] The term "antibody", also known in the art as "immunoglobulin" (Ig), as used herein, refers to a protein constructed from paired heavy and light polypeptide chains; there are various Ig isotypes, including IgA, IgD, IgE, IgG, and IgM. When an antibody is properly folded, each chain folds into multiple distinct globular domains connected by more linear polypeptide sequences. For example, an immunoglobulin light chain folds into a variable (V L ) and constant (CL) domains, while the heavy chain folds into a variable (V H ) and three constants (C H , C H2 , C H3 ) domains. Heavy and light chain variable domains (V H and V L ) results in the formation of an antigen binding region (Fv). Each domain has a well-defined structure familiar to those skilled in the art.

[0126] The light and heavy chain variable regions are responsible for binding to the target antigen and can therefore display significant sequence diversity between antibodies. The constant regions display less sequence diversity and are responsible for binding to many natural proteins to initiate important immune events. The variable regions of an antibody contain the antigen-binding determinants of the molecule and therefore determine the specificity of the antibody for its target antigen. Most of the sequence variation occurs in six hypervariable regions, three in each variable heavy and light chain; the hypervariable regions combine to form the antigen-binding site and aid in the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for its antigen depends on the structure of the hypervariable regions, as well as the size, shape, and chemical nature of the surface they present to the antigen.

[0127] "Antibody fragments" referred to herein may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments, or may be obtained by manipulating naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include, but are not limited to, Fv, single-chain Fv (scFv; a fragment consisting of a V linked to a peptide linker, L and V H molecules composed of a single V L or V H fragments thereof) and multivalent display of any one of them.

[0128] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology. The term should also be interpreted to refer to an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein, or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technology available and well known in the art.

[0129] The term "epitope" refers to an antigenic determinant. An epitope is a specific chemical group or peptide sequence on a molecule that is antigenic (i.e., triggers a specific immune response). Antibodies specifically bind to specific antigenic epitopes, such as antigenic epitopes on polypeptides. Epitopes can be formed by continuous amino acids or non-continuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes usually contain at least 3 amino acids in a unique spatial conformation, more usually at least 5, about 9, about 11, or about 8 to about 12 amino acids. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. For example, see "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol.66, Glenn E.Morris, Ed (1996).

[0130] The terms "antigen" and "immunogenic peptide" are used interchangeably herein and are defined as molecules that elicit an immune response as used herein. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both. It will be understood by those skilled in the art that any macromolecule, including almost all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA or RNA. It will be understood by those skilled in the art that any DNA or RNA (e.g., mRNA) containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes the term "antigen" used herein. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. Obviously, the various aspects described herein include, but are not limited to, using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to elicit a desired immune response. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded by a "gene" at all. Obviously, antigens can be synthesized or derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, cells, or biological fluids.

[0131] "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (such as rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand used as a template for transcription of a gene or cDNA can be referred to as encoding a protein or other product of the gene or cDNA.

[0132] It will be appreciated by those skilled in the art that the immunogens described herein can be provided as protein or viral vector vaccines or mRNA vaccines or any other known vaccine forms. For example, COVID-19 mRNA vaccines are described in Gaviria, M., Kilic, BA network analysis of COVID-19 mRNA vaccine patents. Nat Biotechnol 39, 546–548 (2021). https: / / doi.org / 10.1038 / s41587-021-00912-9 , which are incorporated herein by reference, those skilled in the art will appreciate that the immunogens described herein can be formulated for administration in a similar manner.

[0133] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0134] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment (e.g., a host cell).

[0135] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate and encode the same amino acid sequence. Nucleotide sequences encoding proteins or RNAs may also contain introns to some extent, and nucleotide sequences encoding proteins may contain introns in some forms.

[0136] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a subject's response compared to the level of a subject without the treatment or compound, and / or compared to the level of response of an otherwise identical but untreated subject. The term includes interfering with and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject (usually a human).

[0137] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleotide sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if a promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous, connecting two protein coding regions in the same reading frame if necessary.

[0138] "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion.

[0139] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides used herein are interchangeable. It is generally known to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides" and monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides used herein include, but are not limited to, all nucleic acid sequences obtained by any available method in the art, including but not limited to recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or a cell genome using common cloning techniques and PCR, and by synthetic methods.

[0140] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0141] The term "specific binding" used when referring to antibodies means that the antibody recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one genus may also bind to antigens from one or more genus. However, this cross-genus reactivity itself does not change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody. In some cases, the term "specific binding" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance, referring to the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, antibodies generally recognize and bind to specific protein structures rather than proteins. If the antibody is specific to epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and an antibody will reduce the number of labeled A bound to the antibody.

[0142] The term "therapeutically effective amount", "effective amount" or "sufficient amount" refers to an amount sufficient to achieve a desired result when administered to a subject (including a mammal, such as a human), such as an amount effective to induce a protective immune response. The effective amount of the compound described herein may vary depending on factors such as the immunogen, the age, sex and weight of the subject. As will be appreciated by those skilled in the art, the dosage or treatment regimen may be adjusted to provide the optimal therapeutic response. For example, in certain aspects, administration of an effective amount of an immunogen described herein is sufficient to increase immunity to a pathogen (e.g., Plasmodium).

[0143] In addition, the subject immunization scheme using an effective amount can consist of a single administration or alternatively include a series of administrations. The length of the immunization period depends on a variety of factors, such as the immunogen, the age of the subject, the dosage of the reagent, the patient's responsiveness to the reagent, or a combination of these factors. It should also be understood that during a specific immunization scheme, the effective dose of the reagent used for treatment can be increased or decreased. By standard tests well known in the art, it is apparent that the dosage can be changed. In some aspects, the immunogen described herein can be used before, during, or after conventional therapy is used to treat the disease or disorder (such as malaria).

[0144] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell refers to a cell that has been transfected, transformed or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny cells.

[0145] As used herein, the phrases "transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to the polynucleotide to control transcription initiation by RNA polymerase and expression of the polynucleotide.

[0146] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0147] As used herein, the term "subject" refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is a human.

[0148] Administration "in combination with" one or more other agents includes simultaneous (concurrent) and sequential administration in any order.

[0149] The term "pharmaceutically acceptable" means that the compound or combination of compounds is compatible with the remaining ingredients of the formulation for pharmaceutical use and is generally safe for human administration according to established governmental standards, including those promulgated by the US Food and Drug Administration.

[0150] The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and / or absorption delaying agents, etc. The use of pharmaceutically acceptable carriers is well known.

[0151] The term "adjuvant" refers to a compound or mixture that is present in a vaccine and enhances the immune response to an antigen present in the vaccine. For example, an adjuvant can enhance the immune response to a polypeptide or immunogenic fragment or variant thereof present in the vaccine described herein. An adjuvant can act as a tissue reservoir that slowly releases the antigen, or as an immune activator that non-specifically enhances the immune response. Examples of adjuvants that can be used include MPL-TDM adjuvant (monophosphoryl lipid A / synthetic trehalose dicorynomycolate, available, for example, from GSK Biologics). Another suitable adjuvant is the immunostimulatory adjuvant AS01 / AS02 (GSK). These immunostimulatory adjuvants are formulated to produce a strong T cell response and include QS-21 (a saponin from Quillay saponaria), TL4 ligand (a monophosphoryl lipid A), together in a lipid or liposome carrier. Other adjuvants include, but are not limited to, nonionic block copolymer adjuvants (e.g., CRL 1005), aluminum phosphate (e.g., AlPO . sub.4), R-848 (Th1-like adjuvant), imiquimod, PAM3SYS, polyinosinic acid-cytidylic acid, loxolibin, BCG (bacillus Calmette-Guérin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin-derived antigens (e.g., CTA1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (e.g., MF59 or Montanide ISA 720 obtained from Novartis Vaccines), keyhole limpet Hemocyanin and dinitrophenol.

[0152] "Variant" is a biologically active immunogen, fusion protein, antibody or fragment thereof, which has an amino acid sequence different from the comparison sequence due to the insertion, deletion, modification and / or substitution of one or more amino acid residues in the comparison sequence. The sequence identity of the variant to the comparison sequence is generally less than 100%. However, in general, the amino acid sequence of the biologically active variant has at least about 70% amino acid sequence identity to the comparison sequence, for example, at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Variants include peptide fragments of at least 10 amino acids that retain a certain level of biological activity of the comparison sequence. Variants also include polypeptides in which one or more amino acid residues are added at or within the N- or C-terminus of the comparison sequence. Variants also include polypeptides in which a number of amino acid residues are deleted and optionally substituted with one or more amino acid residues. Variants may also be covalently modified, for example by substitution with a moiety other than a natural amino acid, or by modifying amino acid residues to produce a non-natural amino acid.

[0153] "Percent amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the residues in a sequence of interest (e.g., a polypeptide of the invention), after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions in a candidate sequence shall not be construed as affecting sequence identity or homology. Methods and computer programs for comparison are well known in the art, such as "BLAST".

[0154] "Active" or "activity" herein refers to the biological and / or immunological activity of the immunogens described herein, wherein "biological" activity refers to the (inhibitory or stimulatory) biological function caused by the immunogen.

[0155] The immunogens described herein may include modifications. Such modifications include, but are not limited to, coupling to effector molecules (such as antimalarial drugs or adjuvants). Modifications further include, but are not limited to, coupling to detectable reporter groups. Modifications that extend half-life (such as pegylation) are also included. Proteins and non-protein agents can be coupled to immunogens by methods known in the art. Coupling methods include direct connection, connection by covalently linked linkers and specific binding pair members (such as avidin-biotin). These methods include, for example, Greenfield et al., Cancer Research 50, 6600-6607 (1990) (incorporated herein by reference) and Amon et al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and Kiseleva et al., Mol. Biol. (USSR) 25, 508-514 (1991) (both incorporated herein by reference) methods described.

[0156] "Pan-DR binding" peptides or The peptide (Epimmune, San Diego, CA) is a member of a family of molecules that bind to multiple HLA class II DR molecules. The pattern of the molecule family can be called the HLA class II supertope. Molecules that bind to HLA-DR molecules and stimulate human helper T lymphocyte (HTL) responses in vitro and in vivo can be referred to as providing exogenous T cell help. For further definition of families, see, e.g., U.S. Serial No. 09 / 709,774, Serial No. 09 / 707,738, PCT Publication Nos. WO 95 / 07707 and WO 97 / 26784, U.S. Pat. No. 5,736,142, U.S. Pat. No. 5,679,640, and U.S. Pat. No. 6,413,935, the entire contents of which are incorporated herein by reference.

[0157] Immunogen

[0158] Malaria immunogens are described herein. Typically, the malaria immunogen comprises at least a portion of the wild-type PfCSP amino acid sequence, but lacks the KQ motif, more specifically, lacks the KQ motif found at or near the N-terminal connecting region of the wild-type sequence. In other aspects, the malaria immunogen lacks the KQP motif, more specifically, lacks the KQP motif found at or near the N-terminal connecting region of the wild-type sequence.

[0159] Surprisingly, it was found that in the context of the human immunoglobulin repertoire, sequences lacking the KQ or KQP motif resulted in an antibody response that was more focused on the junction / repeat region. In contrast, PfCSP-based immunogens containing the KQ or KQP motifs elicited a strong B cell response that was non-sporozoite reactive and non-suppressive. The examples herein demonstrate that this unexpected and highly desirable improvement in immunogenicity is clearly associated with the lack of the KQ or KQP motifs.

[0160] In other aspects, the malarial immunogen may lack the N-terminal ETG sequence.

[0161] For example, in some aspects, the malarial immunogen comprises at least one of the following motifs: NPDP, NANP, NVDP and NANP, which may be present in any order, may be repeated in sequence or may be repeated out of sequence to a certain extent. As described above, the malarial immunogen lacks a KQ or KQP motif, particularly a motif that may be present upstream of the NPDP, NANP, NVDP and / or NANP motifs. The malarial immunogens described herein are immunogenic and can be used to treat and / or prevent malaria. In some aspects, the malarial immunogen is used in a vaccine for the prevention of malaria.

[0162] In certain aspects, the malarial immunogen comprises one or more repeating motifs derived from PfCSP, such as:

[0163] -NPDP a ,PDPN a DPNP a or PNPD a ;

[0164] -NANP b ,ANPN b NPNA b or PNAN b ;

[0165] -NVDP c 、VDPN c DPNV c or PNVD c ;and

[0166] -NANP d ,ANPN d NPNA d or PNAN d .

[0167] The letters a, b, c and d indicate the number of times a given motif is repeated, each of a, b, c and d being independently present or absent, and if present, may be repeated any desired number of times as long as the resulting malarial immunogen retains immunogenicity. Typically, at least two motifs are present such that a+b+c+d is at least 2.

[0168] Typically, a, b, c, d or any combination thereof are each independently at least about 1, more typically, a, b, c, d or any combination thereof are each independently about 1 to about 40. This means that a, b, c and d are generally present, and generally do not repeat, or repeat no more than about 40 times. Fractional repetitions are understood to be included herein, because each motif comprises 4 amino acids, and therefore 1.25 repetitions will be understood to include the original motif of the first amino acid repetition, such as NANPN. Similarly, 1.5 repetitions will represent, for example, NANPNA, and 1.75 repetitions will represent, for example, NANPNAN.

[0169] It can be understood from the above that a, b, c and d are each independently present or absent, and are optionally repeated any number of times. Typically, however, a, b, c and d are each present and independently range from about 1 to about 100, e.g., from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 75, about 80, about 90, or about 100, e.g., from about 1 to about 40, or from about 1 to about 20, or from 1 to about 10. Typically, a is 1, b is 1, c is 3, and d is 5 or 18.5.

[0170] It should be understood that when any given motif is repeated in whole or in part, i.e. when a, b, c, d or e is greater than 1, the repeating motif may be continuous or discontinuous with the original motif. For example, for NANP2, this may be NANPNANP or NANP-middle sequence-NANP. It may also be a combination of continuous or discontinuous repeating motifs, such as NANPNANP-middle sequence-NANP.

[0171] It will be appreciated that the motifs listed above and their repeating motifs (if present) may be in any order, however, the motif is typically NPDP a -NANP b -NVDP c -NANP d order.

[0172] In some aspects, the motifs described herein may be preceded by other motifs, such as ADG or PADG motifs. If these motifs are included, they are typically located at or near the N-terminus. In some aspects, the ADG or PADG motifs may be preceded by the NPDP motifs described above. a , NANPb NVDP c or NANP d One or more of the motifs are repeated together. For example, ADGNPDP can be a repeating unit, PADGNPDP can be a repeating unit, and any other motif arrangement described herein can be similarly combined with ADG or PADG.

[0173] Variant sequences or fragments described herein may have any desired sequence identity with the comparison sequences herein, as long as they retain at least a certain level of the desired function of the comparison sequence. For example, the malarial immunogens described herein are immunogenic, and variants of these peptides will retain at least some immunogenicity. Typically, variants have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity with the immunogenic peptide or with the wild-type PfCSP amino acid sequence lacking the KQ motif.

[0174] In some aspects, the malarial immunogen described herein is directly or indirectly fused to a nanocage monomer peptide. The nanocage monomer described herein may be, for example, any nanocage monomer described in WO / 2019 / 023811 (incorporated herein by reference). Typically, the nanocage monomer is ferritin, apoferritin, capsule protein, SOR, dioxotetrahydropteridine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P or MS2 coat protein or a fragment or variant thereof, and may be provided with two or more self-assembling subunits.

[0175] The nanocage monomers can be from any species, but are usually from Helicobacter pylori and are usually ferritin, or HpFerr for short. Also, usually the nanocage monomer peptides are not human. In this way, anti-autoimmune responses can be mitigated.

[0176] Typically, the nanocage monomer peptide comprises or consists of the following amino acid sequence:

[0177]

[0178] Similar to the above, functional variants or fragments are also included. Typically, variants have at least 70% sequence identity with the reference sequence, and variants and fragments are capable of self-assembling into nanocages.

[0179] It will be appreciated that the nanocage monomer peptides may be modified in a variety of different ways to reduce anti-nanocage monomer peptide immune responses and enhance responses to desired antigenic components. For example, the nanocage monomer peptides may be at least partially or completely covered, such as partially or completely covered with polysaccharides, to mask the B cell epitopes of the nanocage. Thus, the nanocage monomer may comprise at least one NXT and / or NXS glycosylation motif. For example, the above sequence (or variant or fragment thereof) may be modified to comprise one or more of the K77N, E79T, E99N and I101T mutations. For example, the nanocage monomer may be glycosylated at N79 and / or N99, such as N79 and N99.

[0180] Thus, in certain aspects, it is understood that the nanocage monomer is glycosylated. It can be glycosylated with N-linked glycans, O-linked glycans, combinations of different glycans, and / or glycans produced in various other expression systems (e.g., yeast, insect cells, plants, etc.). In typical aspects, the nanocage monomer is glycosylated with high mannose glycans.

[0181] Typically, the nanocage monomer peptides are selected so that a plurality of nanocage monomer peptides self-assemble into a nanocage. It should be understood that the immunogenic peptide may decorate the interior and / or exterior surface of the nanocage.

[0182] The malarial immunogens described herein may comprise other peptide sequences. For example, peptides providing exogenous T cell assistance and / or peptides providing autologous T cell assistance may be fused with other peptides described herein in any order. In certain aspects, peptides providing exogenous T cell assistance include PADRE peptides and / or peptides derived from pathogenic molecules, such as tetanus toxoid peptides. If a PADRE peptide is used, it typically comprises the amino acid sequence AKFVAAWTLKAAA, or a functional variant with at least 70% sequence identity thereto, or a fragment of either of them. In alternative or additional aspects, peptides providing autologous T cell assistance may be included herein. Typically, peptides providing autologous T cell assistance include PfCSP T cell peptide epitopes.

[0183] As described above with respect to immunogenic peptides, peptides that provide exogenous T cell help and / or peptides that provide autologous T cell help can independently decorate the interior and / or exterior surfaces of assembled nanocages, which may be the same or different than the manner in which the immunogenic peptides decorate the nanocages.

[0184] In certain aspects, the malarial immunogens described herein include one or more flexible or non-flexible linkers between one or more motifs, nanocage monomers, and any other peptides (e.g., peptides that provide exogenous T cell help and / or peptides that provide autologous T cell help). Typically, the linker is flexible enough to allow the immunogenic peptide to adopt a favorable conformation once the protein is expressed.

[0185] The linker is generally long enough to impart some flexibility to the antigen, although it will be appreciated that the linker length will vary depending on the antigen and antibody sequences and the three-dimensional conformation of the malarial immunogen. Thus, the linker is generally from about 1 to about 30 amino acid residues, such as from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues, such as from about 8 to about 12 amino acid residues, such as 8, 10 or 12 amino acid residues.

[0186] The linker can be any amino acid sequence that does not interfere with the immunogenic binding of the immunogenic peptide. In a typical example, the flexible linker includes GGS or a GGS repeat sequence, such as GGSGGSGGSG, GGGGSGGSGGS or GGGGGSGGSGGSGGS.

[0187] Specific examples of malarial immunogens described herein include, for example, fusion proteins comprising or consisting of the following sequence:

[0188] ADG-NPDP-NANPNVDP3-NANP5-Hpferr-PADRE;

[0189] ADG-NPDP-NANPNVDP3-NANP18-Hpferr-PADRE;

[0190] ADG-NPDP-NANPNVDP3-NANP18.5-Hpferr-PADRE;

[0191] ADG-NPDP-NANPNVDP3-NANP5-LS-PADRE;

[0192] ADG-NPDP-NANPNVDP3-NANP18-LS-PADRE; and / or

[0193] ADG-NPDP-NANPNVDP3-NANP18.5-LS-PADRE.

[0194] More specifically, some examples include fusion proteins comprising or consisting of the following amino acid sequence: 203:

[0196] 205:

[0198] 206:

[0200] 207:

[0202] 208:

[0204] 209:

[0206] 210:

[0208]

[0209] 211:

[0211] 212:

[0213] 213:

[0215]

[0216] or a functional variant thereof having at least 70% sequence identity thereto or a functional fragment of either of them.

[0217] Also described herein are nucleic acid molecules encoding the malaria immunogens described herein, vectors, host cells and vaccines comprising the malaria immunogens described herein.As will be appreciated, the vaccine may comprise an adjuvant, for example as further described above.

[0218] The malarial immunogens described herein are immunogenic and are capable of eliciting an immune response in a subject. Thus, in certain aspects, antibodies that bind to the malarial immunogens described herein are also contemplated. Methods for immunizing subjects, including humans and animals, to generate and characterize such antibodies are well known. Such antibodies can then be used in detection, therapeutic or prophylactic compositions, etc.

[0219] In some aspects, the malarial immunogens described herein can be used as highly effective preerythrocytic subunit malarial vaccines. In some aspects, these can alternatively be C-terminally truncated PfCSP antigens. Additionally or alternatively, the fusion proteins described herein can be used to provide a malarial vaccine that can be enhanced based on PfCSP.

[0220] Further described herein are polysaccharide-covered nanocage monomer peptides, which may be as described above. For example, the nanocage monomer is typically ferritin, apoferritin, encapsulin, SOR, dioxotetrahydropteridine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P or MS2 coat protein or a fragment or variant thereof. The nanocage monomer may be provided as two or more self-assembling subunits. Typically, the nanocage monomer peptide is from Helicobacter pylori and / or is not human.

[0221] It should be understood that the nanocage monomer peptide may also comprise a biologically active moiety, such as an antibody or fragment thereof, an antigen, a detectable moiety, a drug, a diagnostic agent, or a combination thereof. Typically, the biologically active moiety comprises an antigen.

[0222] Multiple nanocage monomer peptides are typically self-assembled into a nanocage, and the bioactive moiety decorates the inner and / or outer surface of the nanocage.

[0223] As described above, the nanocage monomer peptide is at least partially or completely covered or masked, typically with a glycan. Typically, the nanocage monomer comprises at least one NXT and / or NXS glycosylation motif and / or comprises the following amino acid sequence:

[0224]

[0225] or a functional variant thereof having at least 70% sequence identity, or a functional fragment of either of them, wherein the sequence comprises at least one of K77N, E79T, E99N and I101T mutations.

[0226] It will be appreciated that the malarial immunogens may be modified as described above in general and / or for immunomodulation or immunofocusing. In addition, linear peptides of T cell epitopes may be included that aid in immunomodulation / increase humoral / antibody responses.

[0227] As described herein, substantially identical sequences may contain one or more conservative amino acid mutations. It is well known in the art that one or more conservative amino acid mutations of a reference sequence may produce a mutant peptide having no substantial changes in physiological, chemical or functional properties compared to the reference sequence; in this case, the reference sequence and the mutant sequence will be considered "substantially identical" polypeptides. Conservative amino acid mutations may include additions, deletions or substitutions of amino acids; conservative amino acid substitutions are defined herein as substitutions of one amino acid residue for another amino acid residue having similar chemical properties (e.g., size, charge or polarity).

[0228] In a non-limiting example, conservative mutations can be amino acid substitutions. This conservative amino acid substitution can replace another amino acid in the same group with a basic, neutral, hydrophobic or acidic amino acid. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7, which is usually positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R) and lysine (Lys or K). The term "neutral amino acid" (also referred to as "polar amino acid") refers to a hydrophilic amino acid whose side chain is uncharged at physiological pH, but has at least one bond in which a pair of electrons shared by two atoms is more tightly held by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N) and glutamine (Gln or Q). The term "hydrophobic amino acid" (also referred to as "non-polar amino acid") refers to amino acids whose hydrophobicity is greater than zero according to the standardized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A) and glycine (Gly or G). "Acidic amino acids" refer to hydrophilic amino acids with a side chain pK value of less than 7, which are generally negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0229] Sequence identity is used to assess the similarity of two sequences; maximum correspondence between residue positions is determined by calculating the percentage of residues that match when the two sequences are aligned. Sequence identity can be calculated using any known method; for example, computer software can be used to calculate sequence identity. Without wishing to be limited, sequence identity can be calculated using software such as the NCBI BLAST2 service maintained by the Swiss Bioinformatics Institute (and as shown at ca.expasy.org / tools / blast / ), BLAST-P, Blast-N or FASTA-N, or any other suitable software known in the art.

[0230] The substantially identical sequences of the present invention may be at least 85% identical; in another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identical (or any percentage therebetween) to the amino acid level of the sequences described herein. In specific aspects, the substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity may be due to conservative amino acid mutations.

[0231] The malarial immunogen of the present invention may also comprise additional sequences to facilitate its expression, detection or purification. Any such sequence or tag well known to those skilled in the art may be used. For example, and without wishing to be limited, the malarial immunogen may comprise a targeting or signal sequence (such as but not limited to ompA), a detection tag, an exemplary tag box (comprising a Strep tag) or any variant thereof; for example, see U.S. Patent No. 7,981,632, a His tag, a Flag tag with a sequence motif DYKDDDDK, an Xpress tag, an Avi tag, a calmodulin tag, a polyglutamic acid tag, an HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, Softag 1, Softag 3, a V5 tag, a CREB binding protein (CBP), a glutathione S-transferase (GST), a maltose binding protein (MBP), a green fluorescent protein (GFP), a thioredoxin tag or any combination thereof; a purification tag (such as but not limited to His5 or His6) or a combination thereof.

[0232] In another example, the additional sequence may be a biotin recognition site, such as described by Cronan et al. in WO 95 / 04069 or Voges et al. in WO / 2004 / 076670. As is well known to those skilled in the art, linker sequences may be used in combination with additional sequences or tags.

[0233] More specifically, the tag box can include an extracellular component that can specifically bind to an antibody with high affinity or avidity. In a single-chain fusion protein structure, the tag box can be located (a) adjacent to the amino terminus of the connecting region, (b) between the linker modules and connected to the linker, (c) adjacent to the carboxyl terminus of the binding domain, (d) between the binding domain (e.g., scFv) and the effector domain and connect the binding domain to the effector domain, (e) between the subunits of the binding domain and connect the subunits, or (f) at the amino terminus of the single-chain fusion protein. In certain embodiments, one or more connecting amino acids can be located between tag boxes with hydrophobic portions and connected to the tag boxes, or between tag boxes with connecting regions and connected to the tag boxes, or between tag boxes with linker modules and connected to the tag boxes, or between tag boxes with binding domains and connected to the tag boxes.

[0234] Malaria immunogens may also be displayed polyvalently. Multimerization may be achieved by any suitable method known in the art. For example, and without wishing to be limited in any way, multimerization may be achieved using self-assembling molecules as described in Zhang et al. (2004a, 2004b) and WO2003 / 046560.

[0235] Also included herein are isolated or purified malarial immunogens, polypeptides or fragments thereof immobilized on a surface using various methods; for example and without wishing to be limited, the polypeptides may be attached or coupled to the surface via His-tag coupling, biotin binding, covalent binding, adsorption, etc. The solid surface may be any suitable surface, such as, but not limited to, the surface of a well of a microtiter plate, a channel of a surface plasmon resonance (SPR) sensor chip, a membrane, beads (e.g., magnetic-based or agarose gel-based beads or other chromatography resins), glass, a thin film, or any other useful surface.

[0236] In other aspects, the malarial immunogen can be connected to a cargo molecule, or the assembled nanocage can accommodate cargo molecules; the fusion protein can deliver the cargo molecule to the desired site, and can be connected to the cargo molecule using any method known in the art (recombinant technology, chemical coupling, chelation, etc.). The cargo molecule can be any type of molecule, such as a therapeutic agent or a diagnostic agent. For example, and without wishing to be limited in any way, the therapeutic agent can be a radioactive isotope that can be used for radioimmunotherapy; a toxin, such as an immunotoxin; a cytokine, such as an immunocytokine; a cytotoxin; an apoptosis inducer; an enzyme; or any other suitable therapeutic molecule known in the art. In an alternative, the diagnostic agent can include, but is not limited to, a radioactive isotope, a paramagnetic label such as gadolinium or iron oxide, a fluorophore, a near-infrared (NIR) fluorescent dye or dye (such as Cy3, Cy5.5, Alexa680, Dylight680 or Dylight800), an affinity label fused to a detectable protein-based molecule (e.g., biotin, avidin, etc.) or any other suitable agent that can be detected by an imaging method. In one specific, non-limiting example, the malarial immunogen may be linked to a fluorescent agent such as FITC, or may be genetically fused to enhanced green fluorescent protein (EGFP).

[0237] The antibodies to the malarial immunogens described herein specifically bind to the malarial immunogens. Antibody specificity, which refers to the selective recognition of a specific epitope of an antigen by an antibody, can be determined for the malarial immunogens described herein based on affinity and / or avidity. Affinity is determined by the equilibrium constant (K) for the dissociation of the antigen and the antibody. D ) is a measure of the strength of the binding between an antigenic determinant (epitope) and an antibody binding site. Avidity is a measure of how strongly an antibody binds to its antigen. Antibodies are usually expressed in 10 -5 Up to 10 -11 M of K D Combine. Any value greater than 10 -4 M of K D Usually considered to indicate nonspecific binding. D The smaller the value, the higher the binding strength between the antigenic determinant and the antibody binding site. In certain aspects, the K of the antibodies described herein isD Less than 10 -4 M, 10 -5 M, 10 -6 M, 10 - 7 M, 10 -8 M or 10 -9 M.

[0238] Also described herein are nucleic acid molecules encoding the malarial immunogens and polypeptides described herein, as well as vectors comprising the nucleic acid molecules and host cells comprising the vectors.

[0239] The polynucleotides encoding the malarial immunogens described herein include polynucleotides having nucleic acid sequences substantially identical to the nucleic acid sequences of the polynucleotides of the present invention. "Substantially identical" nucleic acid sequences are defined herein as sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity to another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact match of nucleotides between the two sequences.

[0240] In addition, the expression vector provided comprises a polynucleotide sequence operably connected to the expression sequence, promoter and enhancer sequence described previously. Various expression vectors for effectively synthesizing antibody polypeptides in prokaryotes have been developed, such as bacteria and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. The vector of the present invention can comprise fragments of chromosomes, non-chromosomal and synthetic DNA sequences.

[0241] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from Escherichia coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA, such as M13 and other filamentous single-stranded DNA phages. An example of a vector that can be used for yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known SV-40 derivatives, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from functional mammalian vector combinations, such as those described above, as well as functional plasmids and phage DNA.

[0242] Other eukaryotic expression vectors are well known in the art (e.g., P J. Southern & P. ​​Berg, J. Mol. Appl. Genet, 1: 327-341 (1982); Subramani et al., Mol. Cell. Biol, 1: 854-864 (1981); Kaufinann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159: 601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol, 159: 601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), all of which are incorporated herein by reference).

[0243] The expression vector generally comprises at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of the lambda phage, the control region of the fd coat protein, the glycolytic promoter of yeast (e.g., the promoter of 3-phosphoglycerate kinase), the promoter of yeast acid phosphatase (e.g., Pho5), the promoter of yeast alpha mating factor, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus (e.g., early and late promoters or SV40), and other sequences known to control gene expression of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0244] Also described herein are recombinant host cells comprising the expression vectors described above. The malarial immunogens described herein can be expressed in cell lines other than hybridomas. Nucleic acids comprising sequences encoding the polypeptides of the invention can be used to transform suitable mammalian host cells.

[0245] Particularly preferred cell lines are selected based on high expression levels, constitutive expression of the protein of interest, and minimal host protein contamination. Mammalian cell lines that can be used as expression hosts are well known in the art, including many immortalized cell lines, such as, but not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and many other cells. Suitable other eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, Escherichia coli (e.g., Escherichia coli SG-936, Escherichia coli HB 101, Escherichia coli W3110, Escherichia coli X1776, Escherichia coli X2282, Escherichia coli DHI, and Escherichia coli MRC1), Pseudomonas, Bacillus (e.g., Bacillus subtilis), and Streptomyces.

[0246] These recombinant host cells of the present invention can be used to produce malarial immunogens by culturing cells under conditions that allow polypeptide expression and purifying polypeptides from the host cells or the culture medium surrounding the host cells. By inserting a signal or secretory leader peptide coding sequence at the 5' end of the antibody encoding gene of interest, the targeting of the expressed polypeptide secretion in the recombinant host cell can be promoted (see Shokri et al., (2003) Appl Microbiol Biotechnol. 60 (6): 654-664, Nielsen et al., Prot. Eng., 10: 1-6 (1997); von Heinje et al., Nucl. Acids Res., 14: 4683-4690 (1986), all of which are incorporated herein by reference). These secretory leader peptide elements can be derived from prokaryotic or eukaryotic sequences. Therefore, a secretory leader peptide (which is an amino acid attached to the N-terminus of the polypeptide) is appropriately used to guide the polypeptide to be removed from the host cell cytosol and secreted into the culture medium.

[0247] The malarial immunogens described herein may be fused with additional amino acid residues. For example, such amino acid residues may be peptide tags to facilitate separation. Other amino acid residues for homing malarial immunogens to specific organs or tissues are also contemplated.

[0248] In another aspect, described herein is a method of vaccinating a subject by administering an effective amount of a malarial immunogen as described herein to a mammal in need thereof (typically an adult, young, juvenile or newborn mammal). As described above, an effective amount refers to an amount effective to produce a desired effect, such as providing a protective immune response to the antigen in question that mediates prevention of infection with Plasmodium falciparum.

[0249] Any suitable method or route can be used to administer the malarial immunogens and vaccines described herein. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.

[0250] It should be understood that the malarial immunogens described herein, when used for prevention or treatment in mammals, will be administered in the form of a composition that additionally comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc. and combinations thereof. Pharmaceutically acceptable carriers may also contain small amounts of auxiliary substances that extend the shelf life or effectiveness of the binding protein, such as wetting agents or emulsifiers, preservatives or buffers. As is well known in the art, injectable compositions may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

[0251] Although human antibodies are particularly useful for administration to humans, they may also be generated using the malarial immunogens described herein for administration to other mammals. As used herein, the term "mammal" includes, but is not limited to, humans, laboratory animals, domestic pets, and livestock.

[0252] Also included herein are kits for vaccination, comprising a therapeutically or prophylactically effective amount of a malaria immunogen as described herein. For example, the kit may also comprise any suitable adjuvant. The kit may comprise instructions.

[0253] The above disclosure generally describes the present invention. A more complete understanding may be obtained by reference to the following specific examples. Unless otherwise indicated, these examples are for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be construed as being limited to the following examples, but rather should be construed as including any and all variations apparent from the teachings provided herein.

[0254] The following examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert genes encoding polypeptides into such vectors and plasmids, or introduce plasmids into host cells. These methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0255] Without further explanation, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to prepare and utilize the compounds of the present invention and implement the claimed methods. Therefore, the following working examples specifically point out typical aspects of the present invention, but should not be interpreted as limiting the rest of the present invention in any way.

[0256] Example

[0257] Example 1

[0258] Summarize

[0259] PfCSP is a malaria vaccine that elicits immunity against infection. We designed a PfCSP-based immunogen in which the KQP amino acids in the N-terminal linkage of the native PfCSP sequence were removed and the N-terminus of the immunogen was optimized. This immunogen induced a strong protective immune response in WT mice and, interestingly, in the context of the human immunoglobulin repertoire, the immune response was much stronger than that of an immunogen containing these KQP amino acids. These data suggest that PfCSP immunogens lacking these Lys and / or Gln and / or Pro amino acids can elicit stronger and more protective malaria immunity in humans following vaccination.

[0260] background

[0261] Malaria is a major global health problem, causing more than 400,000 deaths and 228 million cases each year, the majority of which are caused by Plasmodium falciparum (Pf) (1). In recent years, progress in the prevention and control of the disease has stalled, primarily due to increasing insecticide resistance among mosquito vectors (2) and the emergence of multidrug-resistant parasites (3).

[0262] The circumsporozoite protein (PfCSP) tightly covers the surface of Plasmodium sporozoites. It plays a crucial role in parasite development in the malaria mosquito vector and in the establishment of human hepatocyte infection (4-7). PfCSP contains a largely disordered central region consisting of only five amino acids (aa; asparagine (Asn, N), alanine (Ala, A), valine (Val, V), aspartic acid (Asp, D), and proline (Pro, P)) arranged in a large number of repeated NANP and NANP-like motifs (NPDP and NVDP) (8, 9). The central region is flanked by an N-terminal domain containing a conserved five-amino acid motif, termed region I (KLKQP at positions 93-97), followed by residues ADG, and a C-terminal thrombospondin repeat (TSR) domain that anchors the protein to the sporozoite surface via a glycosylphosphatidylinositol (GPI) anchor. Unlike the N- and C-terminal domains, which have a great deal of sequence diversity, the central region of the repeats shows only slight variations in the number of NANP and NVDP motifs ( 10 , 11 ).

[0263] The NANP repeat sequences exposed on sporozoites are immunodominant, and antibodies against the central repeat sequences can protect humans from infection (12). Infection with Pf sporozoites appears to drive anti-NANP antibody responses in humans through clonal selection of B cells with high-affinity germline B cell receptors (BCRs), with cells expressing the VH3-33 / VK1-5 genes predominating (13). In addition to antibodies against the NANP repeat sequences, several studies have identified potent antibodies that preferentially target the linker region connecting the PfCSP N-terminus and the NANP repeat sequence region, which contains a single NPDP motif and several interspersed NVDP motifs (14, 15). Cross-reactivity between repeat motifs is characteristic of antibodies encoded by different Ig gene combinations and has also been observed with mouse monoclonal antibodies against CSP sequences of other Plasmodium species, including Plasmodium berghei (16) and Plasmodium vivax (17). Furthermore, cross-reactivity appears to be associated with high binding affinity and parasite inhibitory activity, and thus repeated antigen exposure has been shown to result in enrichment of cross-reactive antibodies (18, 19). Due to the large number of NANP repeats in PfCSP and differences in the immunogenicity and accessibility of PfCSP epitopes (20, 21), selection of antibodies from sporozoite exposure in humans may be driven by affinity for NANP rather than for NVDP or NPDP motifs (18, 19).

[0264] An unresolved question is how to design a PfCSP-based immunogen that elicits the most potent inhibitory antibody response, resulting in protection from malaria parasite infection and disease. Indeed, a broadly effective malaria vaccine against Pf remains elusive, with only one vaccine, RTS,S / AS01, recently approved for use following pilot implementation studies in Malawi, Ghana, and Kenya. RTS,S / AS01 and the similar R21 / Matrix M vaccine are based on a number of PfCSP NANP repeats and a C-terminal domain fused to human hepatitis B surface antigen (HBsAg) and do not contain PfCSP N-terminal or linker sequences. Although these vaccines elicit protection against severe disease soon after multiple vaccinations, immunity wanes rapidly (22, 23), highlighting the need for improved PfCSP immunogens. Antibody studies such as those described above have demonstrated that inclusion of upstream linker epitopes (NPDP and NVDP motifs) in addition to the NANP repeats contained in immunogens such as RTS,S and R21 results in antibody responses that are more inhibitory. However, it is unclear which region upstream of the NPDP / NVDP / NANP junction / repeat sequence should be included in the next generation of PfCSP immunogens.

[0265] Unexpectedly, in the present work, it was found that a PfCSP-based immunogen containing the amino acids "KQP" (which is part of the highly conserved region I immediately upstream of the central region) elicited a strong non-sporozoite reactive and non-inhibitory antibody response after immunization against a human immunoglobulin repertoire. Guided by the newly obtained atomic precision structure-function relationship, an optimized PfCSP-based immunogen was designed with this unwanted epitope removed. It was demonstrated that removal of the KQP motif in the PfCSP immunogen helped to focus the antibody response in both the human immunoglobulin repertoire and the non-human repertoire. Cells dominated the response, particularly in the germinal centers that generated high affinity responses to the vaccine.

[0266] Thus, the PfCSP immunogen described herein is a careful combination of not only the regions associated with the strongest antibody recognition (junctions / repeats), but also the removal of unwanted epitopes associated with recognition by non-sporozoite reactive, non-inhibitory antibodies (e.g., N-terminus, C-terminus, KQP) that distract from the immune response. This immunogen can elicit the strongest inhibitory immune response to Plasmodium infection in humans.

[0267] Materials and methods

[0268] Expression and purification of immunogens

[0269] The genes encoding the immunogens were synthesized by GeneArt (Life Technologies) and cloned into pHLsec or pcDNA3.4 expression vectors. The secreted immunogens were expressed in CHO-M or HEK 293 cells (ThermoFisher Scientific) at a 1:1 ratio from stable cell pools or transient transfection using 50 μg of filtered DNA and the transfection reagent FectoPRO (Polyplus Transfections). The collected supernatant was filtered through a 100 MWCO LVCentramate TMLab tangential flow filtration system (Pall Laboratory), while switching the buffer from the expression medium to 20mM sodium acetate pH4 buffer. The resulting protein solution was loaded into a HiTrap Q HP column (anion exchange) (Cytiva) and eluted with 1M NaCl. The protein-containing components were desalted into 1×PBS using a HiTrap desalting column (Cytiva). Ammonium sulfate was added to the solution to a final concentration of 1.5M, and further purified using a HiTrap Phenyl HP column (hydrophobic purification) (Cytiva) balanced with 1×PBS, 1.5M ammonium sulfate buffer. The protein was eluted with a 1×PBS buffer gradient. The protein-containing components were desalted into 1×PBS using a HiTrap desalting column (Cytiva) again. The purified protein was concentrated to 2mg / ml and filtered with a 0.22μm membrane to ensure sterility. SDS-PAGE was run with 1μg protein under reducing and acidic (glycine pH2.2) conditions.

[0270] Fab production and purification

[0271] The VH and VL regions of monoclonal antibodies 4493 (Murugan et al., 2020), 317 (Oyen et al., 2017), 1210 (Imkeller et al., 2018), and 5D5 (Thai et al., 2020) were cloned into the pcDNA3.4-TOPO expression vector upstream of the human Igγ1-CH1 and Igκ domains, respectively. The VH and VL regions of monoclonal antibody 509893 were cloned into the pcDNA3.4-TOPO expression vector upstream of the human Igγ1-CH1 and Igλ domains, respectively. Paired Fab heavy and light chain plasmids were co-transfected into HEK293F cells (Thermo Fisher Scientific) for transient expression and purified by KappaSelect (4493, 317, 1210, 5D5) or LambdaFabSelect (509893) affinity chromatography (Cytiva) and cation exchange chromatography (MonoS; Cytima). 4493, 317, 1210 and 5D5 Fabs were purified by an additional size exclusion chromatography step (Superdex200Increase 10 / 300GL; Cytiva).

[0272] IgG production and purification

[0273] 1710 IgG (Scally et al., 2018) was transiently expressed in HEK293F cells by co-transfection of paired Ig heavy and light chains and purified by protein A affinity chromatography (Cytiva) followed by size exclusion chromatography (Superdex 200 Increase 10 / 300 GL; Cytiva).

[0274] Biolayer Interferometry (BLI)

[0275] BLI (Octet RED96, ForteBio) experiments were performed to evaluate the binding of immunogens to anti-CSP IgG / Fab 4493, 317, 1210, 1710, 5D5 and to anti-KQP Fab 509893 and 511205. All samples were diluted to 10 μg / mL in kinetic buffer (PBS, pH 7.4, 0.01% [w / v] BSA, 0.002% [v / v] Tween-20) and Fab was immobilized on anti-human Fab-CH1 biosensors (ForteBio). After the baseline was stabilized, the biosensor was immersed in a well containing the kinetic buffer of the immunogen.

[0276] Negative staining electron microscopy

[0277] Immunogens were deposited at a concentration of approximately 0.03 μg / mL onto carbon film coated grids and stained with 2% uranyl formate. Grids were imaged using a FEI Tecnai T20 electron microscope, operating at 200 kV with an Orius charge coupled device (CCD) camera (Gatan Inc).

[0278] Mice and immunization

[0279] Transgenic human immunoglobulin heavy, lambda, and kappa gene fragments Kymab mice (Lee et al. Nat Biotechnol 32, 356–363 (2014)) were used for the study, all female or male and female mice, usually 8-12 weeks of age (all mice within 2 weeks of age) at the time of immunization (defined as the day of administration of dose 1), or 7 to 9 weeks of age female C57BL / 6J mice purchased from commercial suppliers. Immunogens were mixed 1:1 with Sigma adjuvant system (SAS, Sigma) or TLR-4 agonist-based adjuvants according to the manufacturer's instructions. Mice were immunized according to the immunization schedule and immunogen doses mentioned in the figure. Live single B cells identified as germinal center B cells (CD95+GL7+) or plasma cells (TACI+CD138+) by flow cytometry were sorted from the lymph nodes and bone marrow of immunized mice. For both cell populations, PfCSP positivity was determined based on B cells that bound to fluorescent dye-labeled recombinant PfCSP. Paired Ig gene sequences for heavy and light chains were obtained by single-cell RT-PCR and next-generation sequencing of indexed amplicons.

[0280] Sequence analysis:

[0281] Ig sequence assembly and annotation were performed using sciReptor, a bioinformatic pipeline for integration of flow cytometry index data and sequence data (Imkeller et al., BMC Bioinformaticps: / / doi.org / 10.1186 / s12859-016-0920-1). Downstream sequence analysis and data visualization were performed using R 4.1.1.

[0282] Monoclonal Antibody Production:

[0283] The selected Ig genes were synthesized, cloned into the corresponding IGH, IGK or IGL expression vectors, and monoclonal antibodies were recombinantly expressed using HEK293 cells.

[0284] Crystallization and structure determination

[0285] The purified 509893Fab was concentrated and diluted to 10 mg / mL with KQPA peptide at a molar ratio of 1:3. The 509893Fab / KQPA complex was then mixed with 0.2M lithium sulfate, 25% (w / vol) PEG 3350, 0.1M bis-Tris (pH 5.5) at a ratio of 1:1. Crystals appeared after about 3 days and were flash frozen in liquid nitrogen. Data were collected on the 23-IDB beamline of the Advanced PhotonSource and processed and amplified using XDS (Kabsch, 2010). The structure was determined by molecular replacement using Phaser. Resolution (MMcCoy et al., 2007). Structures were refined using phenix.refine (Adams et al., 2010) and refinements were iteratively performed using Coot (Emsley et al., 2010). Buried surface area (BSA) was calculated using the Protein Interfaces, Surfaces and Assemblies (PISA) service of European Bioinformatics (Instp: / / www.ebi.ac.uk / pdbe / prot_int / pistart.html; Krissinel and Henrick, 2007).

[0286] Measuring antibody affinity using SPR

[0287] The Biacore T200 system and Biacore sensor chip CM5 were used to perform SPR-based assays to determine the affinity of the antibody. According to the manufacturer's instructions, two flow cells were fixed with anti-human IgG antibodies using a human Fab capture kit. Antibody samples (10 μg / ml) and negative control mGO53 (10 μg / ml) were captured in the sample and reference flow cells, respectively. The two flow cells were stabilized for 10 minutes by SPR running buffer at a flow rate of 10 μl / min. The designated peptides were serially diluted in the SPR running buffer solution, and the following concentration samples were injected into the two flow cells at a flow rate of 30 μl / min: 0 nM, 15.4 nM, 92.6 nM, 555.5 nM, 3333.3 nM and 20000 nM. Dissociation and binding were performed at 25 ° C for 60 seconds and 180 seconds, respectively. Between the injections of different sample antibodies, the flow cell was regenerated using a 10 mM glycine HCl solution. Data were analyzed using a 1:1 binding model or steady-state kinetic analysis using Biacore T200 software V2.0.

[0288] Plasmodium

[0289] Plasmodium falciparum NF54 (gift from R. Sauerwein) was cultured in O+ human erythrocytes in a Heracell 150i tri-gas incubator (ThermoScientific) at 37°C, 4% CO2, 3% O2. For gametocyte production, asynchronous parasite cultures were diluted to 1% parasitemia and replaced daily with RPMI-1640 medium (Thermo Scientific) supplemented with 10% human A+ serum and 10 mM hypoxanthine (cc-Pro) for 15-16 days until mosquito infection. Pb-PfCSP, an alternative P. berghei line expressing PfCSP (NF54) under the control of the Pb CSP regulatory sequence 19 , obtained from Chris J. Janse and Shahid M. Khan, and passaged every 3-4 days in CD1 female mice.

[0290] mosquito

[0291] All mosquitoes were maintained at 28-30 °C and 70-80% humidity. Anopheles coluzzii Ngousso S1 strain was used to produce Pf NF54 sporozoites. Anopheles gambiae 7b line, an immunocompromised transgenic mosquito from the G3 laboratory strain, was used for Pb-PfCSP sporozoite production and in vivo mosquito challenge experiments.

[0292] Pb-PfCSP sporozoites FACS

[0293] Anopheles gambiae 7b were fed female CD1 mice infected with Pb-PfCSP parasites (0.1-0.8% gametocytes) and kept at 20°C and 80% humidity until further use. Infected mosquitoes were provided with additional uninfected blood food 7 days post infection (dpi), and 20 mosquitoes were dissected for oocyst counts 17 days post infection. Pb-PfCSP sporozoites were isolated from mosquito salivary glands 18 days post infection. Siliconized microtubes (Alpha Laboratories) and pipette tips (VWR) were used to minimize the binding of sporozoites to the surface. The indicated concentrations of monoclonal antibodies were incubated with 150,000 sporozoites in a total volume of 100 μl of PBS containing 1% FCS for 30 minutes at 4°C. After washing, the sporozoites were incubated with 2 μg / ml of anti-human IgG1-Cy5 (DRFZ Berlin) in PBS containing 1% BSA for 30 minutes at 4°C. After washing, live sporozoites were identified by GFP expression and monoclonal antibody binding was quantified using a FACS LSR II instrument (BD Biosciences). Data analysis was performed using FlowJo V.10.0.8 (Tree Star).

[0294] Pf hepatocyte crossing assay

[0295] Anopheles coluzzii mosquitoes were infected with mature cultures of Pf gametocytes (NF54 strain) using artificial midi feeders (Glass Instruments, The Netherlands) and maintained in a controlled S3 facility in accordance with local safety authorization (Landesamt für Gesundheit und Soziales, LAGeSo, Berlin, Germany, project number 411 / 08). 13–15 days after infection, Pf sporozoites were collected from mosquito salivary glands in HC-04 medium and used in hepatocyte crossing assays as described previously ( 24 ). Briefly, salivary gland Pf sporozoites in HC-04 medium were preincubated with 27.5 μL of the indicated concentrations of monoclonal antibodies on ice for 30 min and incubated at 0.5 mg mL –1 Dextran rhodamine (Molecular Probes) was added to human hepatocytes (HC-04; (25)) for 2 hours at 37°C and 5% CO2. Cells were washed and fixed with PBS containing 1% PFA before dextran-positive flow cytometry measurements using a FACS LSR II instrument (BD Biosciences). Data were analyzed by subtracting the background (dextran-positive in cells treated with uninfected mosquito salivary gland material) and normalizing to the maximum Pf crossing capacity (dextran-positive in cells treated with salivary gland Pf sporozoites) using FlowJo V.10.0.8 (Tree Star). Chimeric humanized PfCSP-reactive mAb 2A10 (24) and non-PfCSP-reactive mAb mGO53 (26) were used as positive and negative controls, respectively.

[0296] Internal mosquito attack

[0297] Anopheles gambiae 7b was fed with female CD1 mice infected with Pb-PfCSP parasites (0.1-0.8% gametocytes), kept at 20°C and 80% humidity for one week, and provided with additional uninfected blood food. On the 18th day, mosquitoes with fluorescence in the salivary gland area were selected by a fluorescent stereoscope, and 3 female mosquitoes were placed in separate cups. The next day, mice injected with immunogens were anesthetized and placed on cups until all female mosquitoes had fed with blood food. Blood was collected from mice 3 days after the attack, and blood samples were examined for infected erythrocytes by flow cytometry using a FACS LSR II instrument (BD Biosciences). Mice injected with adjuvant alone were used as negative controls.

[0298] Enzyme-linked immunosorbent assay (ELISA)

[0299] ELISA was performed according to standard procedures. Briefly, ELISA plates were coated with antigen in PBS at 4°C overnight. The plates were washed three times with 0.05% Tween in PBS and then blocked with 4% BSA in PBS for 1 h at room temperature. After washing with 0.05% Tween in PBS, diluted serum samples in 1% BSA / PBS were added and incubated for 1.5 h at room temperature. After washing with 0.05% Tween in PBS, goat-anti-mouse IgG or IgM-HRP secondary antibodies in blocking buffer were added and incubated for 1 h at room temperature. After washing with 0.05% Tween in PBS, ABTS substrate solution was added. Absorbance was measured at 405 nm. Binding of recombinant monoclonal antibodies to antigens was performed according to similar procedures, using 0.01, 0.06, 0.25, and 1.0 μg / ml of serial dilutions of monoclonal antibodies to bind to antigens, and goat anti-hIgG-HRP was used as a secondary antibody.

[0300] Results and Discussion

[0301] Immunizations were performed in wild-type mice and Kymice (transgenic human Ig antibody library) with different immunogens and administration schedules, such as 1) PfCSP sequences fused to different nanoparticles (dioxotetrahydropterin synthase or Helicobacter pylori (Hp) ferritin); 2) PfCSP-based immunogens with or without PfCSP cross-reactive epitopes (NPDP, NVDP), and with or without effective T cell helper (such as PADRE epitope), and different NANP repeat lengths; 3) Immunogens with or without two non-native N-linked glycosylation sites on the Hp ferritin promoter (N79 and N99) in order to reduce anti-carrier antibody responses and increase anti-PfCSP titers; and 4) homologous or heterologous prime-boost immunizations with different administration schedules, routes of administration, schedules and adjuvants. The immune cell frequency, sequencing data and expression of selected monoclonal antibodies recovered from these experiments were analyzed. The affinity / avidity of sera and individual mAbs to different PfCSP sequences was assessed in a crossing inhibition assay and in vivo liver burden measurements and parasitemia challenges were performed using PfCSP transgenic P. berghei.

[0302] In transgenic human Ig antibody repertoire mice, B cells with characteristic IGHV3-33-encoded human antibodies against PfCSP repeat sequences were obtained in response to most immunogens. Monoclonal antibodies from these experiments generally have high affinity for repeat sequences. For immunogens containing PfCSP cross-reactive epitopes (NPDP, NVDP motifs) and effective T cell help (PADRE epitope), high affinity for the connecting peptide was observed, and it was similar to the high affinity for the NANP peptide. Unexpectedly, we observed that IGHV3-15 expressing B cells were also recruited to germinal center reactions and differentiated into plasma cells in the lymph nodes ( Figure 1 A). The enrichment of IGHV3-15 expressing cells was more pronounced in CSP+ germinal center (GC) B cells compared to CSP- GC B cells, confirming that these gene segments are used by B cells actively involved in anti-immunogen responses ( Figure 1 B). Almost all of these cells have the light chain encoded by IGLV3-10 ( Figure 2 ).

[0303] Binding of the VH3-15 / VL3-10 mAb to the KQPA peptide was detected in an ELISA when the biotinylated peptide was captured onto a streptavidin pre-coated plate, but not when the KQPA peptide was directly coated onto a high binding plate ( Figure 3 ). Therefore, direct immobilization of the KQPA peptide to the plate masked the specific residues in the KQPA peptide recognized by the VH3-15 / VL3-10 mAb. In contrast, the VH3-33 / VK1-5 mAb showed binding only when the KQPA peptide was directly coated onto the plate, indicating that the mAb had cross-reactivity with fewer repeating units of NPDP in the peptide. We independently validated the binding properties of the mAbs by measuring their affinity for the target peptide using surface plasmon resonance. From the several mAbs tested, it was found that the VH3-15 / VL3-10 mAb was specific for the KQPA peptide with an affinity as high as nanomolar ( ) compared to the VH3-33 / VK1-5 mAb. Figure 3 ), but not bound to sporozoites ( Figure 4 Two representative high-affinity VH3-15 / VL3-10 monoclonal antibodies (mAb 509893_121 and mAb 511205_132) showed low inhibition of hepatocyte Pf crossing in vitro ( Figure 4 Next, various peptides were screened to better understand the specificity of the VH3-15 / VL3-10 antibodies. It was found that the VH3-15 monoclonal antibodies recognized some amino acids in the N-terminal I region / link of PfCSP with high affinity. Removal of the KQ motif in the PfCSP N-terminal link abolished the monoclonal antibody binding of these VH3-15 / VL3-10 monoclonal antibodies ( Figure 5 The crystal structure of Fab 509893_21 (VH3-15 / VL3-10) in complex with a peptide containing KQPA was solved. The structure confirmed that the KQ residues are extensively contacted by the VH3-15 / VL3-10 antibody ( Figure 6 The combined three-residue KQP contributes 40% of the total peptide interactions ( Figure 6 ).

[0304] This data suggests that KQ and / or more substantially KQP residues can be removed from the immunogen to avoid inducing a B cell response dominated by cells expressing the high affinity VH3-15 mAb, which, as described, is non-sporozoite reactive and non-inhibitory. Different strategies were used to fine-tune the N-terminus of the immunogen: for example, deleting the KQP residue, priming the immunogen with a PfCSP "ADG" sequence or a PfCSP "NPDP" sequence and / or a new signal peptide secretion sequence (e.g. derived from human IgKVIII: MDMRVPAQLLGLLLLWLRGARC). Compared to previous immunogens containing KQP residues with high affinity for non / low parasite inhibitory VH3-15 / VL3-10 mAbs (e.g., 155), these immunogens (e.g., 203) abolished binding of low parasite inhibitory VH3-15 / VL3-10 mAbs ( Figure 7 and Figure 8 Like previous immunogens (e.g., 155), these 155-N-opt immunogens (e.g., 203) expressed well and displayed good biophysical properties: they were pure and well-assembled nanoparticles that bound with high affinity to strongly inhibitory linked / repeat mAbs (4493 and 317), with moderate affinity to a moderate inhibitory mAb (1210), and did not bind to non-inhibitory mAbs or low inhibitory mAbs (1710, 5D5, and 509893_21) ( Figure 8 ).

[0305] Mice transgenic for human Ig antibody repertoires (e.g. Kymice) were immunized with these 155-N-opt immunogens (e.g. 203). Compared to previous immunogens containing KQP residues (e.g. 155), these new immunogens did not induce responses against non-protective N-terminally linked KQP epitopes ( Fig. 9 Surprisingly, much stronger anti-PfCSP responses were observed in human immunoglobulin repertoire mice immunized with 155-N-opt immunogens (e.g., 203) compared to previous immunogens containing KQP residues (e.g., 155) ( Fig.10). It was confirmed that in the mosquito bite challenge model of wild-type mice, the strong immune response elicited by the 155-N-opt immunogen (such as 203) did protect against infection with the Pb-PfCSP transgenic parasite ( Fig.11 Together, these unexpected findings demonstrate the superiority of PfCSP immunogens lacking these Lys and / or Gln and / or Pro amino acids in driving the most potent responses to the highly inhibitory PfCSP junction / repeat epitopes in the context of the human immunoglobulin repertoire.

[0306] Example 2

[0307] The immunogenicity of high mannose glycosylated (203F+Kif) or non-glycosylated (211F) immunogens without KQP residues was tested in mice. Wild-type mice (n=5 per group) were immunized intramuscularly with two doses (0.5 μg / mouse or 10 μg / mouse) of immunogen and adjuvant (LMQ, TLR4 stimulation) or adjuvant alone. The 203 and 211 sequences are as described herein and the method is as described in Example 1 above. In brief, serum IgG responses against the indicated peptides were measured by ELISA at different time points after the first (d0) or second (d28) immunization. The data show the mean and standard deviation of three independent experiments with 5 mice per group.

[0308] Fig.12 Glycosylation was shown to increase immunogenicity to amino acid sequences representing the N-terminal link and NANP repeat domain (P126-bio; upper left), NANP repeat domain (NANP5.5-bio; lower left), and NPDP domain (NPDP-bio; lower right), but not to the Hp ferritin backbone (Hp ferritin; upper right). This suggests that the increase in IgG serum responses is specific to malarial immunogens.

[0309] Fig.13 It was also shown that glycosylation reproducibly increased the immunogenicity of the NANP repeat domain (NANP5.5-bio; upper panel) and the NPDP domain (NPDP-bio; lower panel).

[0310] Next, sporozoite crossing inhibition assays were performed as described in Example 1 using either high mannose glycosylated (203F+Kif) or non-glycosylated (211F) immunogens (both lacking KQP residues). Fig.14 A and 14B show that the percent inhibition of crossing was higher for the glycosylated immunogen compared to the non-glycosylated immunogen at both doses tested.

[0311] Fig.14C shows that both doses of the glycosylated immunogen protected 100% of mice from parasite infection 10 days after exposure to three infectious mosquito bites at day 52 after immunization. The non-glycosylated immunogen protected 50% of the mice, and the control group provided no protection, with all mice becoming infected by day 4 after exposure. These data suggest that while removal of KQP residues protects against malarial infection, the combination of removal of KQP residues and glycosylated immunogens improves protection.

[0312] Example sequence: 153:

[0314] 154:

[0316]

[0317] 155:

[0319] 156:

[0321] 203:

[0323] 205:

[0325] 206:

[0327] 207:

[0329] 208:

[0331] 209:

[0333] 210:

[0335] 211: 212:

[0338] 213:

[0340]

[0341] References

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Claims

1. A malarial immunogen or variant thereof comprising at least a portion of a wild-type PfCSP amino acid sequence lacking a KQ motif.

2. The malarial immunogen of claim 1, lacking the KQP motif.

3. A malarial immunogen according to claim 1 or 2, lacking the N-terminal ETG motif.

4. The malarial immunogen according to any one of claims 1 to 3, comprising the following motif: -NPDP a 、PDPN a 、DPNP a 或PNPD a ; -NANP b 、ANPN b 、NPNA b 或PNAN b ; -NVDP c 、VDPN c DPNV c or PNVD c ;and -NANP d 、ANPN d 、NPNA d 或PNAN d 。 - wherein a, b, c and d are each independently 0 or greater, and wherein a+b+c+d is at least 2.

5. The malarial immunogen of claim 4, wherein a, b, c, d, or any combination thereof, are each independently at least about 1.

6. The malaria immunogen of claim 5, wherein a, b, c, d, or any combination thereof, are each independently from about 1 to about 40.

7. A malarial immunogen according to claim 6, wherein a, b, c and d are each independently from about 1 to about 100, such as from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 75, about 80, about 90 or about 100, such as from about 1 to about 40, or from about 1 to about 20, or from 1 to about 10.

8. A malarial immunogen according to any one of claims 4 to 7, wherein a is 1.

9. A malarial immunogen according to any one of claims 4 to 8, wherein b is 1.

10. The malaria immunogen according to any one of claims 4 to 9, wherein c is 3.

11. A malarial immunogen according to any one of claims 4 to 10, wherein d is 5.

12. A malarial immunogen according to any one of claims 4 to 11, wherein d is 18.

5.

13. A malarial immunogen according to any one of claims 4 to 12, wherein when a, b, c and / or d is greater than 1 such that the corresponding motif is at least partially repeated, each of the repeated motifs is independently continuous.

14. A malarial immunogen according to any one of claims 4 to 13, wherein when a, b, c and / or d is greater than 1 such that the corresponding motif is at least partially repeated, the repeated motifs are each independently discontinuous.

15. A malarial immunogen according to any one of claims 4 to 14, wherein the sequence of the motif is NPDP a -NANP b -NVDP c -NANP d .

16. The malarial immunogen according to any one of claims 1 to 15, further comprising an N-terminal ADG or PADG motif, wherein the ADG or PADG motif is optionally repeated alone or in combination with NPDP a , NANP b NVDP c and NANP d At least one combination of the motifs is repeated.

17. The malarial immunogen of any one of claims 1 to 16, wherein the variant has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the wild-type PfCSP amino acid sequence lacking the KQ motif.

18. The malarial immunogen according to any one of claims 1 to 17, wherein the malarial immunogen is fused directly or indirectly to the nanocage monomer peptide.

19. The malaria immunogen of claim 18, wherein the nanocage monomer is ferritin, apoferritin, capsular protein, SOR, dioxotetrahydropterin synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P or MS2 coat protein or a fragment or variant thereof.

20. The malarial immunogen of claim 19, wherein the nanocage monomer is provided as two or more self-assembling subunits.

21. The malaria immunogen according to any one of claims 18 to 20, wherein the nanocage monomer peptide is from Helicobacter pylori.

22. The malaria immunogen according to any one of claims 18 to 20, wherein the nanocage monomer peptide is not human.

23. The malarial immunogen according to any one of claims 18 to 22, wherein the nanocage monomer peptide is modified to reduce anti-nanocage monomer peptide immune response.

24. The malarial immunogen according to any one of claims 18 to 23, wherein the nanocage monomer peptide is modified to enhance the antigen immune response.

25. A malaria immunogen according to claim 23 or 24, wherein the nanocage monomer peptide is at least partially or completely covered.

26. The malaria immunogen of claim 25, wherein the nanocage monomer peptide is at least partially covered with glycans.

27. The malaria immunogen of claim 26, wherein the nanocage monomer peptide is completely covered with glycans.

28. The malarial immunogen according to any one of claims 21 to 27, wherein the nanocage monomer comprises at least one NXT and / or NXS glycosylation motif.

29. The malarial immunogen according to any one of claims 21 to 27, wherein the nanocage monomer is glycosylated at N79 and / or N99.

30. The malarial immunogen according to any one of claims 21 to 29, wherein the nanocage monomers are glycosylated with high mannose glycans.

31. The malarial immunogen according to any one of claims 18 to 30, wherein a plurality of nanocage monomer peptides self-assemble into a nanocage.

32. A malaria immunogen according to claim 31, wherein the immunogenic peptide decorates the inner and / or outer surface of the nanocage.

33. The malaria immunogen according to any one of claims 1 to 32, further comprising a peptide providing exogenous T cell help and / or a peptide providing autologous T cell help.

34. A malaria immunogen according to claim 33, wherein the peptide providing exogenous T cell help comprises a PADRE peptide and / or a peptide derived from a pathogenic molecule, such as a tetanus toxoid peptide.

35. A malaria immunogen according to claim 34, wherein the PADRE peptide comprises the amino acid sequence AKFVAAWTLKAAA, or a functional variant thereof having at least 70% sequence identity thereto, or a fragment of either of them.

36. A malaria immunogen according to any one of claims 33 to 35, wherein the peptide providing autologous T cell help comprises a PfCSP T cell peptide epitope.

37. A malaria immunogen according to any one of claims 33 to 36, wherein the peptide providing exogenous T cell help and / or the peptide providing autologous T cell help independently decorates the inner surface and / or the outer surface of the assembled nanocage.

38. The malaria immunogen according to any one of claims 1 to 37, further comprising a linker between any one or more of the motifs, the nanocage monomer and any other peptide, such as a peptide providing exogenous T cell help and / or a peptide providing autologous T cell help.

39. A malarial immunogen according to claim 38, wherein the linker is a GGS linker.

40. The malaria immunogen of claim 39, wherein the linker comprises the amino acid sequence: GGS; GGGGSGGSGGSGGS; and / or GGGGGSGGSGGSGGS.

41. The malarial immunogen according to any one of claims 1 to 40, comprising or consisting of the following sequence: ADG-NPDP-NANPNVDP3-NANP5-Hpferr-PADRE; ADG-NPDP-NANPNVDP3-NANP18-Hpferr-PADRE; ADG-NPDP-NANPNVDP3-NANP18.5-Hpferr-PADRE; ADG-NPDP-NANPNVDP3-NANP5-LS-PADRE; and / or ADG-NPDP-NANPNVDP3-NANP18-LS-PADRE; ADG-NPDP-NANPNVDP3-NANP18.5-LS-PADRE.

42. The malarial immunogen according to any one of claims 1 to 41, comprising any one of the following amino acid sequences: 203 205 206 207 208 209 21O 211 212 213 or a functional variant thereof having at least 70% sequence identity thereto or a functional fragment of either of them.

43. A nucleic acid molecule encoding the malaria immunogen of any one of claims 1 to 42. A vector comprising the nucleic acid molecule of claim 43 .

45. A host cell comprising the vector of claim 41 and producing the malaria immunogen of any one of claims 1 to 42.

46. ​​A vaccine comprising the malaria immunogen of any one of claims 1 to 42.

47. The vaccine of claim 46, further comprising an adjuvant.

48. An antibody that binds to the malarial immunogen of any one of claims 1 to 47.

49. A method for treating and / or preventing malaria comprising administering the immunogen, nucleotide, vector, cell or vaccine of any one of claims 1 to 47.

50. Use of the immunogen, nucleotide, vector, cell or vaccine of any one of claims 1 to 47 for the treatment and / or prevention of malaria.

51. An immunogen, nucleotide, vector, cell or vaccine as claimed in any one of claims 1 to 47 for use in the treatment and / or prevention of malaria.

52. The method, use or immunogen, nucleotide, vector, cell or vaccine according to any one of claims 49 to 51, wherein the prophylactic and / or therapeutic effect is enhanceable.

53. The method, use, or immunogen, nucleotide, vector, cell, or vaccine of any one of claims 49 to 51, wherein the prophylactic and / or therapeutic effect lasts for at least about 6 months or longer, e.g., about 9 months or longer, about 12 months or longer, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years or longer.

Images