Methods and compositions for treating and preventing malaria

Abstract

To provide methods and compositions for treating and preventing malaria.SOLUTION: The present disclosure provides immunogenic compositions for inducing an immune response against malaria. The present disclosure also provides novel methods of administering the immunogenic compositions. Disclosed herein are methods and compositions for treating malaria by administering an immunogenic composition. The compositions benefit from the use of saponin-based adjuvants which provide particularly enhanced immune responses. In embodiments, provided herein are immunogenic compositions comprising an antigen of a Plasmodium parasite.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 281,917, filed November 22, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Use of electronic sequence listing The contents of the electronic sequence listing (NOVV_095_01WO_SeqList_ST26.xml; size: (17,531 bytes; and creation date: November 22, 2022) are incorporated herein by reference in their entirety.

[0003] Field of Disclosure The present disclosure relates to compositions and methods for inducing an immune response against Plasmodium parasites. [Background technology]

[0004] Background of the Invention Infectious diseases remain a global problem. Malaria is a leading cause of childhood mortality. As of 2019, there were an estimated 229 million cases of malaria worldwide. In 2019, 409,000 people died from malaria. The only vaccine approved to prevent malaria is RTS,S / AS01 (RTS,S) (MOSQUIRIX®). The protection provided by RTS,S against malaria weakens over time.

[0005] Plasmodium vivax (P. vivax) causes severe morbidity and mortality worldwide. P. vivax blood-stage merozoites invade reticulocytes through an essential interaction between the parasite's Duffy binding protein (PvDBP) and the cellular Duffy antigen / chemokine receptor (DARC). There is a need in the art for new vaccines and methods to prevent malaria. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present disclosure provides an immunogenic composition for inducing an immune response against malaria. The present disclosure also provides a novel method for administering the immunogenic composition. Disclosed herein are methods and compositions for treating malaria by administering the immunogenic composition. The composition particularly benefits from the use of a saponin-based adjuvant, which provides an enhanced immune response.

[0007] In embodiments, provided herein is an immunogenic composition comprising an antigen of a Plasmodium parasite. In embodiments, provided herein is a method of stimulating an immune response to a Plasmodium parasite in a subject, the method comprising administering the immunogenic composition. In embodiments, the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. Typically, it is P. vivax.

[0008] In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the adjuvant comprises at least two types of iscom particles, wherein a first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; and a second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not comprise fraction A of Quillaja Saponaria Molina. In an embodiment, fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina are present. Fraction C of Quillaja Saponaria Molina accounts for about 85% by weight and about 15% by weight, respectively, of the combined weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. In an embodiment, Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for about 92% by weight and about 8% by weight, respectively, of the combined weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. In an embodiment, the adjuvant is administered at a dose of about 50 μg.

[0009] In embodiments, the method of stimulating an immune response to a Plasmodium parasite comprises administering about 0.1 μg to about 100 μg of antigen. In embodiments, the method of stimulating an immune response to a Plasmodium parasite comprises administering about 0.1 μg to about 10 μg of antigen. In embodiments, the method of stimulating an immune response to a Plasmodium parasite comprises administering about 0.1 μg to about 5 μg of antigen. In embodiments, the method of stimulating an immune response to a Plasmodium parasite comprises administering about 0.1 μg to about 3 μg of antigen. In embodiments, the method of stimulating an immune response to a Plasmodium parasite comprises administering about 0.1 μg to about 2 μg of antigen. In embodiments, the method comprises administering a first dose and a second dose of the immunogenic composition. In embodiments, the method comprises administering a third dose of the immunogenic composition. In embodiments, the dose of antigen in the second dose is less than the dose of antigen in the first dose. In embodiments, the dose of antigen in the third dose is less than the dose of antigen in the first dose. In embodiments, the first and second doses contain the same amount of antigen. In embodiments, the second and third doses contain the same amount of antigen. In embodiments, the first and third doses contain the same amount of antigen. In embodiments, the third dose contains about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the antigen in the first dose. In embodiments, the second dose contains about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the antigen in the first dose.

[0010] Provided herein is a method of stimulating an immune response to a Plasmodium parasite in a subject, comprising administering an immunogenic composition comprising a Duffy binding protein (DBP) derived from a Plasmodium parasite. In embodiments, the DBP is a Plasmodium vivax ("PvDBP"). In embodiments, the DBP comprises Region I, Region II, Region III, Region IV, Region V, Region VI, Region VII, or any combination thereof of PvDBP. In embodiments, the DBP comprises Region II of PvDBP ("PvDBPII"). In embodiments, the DBP comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-9. In embodiments, the DBP comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, The DBP comprises a polypeptide having at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 4-9. In embodiments, the DBP comprises amino acids 194-521 of SEQ ID NO: 1 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 194-521 of SEQ ID NO: 1. In embodiments, the DBP is expressed in Escherichia coli.In embodiments, the immunogenic composition comprises about 1 μg to about 100 μg of DBP, about 10 μg to about 100 μg of DBP, about 25 to about 100 μg of DBP, or about 35 to about 55 μg of DBP. In embodiments, the immunogenic composition comprises about 2 μg, 5 μg, 10 μg, or 50 μg of DBP. In embodiments, the immunogenic composition comprises an adjuvant. In embodiments, the immunogenic composition comprises about 1 μg to about 100 μg; about 25 μg to about 75 μg; or about 40 μg to about 60 μg of adjuvant. In embodiments, the immunogenic composition comprises about 50 μg of adjuvant. In embodiments, the immunogenic composition comprises about 25 μg of adjuvant. In embodiments, the adjuvant is a saponin adjuvant. In an embodiment, the saponin adjuvant comprises at least two types of iscom particles, wherein: the first iscom particle comprises Quillaja Saponaria Molina Fraction A but not Quillaja Saponaria Molina Fraction C; and the second iscom particle comprises Quillaja Saponaria Molina Fraction C but not Quillaja Saponaria Molina Fraction A. In an embodiment, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant, Quillaja Saponaria Molina Fraction A accounts for 50 to 96% by weight, and Quillaja Saponaria Molina Fraction C accounts for the remainder. In an embodiment, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, Fraction A of Quillaja Saponaria Molina accounts for at least 75% by weight, and Fraction C of Quillaja Saponaria Molina accounts for the remainder.In one embodiment, the total weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C in the adjuvant is about 85% by weight and about 15% by weight, respectively. In one embodiment, the total weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C in the adjuvant is about 92% by weight and about 8% by weight, respectively. In one embodiment, the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. In one embodiment, the method comprises administering the immunogenic composition in a pre-filled syringe. In embodiments, the method comprises administering a first dose and a second dose of the immunogenic composition. In embodiments, the method comprises administering a third dose of the immunogenic composition. In embodiments, the method comprises administering a fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition. In embodiments, the first and second doses comprise about the same amount of DBP. In embodiments, the second and third doses comprise about the same amount of DBP. In embodiments, the first and third doses comprise about the same amount of DBP. In embodiments, the first, second, and third doses comprise about the same amount of DBP. In embodiments, the first, second, third, and fourth doses comprise about the same amount of DBP. In embodiments, the amount of DBP in the second dose is less than the amount of DBP in the first dose. In embodiments, the amount of DBP in the third dose is less than the amount of DBP in the first dose.In embodiments, the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose contains about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the DBP in the first dose. In embodiments, the second dose contains about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the DBP in the first dose. In embodiments, the method includes administering the second dose about one month after the first dose. In embodiments, the method includes administering the second dose about 28 days after the first dose. In embodiments, the method comprises administering the third dose about 56 days after the first dose. In embodiments, the method comprises administering the third dose about 2 months after the first dose. In embodiments, the method comprises administering the third dose about 168 days after the first dose. In embodiments, the method comprises administering the third dose about 6 months after the first dose. In embodiments, the method comprises administering the third dose about 14 months after the first dose. In embodiments, the method comprises administering the third dose about 14 months after the second dose. In embodiments, the method includes administering the third dose at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after the first or second dose. In embodiments, the method includes administering the third dose about 6 to about 18 months, about 9 to about 18 months, about 12 to about 15 months, or about 12 to about 18 months after the first or second dose.In an embodiment, the method comprises: (i) administering a first dose of the immunogenic composition; (ii) administering a second dose of the immunogenic composition about one month after the first dose; and (iii) administering a third dose of the immunogenic composition about 14 months after the second dose. In an embodiment, the method comprises: (i) administering a first dose of the immunogenic composition; (ii) administering a second dose of the immunogenic composition about one month after the first dose; and (iii) administering a third dose of the immunogenic composition about 14 months after the first dose. In an embodiment, the immunogenic composition comprises about 50 μg of DBP. In an embodiment, the immunogenic composition comprises about 50 μg of saponin adjuvant. In embodiments, the method comprises administering the immunogenic composition for up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, up to about 12 months, up to 13 months, up to 14 months, up to 15 months, up to 16 months, up to 17 months, up to 18 months, or up to 19 months after administration of the immunogenic composition. , for up to 20 months, for up to 21 months, for up to 22 months, for up to 23 months, or for up to 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50% effectiveness in preventing malaria.In embodiments, the method comprises administering the immunogenic composition for at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, at least about 6 months, at least about 6.5 months, at least about 7 months, at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least. about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 60% to about 95%, Prevents malaria with about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50% effectiveness. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows the immune responses induced by a viral vector formulation using ChAd63 and an MVA viral vector (vvDBP) and an immunogenic composition comprising PvDBPII and a saponin adjuvant (i.e., MATRIX-M™). The compositions are labeled "DBP / MM." For DBP / MM MD, three doses of the immunogenic composition were administered one month apart. For DBP / MM DD, the first two doses of the immunogenic composition were administered one month apart, with the third dose administered 14 months after the second dose ("delayed dosing approach"). The lines on the graph are shown as medians with interquartile ranges (IQRs). The arrows on the graph indicate the time of immunization. The time points from the last immunization (FV) are indicated. For the vvDBP PPB group, only data from the second or subsequent vaccinations are shown. The DBP / MM delayed dosing regimen demonstrated greater peak IgG responses than the other regimens.

[0012] [Figure 2] Figure 2 illustrates the enhanced immune response with the delayed administration approach of Figure 1. The graph shows the antibody response associated with immunization. Isotype responses are measured by standardized ELISA 14 days after the final vaccination. The dotted line indicates the 5 AU assay cutoff. Lines are shown from minimum to maximum as well as median. Significant pairwise comparisons from Kruskal-Wallis tests and Dunn's multiple comparisons are shown. IgG2 and IgA2 responses (not shown) were near background. The delayed administration approach resulted in up to 40-fold higher IgG4 responses than other regimens. The delayed administration approach also resulted in up to 7-fold higher IgG1 responses than other regimens.

[0013] [Figure 3A]Figures 3A-3B illustrate the enhanced immune response with the delayed administration approach in Figure 1. Figure 3A shows anti-PvDBPII IgG avidity 14 days after the final vaccination. Avidity was measured using a sodium isothiocyanate (NaSCN) chemical displacement ELISA (Kruskal-Wallis test with Dunn's multiple comparisons). Figure 3B shows in vitro growth inhibitory activity (GIA%) using a transgenic P. knowlesi strain expressing the Salvador I strain of PvDBP. The DBP / MM DD regimen was able to induce growth-inhibitory IgG. Lines are shown as medians with interquartile ranges (IQR). Delayed administration improves IgG avidity and functionality. [Figure 3B] Figures 3A-3B illustrate the enhanced immune response with the delayed administration approach in Figure 1. Figure 3A shows anti-PvDBPII IgG avidity 14 days after the final vaccination. Avidity was measured using a sodium isothiocyanate (NaSCN) chemical displacement ELISA (Kruskal-Wallis test with Dunn's multiple comparisons). Figure 3B shows in vitro growth inhibitory activity (GIA%) using a transgenic P. knowlesi strain expressing the Salvador I strain of PvDBP. The DBP / MM DD regimen was able to induce growth-inhibitory IgG. Lines are shown as medians with interquartile ranges (IQR). Delayed administration improves IgG avidity and functionality.

[0014] [Figure 4A]Figures 4A-4E illustrate immune cells induced by immunization with the delayed administration approach of Figure 1. Figure 4A shows that immunization induces antigen-specific IgA+ CD19+ CD20+ memory B cells. Figure 4B shows that immunization induces antigen-specific IgG+ CD19+ CD20+ memory B cells. Figure 4C shows that immunization induces antigen-specific IgM+ CD19+ CD20+ memory B cells. DBP-specific cells were defined as their co-staining with the two PvDBPII probes and are shown in Q14. Responses are shown in blue before vaccination and pink after vaccination. Figures 4D-4E show the percentage of memory CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2) (Figure 4D) or Th2 (IL-5, IL-13) (Figure 4E) cytokines in response to stimulation with medium, PvDBPII ("Med DBP") peptide, or SEB control (right axis) at the indicated time points. Lines are shown as medians with IQRs. The data show that vaccination with PvDBPII induces antigen-specific memory IgA+ and IgG+ B cells and CD4+ T cells. [Figure 4B]Figures 4A-4E illustrate immune cells induced by immunization with the delayed administration approach of Figure 1. Figure 4A shows that immunization induces antigen-specific IgA+ CD19+ CD20+ memory B cells. Figure 4B shows that immunization induces antigen-specific IgG+ CD19+ CD20+ memory B cells. Figure 4C shows that immunization induces antigen-specific IgM+ CD19+ CD20+ memory B cells. DBP-specific cells were defined as their co-staining with the two PvDBPII probes and are shown in Q14. Responses are shown in blue before vaccination and pink after vaccination. Figures 4D-4E show the percentage of memory CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2) (Figure 4D) or Th2 (IL-5, IL-13) (Figure 4E) cytokines in response to stimulation with medium, PvDBPII ("Med DBP") peptide, or SEB control (right axis) at the indicated time points. Lines are shown as medians with IQRs. The data show that vaccination with PvDBPII induces antigen-specific memory IgA+ and IgG+ B cells and CD4+ T cells. [Figure 4C]Figures 4A-4E illustrate immune cells induced by immunization with the delayed administration approach of Figure 1. Figure 4A shows that immunization induces antigen-specific IgA+ CD19+ CD20+ memory B cells. Figure 4B shows that immunization induces antigen-specific IgG+ CD19+ CD20+ memory B cells. Figure 4C shows that immunization induces antigen-specific IgM+ CD19+ CD20+ memory B cells. DBP-specific cells were defined as their co-staining with the two PvDBPII probes and are shown in Q14. Responses are shown in blue before vaccination and pink after vaccination. Figures 4D-4E show the percentage of memory CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2) (Figure 4D) or Th2 (IL-5, IL-13) (Figure 4E) cytokines in response to stimulation with medium, PvDBPII ("Med DBP") peptide, or SEB control (right axis) at the indicated time points. Lines are shown as medians with IQRs. The data show that vaccination with PvDBPII induces antigen-specific memory IgA+ and IgG+ B cells and CD4+ T cells. [Figure 4D]Figures 4A-4E illustrate immune cells induced by immunization with the delayed administration approach of Figure 1. Figure 4A shows that immunization induces antigen-specific IgA+ CD19+ CD20+ memory B cells. Figure 4B shows that immunization induces antigen-specific IgG+ CD19+ CD20+ memory B cells. Figure 4C shows that immunization induces antigen-specific IgM+ CD19+ CD20+ memory B cells. DBP-specific cells were defined as their co-staining with the two PvDBPII probes and are shown in Q14. Responses are shown in blue before vaccination and pink after vaccination. Figures 4D-4E show the percentage of memory CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2) (Figure 4D) or Th2 (IL-5, IL-13) (Figure 4E) cytokines in response to stimulation with medium, PvDBPII ("Med DBP") peptide, or SEB control (right axis) at the indicated time points. Lines are shown as medians with IQRs. The data show that vaccination with PvDBPII induces antigen-specific memory IgA+ and IgG+ B cells and CD4+ T cells. [Figure 4E]Figures 4A-4E illustrate immune cells induced by immunization with the delayed administration approach of Figure 1. Figure 4A shows that immunization induces antigen-specific IgA+ CD19+ CD20+ memory B cells. Figure 4B shows that immunization induces antigen-specific IgG+ CD19+ CD20+ memory B cells. Figure 4C shows that immunization induces antigen-specific IgM+ CD19+ CD20+ memory B cells. DBP-specific cells were defined as their co-staining with the two PvDBPII probes and are shown in Q14. Responses are shown in blue before vaccination and pink after vaccination. Figures 4D-4E show the percentage of memory CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2) (Figure 4D) or Th2 (IL-5, IL-13) (Figure 4E) cytokines in response to stimulation with medium, PvDBPII ("Med DBP") peptide, or SEB control (right axis) at the indicated time points. Lines are shown as medians with IQRs. The data show that vaccination with PvDBPII induces antigen-specific memory IgA+ and IgG+ B cells and CD4+ T cells.

[0015] [Figure 5] FIG. 5 shows two different viral vectors encoding the PvDBPII ("PvDBP_RII") antigen and their administration.

[0016] [Figure 6] FIG. 6 shows an exemplary delayed administration regimen of an immunogenic composition comprising PvDBPII and a saponin adjuvant (eg, MATRIX-M™). DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" may refer to one protein or a mixture of such proteins, reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0018] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response elicited against the immunogen. Modification of the immune response can include enhancing or broadening the specificity of either or both the antibody and cellular immune responses.

[0019] As used herein, the terms "about" or "approximately," when preceding a numerical value, indicate a range of that value plus or minus 10%. For example, "about 100" includes 90 and 110.

[0020] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances such as proteins (including glycoproteins) and peptides that are capable of eliciting an immune response.

[0021] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the generation in the subject of a humoral and / or cellular immune response to the antigen.

[0022] As used herein, a "subunit" composition (e.g., a vaccine) comprises one or more selected antigens from a pathogen, but not all antigens. Such compositions are substantially free of intact virus or lysates of such cells or particles, and are typically prepared from immunogenic polypeptides that have been at least partially purified, often substantially purified, from the pathogen. The antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0023] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance forms a majority percentage of the sample in which it is contained. For example, in a sample, a substantially purified component comprises 85%, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99% of the sample. When a component is substantially replaced, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably about 0.5% to about 1.0%.

[0024] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining a beneficial or desired result, e.g., a clinical result. For purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the onset or progression of an infection or disease; ameliorating or reducing the development of symptoms of an infection or disease, or a combination thereof.

[0025] "Prevention," as used herein, is used interchangeably with "prophylaxis" and can mean preventing an infection or disease altogether or preventing the onset of symptoms of that infection or disease; delaying the onset of an infection or disease or its symptoms; or reducing the severity of an infection or disease or its symptoms that has substantially occurred.

[0026] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

[0027] As used herein, the term "vaccine" refers to an immunogenic composition (e.g., an immunogen derived from a pathogen) used to induce an immune response against a pathogen. The immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen administered to a subject to generate an immune response. Preferably, the vaccine induces an immune response that is effective in preventing infection from Plasmodium parasites.

[0028] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, a teenager, a child (2 to 14 years old), an infant (from birth to 2 years old), or a newborn (up to 2 months old). In certain aspects, the subject is up to 4 months old, or up to 6 months old. In some aspects, the adult is about 65 years old or older, or about 60 years old or older. In some aspects, the subject is a pregnant woman or a woman who intends to become pregnant. In other aspects, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain aspects, the subject can be a pet (e.g., a dog or cat).

[0029] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government, or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in mammals, and more particularly, humans. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0030] The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared. Percent identity can be calculated using the online tool, CLUSTALW2. The following parameters can be used for CLUSTALW2 Pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.

[0031] As used herein, the terms "co-formulation mix," "co-formulation," "co-formulated vaccine composition," "pre-filled syringe," and "premix" refer to a vaccine formulation prepared for short- to long-term storage prior to administration to a subject. Such vaccine formulations include a combination of antigen and adjuvant in the same container and are prepared prior to administration. In an embodiment, a pre-filled syringe contains a vaccine formulation including a DBP polypeptide (e.g., PvDBPII) and an adjuvant (e.g., a saponin adjuvant (e.g., a Matrix adjuvant)).

[0032] antigen In embodiments, the immunogenic compositions disclosed herein comprise some or all of the Duffy binding protein (DBP). In embodiments, the DBP is derived from the Plasmodium parasite. In embodiments, the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi.

[0033] In embodiments, the DBP is derived from the P. vivax parasite. The DBP derived from P. vivax is referred to herein as "PvDBP." In embodiments, PvDBP is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having the amino acid sequence of SEQ ID NO:1.

[0034] PvDBP is divided into seven regions (regions I-VII). In embodiments, the DBP is region I, region II, region III, region IV, region V, region VI, region VII, or a combination thereof of PvDBP. In embodiments, the DBP comprises region II of PvDBP (referred to herein as "PvDBPII"). PvDBPII is the amino-terminal cysteine-rich region of PvDBP, which contains the receptor-binding domain of PvDBP. PvDBPII binds to the Duffy antigen receptor for chemokines (DARC). A key binding motif in PvDBPII has been mapped to a 170-amino acid stretch (amino acids 291-460) containing cysteines 5-8.

[0035] In embodiments, PvDBPII is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide comprising amino acids 194 to 521 of SEQ ID NO:1.

[0036] In embodiments, PvDBPII is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0037] In embodiments, PvDBPII is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having the amino acid sequence of SEQ ID NO:3.

[0038] The amino acid sequences of the polypeptides of SEQ ID NOs: 1-3 are found below. [Table 1-1] [Table 1-2]

[0039] In an embodiment, the DBP is derived from a P. falpicarum parasite. In an embodiment, the DBP derived from a P. falpicarum parasite is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOS: 4 to 9. [Table 2-1] [Table 2-2] [Table 2-3]

[0040] Further suitable antigens are disclosed in the following references, which are incorporated by reference in their entirety for all purposes: Singh et al., "Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial." NPJ vaccines vol. 3 48. 28 Sep. 2018; Bhardwaj, R. et al., Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development. Protein Expr. Purif. 136, 52-57 (2015); U.S. Patent No. 5,993,827; and Chitnis et al., J Exp Med. 1994 Aug 1; 180(2): 497-506.

[0041] In embodiments, the DBP is expressed and purified from E. coli host cells. In embodiments, the DBP is expressed and purified in insect cells. In embodiments, the insect cells are Drosophila A2 cells. In embodiments, the DBP is expressed in host cells using a fed-batch fermentation strategy. In embodiments, the DBP is purified using chromatography. In embodiments, the chromatography is cation exchange chromatography. In embodiments, the chromatography is anion exchange chromatography. In embodiments, the chromatography is affinity chromatography. In embodiments, the DBP comprises a C-tag and is purified via the C-tag. In embodiments, the DBP is filtered on an anion exchange support. In embodiments, the anion exchange support comprises pendant quaternary amine functional groups on a cross-linked polymer coating. The following references describe the expression and purification of DBP and are incorporated by reference herein in their entirety for all purposes: Singh et al., Npj Vaccines, volume 3, article 48 (2018); Hjerrild et al., Scientific Reports, 6: 30357 (2016); Bhardwaj, R. et al., Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development. Protein Expr. Purif. 136, 52-57 (2015).

[0042] In embodiments, the DBPs described herein comprise an N- or C-terminal tag. In embodiments, the N- or C-terminal tag is a polyglutamate tag, a FLAG tag, an HA tag, a polyHis tag (having about 5-10 histidines) (SEQ ID NO: 10), a hexahistidine tag (SEQ ID NO: 11), an 8x-His tag (having 8 histidines) (SEQ ID NO: 12), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, an Fc tag, or a C tag. In embodiments, the extension comprises a C tag. The C tag comprises the sequence EPEA (SEQ ID NO: 13). In embodiments, the N- or C-terminal tag is removed.

[0043] In embodiments, provided herein is a nucleic acid encoding any of the DBPs described herein. In embodiments, the nucleic acid encoding the Plasmodium parasite antigen is extended at the 5' end, the 3' end, or both. In embodiments, the extension encodes a protein used for purification or detection. Non-limiting examples of tags are described throughout this disclosure. In embodiments, the tag is removed.

[0044] Advantageously, the immunogenic compositions described herein can induce robust humoral immune responses capable of inhibiting parasite growth in vitro. The present disclosure utilizes a delayed boost approach to provide significantly enhanced immune responses. Delayed administration of the DBP / MM vaccine results in substantially improved IgG concentrations, avidity, altered isotype profiles, and significantly increased IgG4 and IgG1 responses. Thus, in embodiments, a second immunogenic composition is administered after the first composition to boost the response, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or at least 16 months apart. The first and second compositions can be the same or different.

[0045] Adjuvants In embodiments, the immunogenic composition comprises an adjuvant.

[0046] In certain embodiments, the compositions disclosed herein can be combined with one or more adjuvants to enhance immune responses. In other embodiments, the compositions are prepared without adjuvants and are therefore available for administration as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein can provide a protective immune response when administered as a single dose. Alum-free compositions that induce robust immune responses are particularly useful in adults aged about 60 years and older.

[0047] Aluminum-based adjuvants In an embodiment, the adjuvant may be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the alum. Often, the nanoparticles are 92% to 97% bound to the alum in the composition. The amount of alum is typically present in a range of about 400 μg to about 1250 μg per dose. For example, the alum may be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, the alum is present in an amount of about 400 μg per 120 μg dose of protein nanoparticles.

[0048] In an embodiment, the adjuvant is a saponin adjuvant.

[0049] Saponin adjuvants Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponins are prepared using a multi-step purification process that produces multiple fractions. As used herein, the term "saponin fraction derived from Quillaja saponaria Molina" is used generally to describe a semi-purified or defined saponin fraction of Quillaja saponaria, or a substantially pure fraction thereof.

[0050] Saponin fraction Several approaches to producing saponin fractions are suitable. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows: A lipophilic fraction from Quil A (crude aqueous Quillaja saponaria Molina extract) is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC eluting with a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is or corresponds to the fraction eluted at approximately 39% acetonitrile. The fraction referred to herein as "Fraction B" or "QH-B" is or corresponds to the fraction eluted at approximately 47% acetonitrile. The fraction referred to herein as "Fraction C" or "QH-C" is or corresponds to the fraction eluted at approximately 49% acetonitrile. Further information regarding fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Quillaja saponaria Molina fractions A, B, and C each represent a group or family of closely chemically related molecules with definable properties. The chromatographic conditions under which they are obtained are such that there is a high degree of batch-to-batch reproducibility with respect to elution profile and biological activity.

[0051] Other saponin fractions have been described: fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP 03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22 of EP 0 3632 279 B2 can be used, in particular QA-7, QA-17, QA-18, and QA-21, which can be obtained as described in EP 0 3632 279 B2, in particular on page 6 and in Example 1 on pages 8 and 9.

[0052] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; i.e., the fractions are substantially free of contaminants from other materials. In certain aspects, a substantially pure saponin fraction may contain up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 7%, up to 5%, up to 2%, up to 1%, up to 0.5%, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0053] ISCOM Structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (Immune Stimulating COMplexes). ISCOMs may be prepared as described in EP 0109942 B1, EP 0242380 B1 and EP 0180546 B1. In certain embodiments, transport and / or passenger antigens may be used as described in EP 9600647-3 (PCT / SE97 / 00289).

[0054] Matrix adjuvants In embodiments, the ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol (e.g., cholesterol). In certain aspects, the ISCOM matrix complex also comprises a phospholipid. The ISCOM matrix complex may also comprise one or more other immunomodulatory (adjuvant-active) substances (not necessarily glycosides) and may be produced as described in EP 0 436 620 B1 (which is incorporated by reference in its entirety).

[0055] In other aspects, the ISCOM is an ISCOM complex. The ISCOM complex contains at least one saponin, at least one lipid, and at least one species of antigen or epitope. The ISCOM complex contains antigens associated with detergent treatment, resulting in some of the antigens becoming integrated into the particles. In contrast, ISCOM matrices are formulated as a mixture with antigens and the association between ISCOM matrix particles is mediated by electrostatic and / or hydrophobic interactions.

[0056] According to one embodiment, the ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one further adjuvant (which is also incorporated into or mixed with the ISCOM or ISCOM matrix complex) is selected from fraction A, fraction B, or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any derived subfraction, e.g., QA 1-21.

[0057] In certain aspects, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percents of different saponin fractions may be used. Any combination of weight percents of any two fractions may be used. For example, the particle may contain any weight percent of Fraction A and any weight percent of another saponin fraction (e.g., crude saponin fraction or Fraction C, respectively). Thus, in certain aspects, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60%, or 50% by weight of one saponin fraction (e.g., fraction A), with the remainder being up to 100% of another saponin (e.g., any crude fraction or any other fraction (e.g., fraction C)). The weights are calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.S. Application Publication No. 2013 / 0129770, which is incorporated by reference in its entirety.

[0058] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction (e.g., Fraction A) and the remainder, up to 100%, of another fraction (e.g., crude saponin fraction or Fraction C), calculated as the total weight of the saponin fractions.

[0059] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction (e.g., fraction A) and 1% to 60% by weight of another fraction (e.g., crude saponin fraction or fraction C), the weights being calculated as the total weight of the saponin fractions.

[0060] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction (e.g., Fraction A) and 30% to 5% by weight of another fraction (e.g., crude saponin fraction or Fraction C), calculated as the total weight of the saponin fractions. In another embodiment, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA 1-21.

[0061] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can contain multiple particles, where each particle contains only one saponin fraction. That is, a particular composition can contain one or more different types of ISCOM-matrix complex particles and / or one or more different types of ISCOM complex particles, where each individual particle contains one saponin fraction from Quillaja saponaria Molina, and where the saponin fraction in one complex is different from the saponin fraction in the other complex particle.

[0062] In certain aspects, one type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. A composition or vaccine may comprise at least two types of complexes or particles, each type having one type of saponin incorporated into a physically distinct particle.

[0063] In the above composition, ISCOM matrix complex particles and / or mixtures of ISCOM complex particles may be used, in which one saponin fraction of Quillaja saponaria Molina and another saponin fraction of Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0064] The ISCOM matrices or ISCOM complex particles (each containing one saponin fraction) can be present in the composition in any combination of weight percent. In certain aspects, the composition can contain 0.1% to 99.9%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, 40% to 60%, or 50% by weight of ISCOM matrices or complexes containing a first saponin fraction, with the remainder being ISCOM matrices or complexes containing a different saponin fraction. In some aspects, the remainder is one or more ISCOM matrices or complexes, where each matrix or complex particle contains only one saponin fraction. In other aspects, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[0065] In certain compositions, the only saponin fraction in the first ISCOM matrix or ISCOM complex particle is fraction A and the only saponin fraction in the second ISCOM matrix or ISCOM complex particle is fraction C.

[0066] A preferred composition comprises a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, where the Fraction A ISCOM matrix comprises about 70% by weight of the total saponin adjuvant, and the Fraction C ISCOM matrix comprises about 30% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix comprises about 85% by weight of the total saponin adjuvant, and the Fraction C ISCOM matrix comprises about 15% by weight of the total saponin adjuvant. Thus, in certain compositions, the Fraction A ISCOM matrix will be present in an amount ranging from about 70% to about 85% and the Fraction C ISCOM matrix will be present in an amount ranging from about 15% to about 30% by weight of the total saponin adjuvant. In one embodiment, the Fraction A ISCOM matrix and the Fraction C ISCOM matrix each comprise 50-96% by weight of the combined weight of the adjuvant, with the Fraction C ISCOM matrix comprising the remainder. In one embodiment, the Fraction A ISCOM matrix comprises at least 75% by weight of the combined weight of the Fraction A ISCOM matrix and the Fraction C ISCOM matrix each comprising the remainder. In one embodiment, the Fraction A ISCOM matrix comprises about 92% by weight and the Fraction C ISCOM matrix comprises about 8% by weight of the total weight of the saponin adjuvant in the composition. Matrix-M is a compound referred to herein as a saponin adjuvant. TM In a particularly preferred composition, referred to as "Matrix-M," the Fraction A ISCOM matrix is ​​present at about 85% and the Fraction C ISCOM matrix is ​​present at about 15% by weight of the total saponin adjuvant in the composition. TM may be referred to interchangeably as Matrix-M1.

[0067] Exemplary QS-7 and QS-21 fractions, their production and their uses are described in U.S. Patent Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343, and 8,173,141, which are incorporated herein by reference.

[0068] In some compositions, other adjuvants may be used in addition or instead. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" (incorporated herein by reference in its entirety for all purposes) is contemplated within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds (e.g., thur-MDP and nor-MDP), CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, and RIBI (which contains three components extracted from bacteria: MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / TWEEN® polysorbate 80 emulsion). In embodiments, the adjuvant can be paucilamellar lipid vesicles, such as NOVASOMES®. NOVASOMES® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They contain BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see US Pat. Nos. 5,629,021, 6,387,373, and 4,911,928).

[0069] In embodiments, the immunogenic composition comprises the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), which is described in the following documents, which are incorporated herein by reference in their entireties: Behzad et al. J Infect Dis. 2012 Feb 1;205(3):466-473.

[0070] excipients In embodiments, the immunogenic compositions described herein include various excipients, buffers, etc. For example, the immunogenic compositions may include sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM; in certain cases, about 22 mM sodium phosphate is present. Histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, if present, may be about 150 mM. In certain compositions, the sodium chloride may be present at higher concentrations, e.g., about 200 mM to about 500 mM. In embodiments, the sodium chloride is present at a high concentration, including but not limited to, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0071] Administration and Dosage

[0009] Provided herein are novel methods for administering the aforementioned immunogenic compositions. In embodiments, the immunogenic compositions induce an immune response against malaria in a subject in need thereof. In embodiments, the immunogenic compositions induce an immune response against a parasite selected from Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi.

[0072] In embodiments, the amount of DBP administered per dose ranges from about 0.1 μg to about 100 μg (including all ranges and subranges therebetween), e.g., 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, μg, approximately 12 μg, approximately 13 μg, approximately 14 μg, approximately 15 μg, approximately 16 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21 μg, approximately 22 μg, approximately 23 μg, approximately 24 μg, approximately 25 μg, approximately 26 μg g, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, About 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 In an embodiment, a dose of about 70 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg is administered. In an embodiment, each dose of the immunogenic composition comprises about 50 μg of DBP.

[0073] In embodiments, the immunogenic composition includes an adjuvant. In embodiments, the dose of the adjuvant ranges from about 1 μg to about 100 μg. For example, in embodiments, the dose of the adjuvant ranges from about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21 μg, approximately 22 μg, approximately 23 μg, approximately 24 μg, approximately 25 μg, approximately 26 μg, approximately 27 μg, approximately 28 μg, approximately 29 μg, approximately 30 μg g, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, About 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73

[0034] In embodiments, the adjuvant is about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg. In embodiments, the adjuvant is a saponin adjuvant. In embodiments, the saponin adjuvant comprises 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. In embodiments, the immunogenic composition comprises about 50 μg adjuvant.

[0074] In an embodiment, the immunogenic composition described herein is administered in a single dose. In an embodiment, the immunogenic composition described herein is administered in multiple doses. For example, in an embodiment, 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, or 10 doses of the immunogenic composition are administered. These doses are referred to as the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth doses.

[0075] In embodiments, the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition is administered about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 days after administration of the first dose of the immunogenic composition. weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks After about 41 weeks, after about 42 weeks, after about 43 weeks, after about 44 weeks, after about 45 weeks, after about 46 weeks, after about 47 weeks, after about 48 weeks, after about 49 weeks, after about 50 weeks, after about 51 weeks, after about 52 weeks, after about 53 weeks, after about 54 weeks, after about 55 weeks, after about 56 weeks, after about 57 weeks, after about 58 weeks, after about 59 weeks, after about 60 weeks, after about 61 weeks, after about 62 weeks, after about 63 weeks, after about 64 weeks, after about 65 weeks, after about 66 weeks, after about 67 weeks, after about 68 weeks, after about 69 weeks, after about 70 weeks, after about 71 weeks, after about 72 weeks, after about 73 weeks, after about 74 weeks, after about 75 weeks, after about 76 weeks, after about 77 weeks, after about 78 weeks, after about 79 weeks, after about 80 weeks, after about 81 weeks, after about 82 weeks, after about 83 weeks, after about 84 weeks, after about 85 weeks, after about 86 weeks, after about 87 weeks, after about 88 weeks, after about 89 weeks, after about 90 weeks, after about 91 weeks, after about 92 weeks, after about 93 weeks, after about 94 weeks, after about 95 weeks, after about 96 weeks, after about 97 weeks, after about 98 weeks, after about 99 weeks, after about 100 weeks, after about 101 weeks, after about 102 weeks, after about 103 weeks, after about 104 weeks, after about 105 weeks, after about 106 weeks, after about 107 weeks, after about 108 weeks, after about 109 weeks, after about 110 weeks, after about 111 weeks, after about After 6 weeks, after about 77 weeks, after about 78 weeks, after about 79 weeks, after about 80 weeks, after about 81 weeks, after about 82 weeks, after about 83 weeks, after about 84 weeks, after about 85 weeks, after about 86 weeks, after about 87 weeks, after about 88 weeks, after about 89 weeks, after about 90 weeks, after about 91 weeks, after about 92 weeks, after about 93 weeks, after about 94 weeks, after about 95 weeks, after about 96 weeks, after about 97 weeks, after about 98 weeks, after about 99 weeks, after about 100 weeks, after about 1 month (e.g., after 28 days, after 29 days, after 30 days, or after 31 days), after about 2 months, after about 3 months, after about 4 months, after about 5 months, after about 6 months, after about 7 months, after about 8 months,In an embodiment, the second dose of the immunogenic composition is administered about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months later. In an embodiment, the second dose of the immunogenic composition is administered about 1 month after administration of the first dose. In an embodiment, the third dose of the immunogenic composition is administered about 1 month or about 2 months after administration of the first dose. In an embodiment, the third dose of the immunogenic composition is administered about 14 months or about 15 months after administration of the first or second dose.

[0076] In embodiments, the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of immunogenic composition is administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, at least about 32 weeks, at least about 33 weeks, at least about 34 weeks, at least about 35 weeks, at least about 36 weeks, at least about 37 weeks, at least about 38 weeks, at least about 39 weeks, at least about 40 weeks, at least about 41 weeks, at least about 42 weeks, at least about 43 weeks, at least about 44 weeks, at least about 45 weeks, at least about 46 weeks, at least about 47 weeks, at least about 48 weeks, at least about 49 weeks,After at least about 50 weeks, after at least about 51 weeks, after at least about 52 weeks, after at least about 53 weeks, after at least about 54 weeks, after at least about 55 weeks, after at least about 56 weeks, after at least about 57 weeks, after at least about 58 weeks, after at least about 59 weeks, after at least about 60 weeks, after at least about 61 weeks, after at least about 62 weeks, after at least about 63 weeks, after at least about 64 weeks, after at least about 65 weeks, after at least about 66 weeks, after at least about 67 weeks, after at least about 68 weeks after at least about 69 weeks, after at least about 70 weeks, after at least about 71 weeks, after at least about 72 weeks, after at least about 73 weeks, after at least about 74 weeks, after at least about 75 weeks, after at least about 76 weeks, after at least about 77 weeks, after at least about 78 weeks, after at least about 79 weeks, after at least about 80 weeks, after at least about 81 weeks, after at least about 82 weeks, after at least about 83 weeks, after at least about 84 weeks, after at least about 85 weeks, after at least about 86 weeks, after at least about After 87 weeks, after at least about 88 weeks, after at least about 89 weeks, after at least about 90 weeks, after at least about 91 weeks, after at least about 92 weeks, after at least about 93 weeks, after at least about 94 weeks, after at least about 95 weeks, after at least about 96 weeks, after at least about 97 weeks, after at least about 98 weeks, after at least about 99 weeks, after at least about 100 weeks, after at least about 1 month, after at least about 2 months, after at least about 3 months, after at least about 4 months, after at least about 5 months, after at least about 6 months In some embodiments, the second dose of immunogenic composition is administered at least about 1 month after administration of the first dose. In some embodiments, the third ...In some embodiments, the third dose of immunogenic composition is administered at least about 1 month or at least about 2 months after the first dose. In some embodiments, the third dose of immunogenic composition is administered at least about 14 months or at least about 15 months after the first or second dose.

[0077] In embodiments, the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition is administered about 6 to about 18 months, about 9 to about 18 months, about 12 to about 15 months, about 12 to about 18 months, or about 12 to about 24 months after the first or second dose. In embodiments, the second dose of the immunogenic composition is administered about 6 to about 18 months, about 9 to about 18 months, about 12 to about 15 months, about 12 to about 18 months, or about 12 to about 24 months after the first dose.

[0078] In an embodiment, three doses of the immunogenic composition described herein are administered as a first dose, a second dose, and a third dose. In an embodiment, each dose of the immunogenic composition comprises about 50 μg of DBP. In an embodiment, each dose of the immunogenic composition comprises about 50 μg of adjuvant. In an embodiment, each dose of the immunogenic composition comprises about 50 μg of adjuvant and about 50 μg of antigen. In an embodiment, the second dose is administered about one month after the administration of the first dose, and the third dose is administered about 14 months after the administration of the second dose. In an embodiment, the second dose is administered about one month after the administration of the first dose, and the third dose is administered about 14 months after the administration of the first dose.

[0079] The compositions disclosed herein can be administered to specific tissues via systemic, mucosal, or transdermal routes, or directly. As used herein, the term "systemic administration" includes parenteral administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, or kidney dialytic infusion techniques. Typically, the systemic parenteral administration is intramuscular injection. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal, and ocular administration. Preferably, administration is intramuscular.

[0080] In embodiments, the dose is administered in a volume of about 0.1 mL to about 1.5 mL, for example, about 0.1 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, or about 1.5 mL. In embodiments, the dose is administered in a volume of 0.25 mL. In embodiments, the dose is administered in a volume of 0.5 mL. In embodiments, the dose is administered in a volume of 0.6 mL.

[0081] In embodiments, the immunogenic composition may contain the antigen at a concentration of about 1 μg / mL to about 50 μg / mL, 10 μg / mL to about 100 μg / mL, about 10 μg / mL to about 50 μg / mL, about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL, or about 240 μg / mL to about 260 μg / mL.

[0082] In embodiments, the immunogenic compositions described herein are effective in preventing infection by malaria. The effectiveness can be determined by comparing the number of subjects who have been diagnosed with malaria and who have not received the malaria immunogenic composition (controls diagnosed with malaria) with the number of subjects who have subsequently been diagnosed with malaria and who have received the malaria immunogenic composition. The following formula is used to calculate efficacy: 100 x (percentage of controls diagnosed with malaria - percentage of subjects diagnosed with malaria who received the immunogenic composition) / (percentage of controls diagnosed with malaria).

[0083] In embodiments, the immunogenic compositions described herein have an efficacy of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0084] In embodiments, the methods described herein provide for a period of at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, at least about 6 months, at least about 6.5 months, at least about 7 months, at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36 months, at least about 37 months, at least about 38 months, at least about 39 months, at least about 40 months, at least about 41 months, at least about 42 months, at least about 43 months, at least about 44 months, at least about 45 months, at least about 46 months, at least about 47 months, at least about 48 months, at least about 49 months, at least about 50 months, at least about 51 months, at least about 52 months, at least about 53 months, at least about or at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 6 Prevents malaria with an effectiveness of 5% to about 85%, about 69% to about 81%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%.

[0085] In embodiments, the methods described herein can be used to treat a patient with a HIV-infected or HIV-positive virus (HIV)-positive virus (HIV)-positive virus (HIV)-positive virus (HIV)-positive virus (HIV)-negative virus (NEV)-positive virus (NEV)-negative ... For up to 20 months, for up to 21 months, for up to 22 months, for up to 23 months, or for up to 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 6 Prevents malaria with an effectiveness of 0% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%.

[0086] In some embodiments, the present disclosure provides a co-formulation (i.e., pre-filled syringe or pre-mix) strategy for immunogenic compositions comprising DBP and an adjuvant (e.g., a saponin adjuvant). A typical vaccine administration strategy currently utilized is bedside mix formulation. That is, the vaccine composition and the adjuvant are stored separately and mixed before administration. Premix, co-formulation, or pre-filled syringe strategies for vaccines are less common due to concerns about the stability of the antigens (e.g., DBP) and their subsequent immunogenic potential. The present disclosure provides immunogenic compositions that can be pre-mixed and stored in advance. The vaccination strategies and formulations of the present disclosure can improve vaccination efficiency and reduce the risk of bedside mixing errors while maintaining overall safety and immunogenicity.

[0087] Various containers can be used to store and transport premix formulations, including single-dose syringes and plastic ampoules. In some cases, plastic ampoules can be manufactured using blow-fill-seal manufacturing techniques or methods. Generally, blow-fill-seal (BFS) manufacturing methods involve extruding a plastic material (e.g., a resin) to form a parison, which is then placed into a mold and cut to size. A filling needle or mandrel is then used to inflate the plastic, which then results in a hollow ampoule that substantially matches the shape of the mold. Once inflated, a desired volume of liquid can be injected into the ampoule, the filling needle or mandrel can be removed, and the ampoule can be sealed. Thus, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0088] In some cases, the ability to aseptically produce sterile ampoules containing a desired liquid may make BFS-produced ampoules particularly suitable for the pharmaceutical industry. However, BFS technology has not been compatible with all pharmaceutical liquids, products, and the like. For example, some known BFS manufacturing methods involve delivering the liquid or product to the ampoules while the plastic is still relatively hot, which can have adverse effects on temperature-sensitive liquids and / or products (e.g., vaccines, biologics, and the like). However, advances in cold BFS technology have expanded the variety of suitable products, liquids, and the like, allowing some vaccines, biologics, and / or other temperature-sensitive pharmaceuticals to be included in BFS ampoules.

[0089] In some cases, BFS ampoules can have a size, shape, and / or configuration based at least in part on the desired use and / or the desired pharmaceutical liquid or dosage that the ampoules are configured to contain. For example, some known BFS ampoules can include pierce caps, screw-off caps, caps including male or female luers, etc. Some known BFS ampoules can have a size and / or shape based on the volume or dosage of liquid that is configured to be placed therein. Furthermore, some known BFS ampoules can be manufactured with multiple temporarily connected ampoules in a band. This can increase manufacturing, packaging, and / or storage efficiency, etc.

[0090] In an embodiment, the immunogenic composition described herein is provided in a pre-filled syringe.When the immunogenic composition is prepared in a pre-filled syringe, antigen and adjuvant can be combined before administration.In an embodiment, the pre-filled syringe comprises DBP and adjuvant (for example, saponin adjuvant). In an embodiment, the pre-filled syringe comprises DBP and a saponin adjuvant, wherein the adjuvant comprises at least two types of iscom particles, wherein a first iscom particle comprises Quillaja Saponaria Molina fraction A and does not comprise Quillaja Saponaria Molina fraction C; and a second iscom particle comprises Quillaja Saponaria Molina fraction C and does not comprise Quillaja Saponaria Molina fraction A; wherein Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C account for about 85% by weight and about 15% by weight, respectively, of the total weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C in the adjuvant. In an embodiment, the pre-filled syringe comprises DBP and a saponin adjuvant, wherein the adjuvant comprises at least two types of iscom particles, wherein a first iscom particle comprises Quillaja Saponaria Molina Fraction A and does not comprise Quillaja Saponaria Molina Fraction C; and a second iscom particle comprises Quillaja Saponaria Molina Fraction C and does not comprise Quillaja Saponaria Molina Fraction A; wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant.In an embodiment, the pre-filled syringe comprises DBP and a saponin adjuvant, wherein the adjuvant comprises at least two types of iscom particles, wherein a first iscom particle comprises Quillaja Saponaria Molina Fraction A and does not comprise Quillaja Saponaria Molina Fraction C; and a second iscom particle comprises Quillaja Saponaria Molina Fraction C and does not comprise Quillaja Saponaria Molina Fraction A; wherein Quillaja Saponaria Molina Fraction A accounts for at least about 75% by weight of the combined weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant, and Quillaja Saponaria Molina Fraction C accounts for the remainder. [Example]

[0091] Example Example 1: A delayed administration approach enhanced the immune response to a protein-based vaccine Immunogenic compositions comprising PvDBPII and a saponin adjuvant were administered according to the DBP / MM MD ("monthly administration") or DBP / MM DD ("delayed administration") schedules shown in Figure 6. The saponin adjuvant contained 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. For DBP / MM MD ("monthly administration"), three doses were administered, one month apart. For DBP / MM DD ("delayed administration"), the first two doses were administered one month apart, with the third dose administered 14 months after the second dose. The immunogenicity of the compositions was compared with two viral vector formulations ("vvDBP"): (i) a ChAd63 viral vector encoding PvDBPII and (ii) an MVA viral vector encoding PvDBPII. The vvDBPs were administered according to the schedule shown in Figure 5.

[0092] Figure 1 shows the immune responses induced by each dosing schedule. The immune responses obtained by administering the immunogenic composition containing PvDBPII and a saponin adjuvant were improved compared to those induced by administering the viral vector formulation. Furthermore, the immune responses obtained by waiting 14 months to administer the third dose (i.e., "delayed dosing") were significantly improved compared to monthly dosing. Compare DBP / MM DD to DBP / MM MD in Figure 2. Figures 3A-3B and 4A-4E further confirm that delayed dosing enhances the immune response.

[0093] Numbered Embodiments Embodiment 1. An immunogenic composition comprising a Duffy binding protein (DBP) from a Plasmodium parasite. Embodiment 2. The immunogenic composition of embodiment 1, wherein the DBP is PvDBP. Embodiment 3 The immunogenic composition of embodiment 1 or 2, wherein the DBP comprises Region I, Region II, Region III, Region IV, Region V, Region VI, Region VII, or any combination thereof, of PvDBP. Embodiment 4. The immunogenic composition of any one of embodiments 1 to 3, wherein the DBP comprises region II of PvDBP (“PvDBPII”). Embodiment 5. The immunogenic composition of any one of embodiments 1-4, wherein the DBP comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-9. Embodiment 6. The immunogenic composition of any one of Embodiments 1 to 4, wherein the DBP comprises amino acids 194 to 521 of SEQ ID NO:1 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 194 to 521 of SEQ ID NO:1. Embodiment 7. The immunogenic composition of any one of embodiments 1 to 6, wherein the DBP is expressed in Escherichia coli. Embodiment 8. The immunogenic composition of any one of embodiments 1 to 7, comprising an adjuvant. Embodiment 9 The immunogenic composition of embodiment 8, wherein the adjuvant is a saponin adjuvant. Embodiment 10. The immunogenic composition of any one of Embodiments 8 to 9, wherein the immunogenic composition comprises about 1 μg to about 100 μg of adjuvant. Embodiment 11. The immunogenic composition of any one of Embodiments 8 to 10, wherein the immunogenic composition comprises about 25 μg to about 75 μg of adjuvant. Embodiment 12 The immunogenic composition of any one of Embodiments 8 to 11, wherein the immunogenic composition comprises about 50 μg of adjuvant. Embodiment 13 The immunogenic composition of any one of Embodiments 8 to 12, wherein the immunogenic composition comprises about 25 μg of adjuvant. Embodiment 14. The immunogenic composition of any one of Embodiments 1 to 13, wherein the immunogenic composition comprises about 10 μg to about 100 μg of DBP, about 25 to about 100 μg of DBP, or about 35 to about 55 μg of DBP. Embodiment 15 The immunogenic composition of any one of Embodiments 1 to 14, wherein the immunogenic composition comprises about 50 μg of DBP. Embodiment 16 The immunogenic composition of any one of Embodiments 1 to 15, wherein the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. Embodiment 17 The adjuvant comprises at least two types of iscom particles, wherein: said first iscom particles comprising fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; 17. The immunogenic composition according to any one of embodiments 8 to 16, wherein said second iscom particles comprise fraction C of Quillaja Saponaria Molina and do not comprise fraction A of Quillaja Saponaria Molina. Embodiment 18. The immunogenic composition of embodiment 17, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 85% by weight and about 15% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. Embodiment 19. The immunogenic composition of embodiment 17, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 92% by weight and about 8% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. Embodiment 20. The immunogenic composition of any one of Embodiments 8 to 19, comprising about 50 μg adjuvant. Embodiment 21 The immunogenic composition of any one of claims 1 to 20, comprising about 0.1 μg to about 100 μg of DBP; about 10 μg to about 100 μg of DBP; about 25 μg to about 75 μg of DBP; or about 45 μg to about 55 μg of DBP. Embodiment 22. The immunogenic composition of any one of claims 1 to 21, comprising about 50 μg of DBP. Embodiment 21. A method of stimulating an immune response to Plasmodium parasites in a subject, comprising administering a composition of any one of Embodiments 1-20. Embodiment 22 The method of embodiment 21, comprising administering a first dose and a second dose of the immunogenic composition. Embodiment 23 The method of embodiment 22, comprising administering a third dose of the immunogenic composition. Embodiment 24 The method of any one of Embodiments 22-23, wherein the dose of DBP in the second dose is less than the dose of DBP in the first dose. Embodiment 25 The method of any one of Embodiments 23-24, wherein the dose of DBP in the third dose is less than the dose of DBP in the first dose. Embodiment 26 The method of any one of embodiments 23-25, wherein the dose of DBP in the third dose is less than the dose of DBP in the second dose. Embodiment 27 The method of any one of embodiments 21-26, wherein the first dose and the second dose comprise approximately equal amounts of DBP. Embodiment 28 The method of any one of embodiments 23-27, wherein the first dose and the third dose comprise approximately equal amounts of DBP. Embodiment 29 The method of any one of embodiments 23-27, wherein the second dose and the third dose comprise about the same amount of DBP. Embodiment 30. The method of any one of embodiments 23-29, wherein the third dose is administered at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after the first dose. Embodiment 31. The method of any one of embodiments 23-29, wherein the third dose is administered at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 months after the second dose. Embodiment 32. The method of any one of embodiments 23-29, wherein the third dose is administered about 6 months to about 2 years after the first dose. Embodiment 33. The method of any one of embodiments 23-29, wherein the third dose is administered about 6 months to about 2 years after the second dose. Embodiment 34 The method of any one of embodiments 23-29, wherein the third dose is administered starting about 14 months after the first dose or about 14 months after the second dose.

[0094] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be considered as, an admission or any form of suggestion that it constitutes valid prior art or forms part of the general technical knowledge in any country in the world. This application incorporates by reference the disclosures of U.S. Patent Nos. 10,729,764, 9,821,046, and 8,821,881 in their entireties for all purposes. In certain embodiments, for example, the following are provided: (Item 1) A method of stimulating an immune response to a Plasmodium parasite in a subject, comprising administering an immunogenic composition comprising a Duffy binding protein (DBP) derived from a Plasmodium parasite. (Item 2) Item 2. The method of item 1, wherein the DBP is PvDBP. (Item 3) Item 10. The method of item 1, wherein the DBP comprises region I, region II, region III, region IV, region V, region VI, region VII, or any combination thereof of PvDBP. (Item 4) 4. The method of claim 3, wherein the DBP comprises domain II of PvDBP ("PvDBPII"). (Item 5) 5. The method of any one of items 1 to 4, wherein the DBP comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1 to 9. (Item 6) 5. The method of any one of Items 1 to 4, wherein the DBP comprises amino acids 194 to 521 of SEQ ID NO: 1 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 194 to 521 of SEQ ID NO: 1. (Item 7) 7. The method according to any one of items 1 to 6, wherein the DBP is expressed in Escherichia coli. (Item 8) 8. The method according to any one of items 1 to 7, wherein the immunogenic composition comprises about 1 μg to about 100 μg of DBP, about 10 μg to about 100 μg of DBP, about 25 to about 100 μg of DBP, or about 35 to about 55 μg of DBP. (Item 9) 8. The method of any one of items 1 to 7, wherein the immunogenic composition comprises about 2 μg, 5 μg, 10 μg, or 50 μg of DBP. (Item 10) 10. The method according to any one of items 1 to 9, wherein the immunogenic composition comprises an adjuvant. (Item 11) Item 11. The method according to item 10, wherein the immunogenic composition comprises about 1 μg to about 100 μg; about 25 μg to about 75 μg; or about 40 μg to about 60 μg of adjuvant. (Item 12) 12. The method of claim 10 or 11, wherein the immunogenic composition comprises about 50 μg of adjuvant. (Item 13) 12. The method of claim 10 or 11, wherein the immunogenic composition comprises about 25 μg of adjuvant. (Item 14) 14. The method according to any one of items 10 to 13, wherein the adjuvant is a saponin adjuvant. (Item 15) The saponin adjuvant comprises at least two types of iscom particles, wherein: said first iscom particles comprising fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; The second iscom particles contain fraction C of Quillaja Saponaria Molina and do not contain fraction A of Quillaja Saponaria Molina, Item 15. The method according to item 14. (Item 16) Item 16. The method according to Item 15, wherein, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant, Quillaja Saponaria Molina Fraction A accounts for 50 to 96% by weight, and Quillaja Saponaria Molina Fraction C accounts for the remainder. (Item 17) Item 16. The method according to Item 15, wherein Fraction A of Quillaja Saponaria Molina accounts for at least 75% by weight of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, and Fraction C of Quillaja Saponaria Molina accounts for the remainder. (Item 18) Item 16. The method according to Item 15, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 85% by weight and about 15% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. (Item 19) Item 16. The method according to Item 15, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 92% by weight and about 8% by weight, respectively, of the total weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. (Item 20) 20. The method of any one of items 1 to 19, wherein the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. (Item 21) 21. The method of any one of items 1 to 20, comprising administering the immunogenic composition in a pre-filled syringe. (Item 21) 21. The method of any one of items 1 to 20, comprising administering a first dose and a second dose of the immunogenic composition. (Item 22) 22. The method of claim 21, comprising administering a third dose of the immunogenic composition. (Item 23) 23. The method of claim 22, comprising administering a fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition. (Item 24) 24. The method of any one of items 21 to 23, wherein the first dose and the second dose comprise about the same amount of DBP. (Item 25) 25. The method of any one of items 22 to 24, wherein the second dose and the third dose comprise about the same amount of DBP. (Item 26) 26. The method of any one of items 22 to 25, wherein the first and third doses comprise about the same amount of DBP. (Item 27) 27. The method of any one of items 22 to 26, wherein the first, second, and third doses comprise about the same amount of DBP. (Item 28) 28. The method of any one of items 22 to 27, wherein the first, second, third, and fourth doses comprise about the same amount of DBP. (Item 29) 24. The method of any one of items 21 to 23, wherein the amount of DBP in the second dose is less than the amount of DBP in the first dose. (Item 30) 24. The method of any one of items 22 to 23, wherein the amount of DBP in the third dose is less than the amount of DBP in the first dose. (Item 31) 31. The method of any one of items 21 to 30, wherein the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose comprises about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the DBP in the first dose. (Item 31) 32. The method of any one of items 21-23 or 31, wherein the second dose comprises about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the DBP in the first dose. (Item 32) 32. The method of any one of items 21 to 31, comprising administering the second dose about one month after the first dose. (Item 33) 32. The method of any one of items 21 to 31, comprising administering the second dose about 28 days after the first dose. (Item 34) 34. The method of any one of items 22 to 33, comprising administering the third dose about 56 days after the first dose. (Item 35) 34. The method of any one of items 22 to 33, comprising administering the third dose about 2 months after the first dose. (Item 36) 34. The method of any one of items 22 to 33, comprising administering the third dose about 168 days after the first dose. (Item 37) 34. The method of any one of items 22 to 33, comprising administering the third dose about 6 months after the first dose. (Item 38) 34. The method of any one of items 22 to 33, comprising administering the third dose about 14 months after the first dose. (Item 39) 34. The method of any one of items 22 to 33, comprising administering the third dose about 14 months after the second dose. (Item 31) 34. The method of any one of items 22 to 33, comprising administering the third dose at least 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months after the first dose or the second dose. (Item 32) 34. The method according to any one of items 22 to 33, comprising administering the third dose about 6 months to about 18 months, about 9 months to about 18 months, about 12 months to about 15 months, or about 12 months to about 18 months after the first or second dose. (Item 33) (i) administering a first dose of the immunogenic composition; (ii) administering a second dose of the immunogenic composition about one month after the first dose; (iii) administering a third dose of the immunogenic composition about 14 months after the second dose; The method according to any one of Items 22 to 33, comprising: (Item 34) (i) administering a first dose of the immunogenic composition; (ii) administering a second dose of the immunogenic composition about one month after the first dose; (iii) administering a third dose of the immunogenic composition about 14 months after the first dose; The method according to any one of Items 22 to 33, comprising: (Item 35) 35. The method of claim 33 or 34, wherein the immunogenic composition comprises about 50 μg of DBP. (Item 36) 36. The method of any one of items 33 to 35, wherein the immunogenic composition comprises about 50 μg of a saponin adjuvant. (Item 37) The method may be performed for up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, up to about 12 months, up to 13 months, up to 14 months, up to 15 months, up to 16 months, up to 17 months, up to 18 months, up to 19 months, up to 20 months, up to 21 months after administration of the immunogenic composition. For 1 month, for up to 22 months, for up to 23 months, or for up to 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81%, about 60% to about 95%, about 60% to about 90%, about 37. The method according to any one of items 1 to 36, wherein the method prevents malaria with an efficacy of 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%. (Item 38) The method may include administering the immunogenic composition for at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, at least about 6 months, at least about 6.5 months, at least about 7 months, at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36 months, at least about 37 months, at least about 38 months, at least about 39 months, at least about 40 months, at least about 41 months, at least about 42 months, at least about 43 months, at least about 44 months, at least about 45 months, at least about 46 months, at least about 47 months, at least about 48 months, at least about 49 months, at least about 50 months, at least about 51 months, at least about 52 months, at least about 53 months, at least about 54 or for at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 60% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 69% to about 81 ...9%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65% to about 99%, about 65 37. The method according to any one of items 1 to 36, wherein the method prevents malaria with an efficacy of about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, or about 40% to about 50%.

Claims

[Claim 1] The invention described in the specification.

Images