Malaria vaccine preparations

Abstract

Disclosed herein is an immunogenic composition comprising an antigen of a parasite of the genus Plasmodium. Also disclosed is a method of administering said composition. In an embodiment, provided herein is a method of stimulating an immune response against a parasite of the genus Plasmodium in a subject, comprising administering an immunogenic composition. In an embodiment, the parasite of the genus Plasmodium is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the following applications, each of which is incorporated by reference in its entirety for all purposes: U.S. Provisional Patent Application No. 63 / 271,420, filed October 25, 2021, and U.S. Provisional Patent Application No. 63 / 281,332, filed November 19, 2021.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NOVV_094_02WO_SeqList_ST26.xml; size: (10,386 bytes; and creation date: October 25, 2022) are incorporated by reference herein in their entirety.

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

[0004] 2. Background of the Invention Malaria is a leading cause of childhood death. As of 2019, there were an estimated 229 million cases of malaria worldwide. In 2019, 409,000 people died from malaria. Only one vaccine is approved to prevent malaria (RTS,S / AS01 (RTS,S) (MOSQUIRIX®)). The protective ability of RTS,S against malaria weakens over time. There is a need in the art for novel vaccines and methods to prevent malaria. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present disclosure provides immunogenic compositions for inducing an immune response against malaria. The present disclosure also provides novel methods of administering said immunogenic compositions.

[0006] 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 comprising administering the immunogenic composition. In embodiments, the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. In embodiments, the antigen comprises a circumsporozoite (CS) protein or a fragment thereof. In embodiments, the CS protein is from Plasmodium falciparum. In embodiments, the antigen comprises a Hepatitis B surface antigen (HBsAg) or a fragment thereof. In embodiments, the antigen comprises an amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1. In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the adjuvant comprises at least two 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 account for about 85% by weight and about 15% by weight, respectively, based on the total weight of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.In an embodiment, the weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C is about 92% by weight and about 8% by weight, respectively, based on the total weight of Quillaja Saponaria Molina fraction A and Quillaja Saponaria Molina fraction C in the adjuvant. In an embodiment, the adjuvant is administered at a dose of about 50 μg.

[0007] In an embodiment, the method of stimulating an immune response against a Plasmodium parasite comprises administering from about 0.1 μg to about 100 μg of antigen. In an embodiment, the method of stimulating an immune response against a Plasmodium parasite comprises administering from about 0.1 μg to about 10 μg of antigen. In an embodiment, the method of stimulating an immune response against a Plasmodium parasite comprises administering from about 0.1 μg to about 5 μg of antigen. In an embodiment, the method of stimulating an immune response against a Plasmodium parasite comprises administering from about 0.1 μg to about 3 μg of antigen. In an embodiment, the method of stimulating an immune response against a Plasmodium parasite comprises administering from about 0.1 μg to about 2 μg of antigen. In an embodiment, the method comprises administering a first dose and a second dose of the immunogenic composition. In an embodiment, the method comprises administering a third dose of the immunogenic composition. In an embodiment, the dose of antigen in the second dose is less than the dose of antigen in the first dose. In an embodiment, the dose of antigen in the third dose is less than the dose of antigen in the first dose. In an embodiment, the first and second doses contain the same amount of antigen. In an embodiment, the second and third doses contain the same amount of antigen. In an embodiment, the first and third doses contain the same amount of antigen. In an embodiment, 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 an embodiment, 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. In an embodiment, the second dose is administered about 28 days after the first dose. In an embodiment, the third dose is administered about 56 days after the first dose. In an embodiment, the third dose is administered about 168 days after the first dose.

[0008] In embodiments, provided herein is a method of stimulating an immune response to a parasite of the genus Plasmodium in a subject, comprising administering: (i) an immunogenic composition comprising a first dose of an antigen of a parasite of the genus Plasmodium; said first dose comprising about 10 μg of said antigen; (ii) a second dose of the immunogenic composition, the second dose comprising about 10 μg of the antigen; and (iii) a third dose of the immunogenic composition, wherein the third dose comprises about 2 μg of the antigen.

[0009] In an embodiment, the second dose is administered about 28 days after the first dose. In an embodiment, the third dose is administered about 56 days after the first dose. In an embodiment, the third dose is administered about 168 days after the first dose. In an embodiment, the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. In an embodiment, the antigen comprises a circumsporozoite (CS) protein or a fragment thereof. In an embodiment, the CS protein is derived from Plasmodium falciparum. In an embodiment, the antigen comprises a Hepatitis B surface antigen (HBsAg) or a fragment thereof. In an embodiment, the antigen comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the adjuvant comprises at least two iscom particles, in which: 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 account for about 85% by weight and about 15% by weight, respectively, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant.In an embodiment, the fraction A of Quillaja Saponaria Molina and the fraction C of Quillaja Saponaria Molina account for about 92% by weight and about 8% by weight, respectively, based on the total weight of the fraction A of Quillaja Saponaria Molina and the fraction C of Quillaja Saponaria Molina in the adjuvant. In an embodiment, the adjuvant is administered at a dose of about 50 μg.

[0010] In an embodiment, provided herein is a method of stimulating an immune response against a Plasmodium parasite in a subject, comprising administering an immunogenic composition comprising an antigen of a Plasmodium parasite, wherein the antigen comprises a circumsporozoite (CS) protein or a fragment thereof and a hepatitis B surface antigen (HBsAg) or a fragment thereof. In an embodiment, the antigen of a Plasmodium parasite comprises an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence 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 at least 99.5% identity to SEQ ID NO:1 or SEQ ID NO:2. In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the immunogenic composition comprises from about 1 μg to about 100 μg of an adjuvant. In an embodiment, the immunogenic composition comprises about 25 μg to about 75 μg of adjuvant. In an embodiment, the immunogenic composition comprises about 50 μg of adjuvant. In an embodiment, the immunogenic composition comprises about 25 μg of adjuvant. In an embodiment, the adjuvant comprises at least two 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 accounts for 50-96% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder, based on the total weight of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.In an embodiment, the weight of the fraction A of Quillaja Saponaria Molina and the weight of the fraction C of Quillaja Saponaria Molina in the adjuvant is at least 75% by weight, and the weight of the fraction C of Quillaja Saponaria Molina is the remainder. In an embodiment, the weight of the fraction A of Quillaja Saponaria Molina and the weight of the fraction C of Quillaja Saponaria Molina in the adjuvant is about 85% by weight and about 15% by weight, respectively. In an embodiment, the weight of the fraction A of Quillaja Saponaria Molina and the weight of the fraction C of Quillaja Saponaria Molina in the adjuvant is about 92% by weight and about 8% by weight, respectively. In an embodiment, the method comprises administering the immunogenic composition in a pre-filled syringe. In an embodiment, the dose of the administered antigen is about 0.1 μg to about 100 μg; about 0.1 μg to about 10 μg; about 0.1 μg to about 5 μg; about 0.1 μg to about 3 μg; or about 0.1 μg to about 2 μg; about 2 μg to about 10 μg; or about 2 μg to about 5 μg. In an embodiment, the immunogenic composition comprises about 2 μg of antigen. In an embodiment, the immunogenic composition comprises about 5 μg of antigen. In an embodiment, the immunogenic composition comprises about 10 μg of antigen. In an embodiment, the immunogenic composition comprises about 50 μg of antigen. In an embodiment, the method comprises administering a first dose and a second dose of the immunogenic composition. In an embodiment, the method comprises administering a third dose of the immunogenic composition. In an embodiment, the method includes administering a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, or a tenth dose of an immunogenic composition. In an embodiment, the amount of antigen in the second dose is less than the amount of antigen in the first dose.In an embodiment, the amount of antigen in the third dose is less than the amount of antigen in the first dose. In an embodiment, the first dose and the second dose contain about the same amount of antigen. In an embodiment, the second dose and the third dose contain about the same amount of antigen. In an embodiment, the first dose and the third dose contain about the same amount of antigen. In an embodiment, the first dose, the second dose, and the third dose contain about the same amount of antigen. In an embodiment, the first dose, the second dose, the third dose, and the fourth dose contain about the same amount of antigen. In an embodiment, the third dose or the fourth dose or the fifth dose or the sixth dose or the seventh dose or the eighth dose or the ninth dose or the tenth 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 an embodiment, 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 antigen in the first dose. In an embodiment, the method comprises administering the second dose about 1 month after the first dose. In an embodiment, the method comprises administering the second dose about 28 days after the first dose. In an embodiment, the method comprises administering the third dose about 56 days after the first dose. In an embodiment, the method comprises administering the third dose about 2 months after the first dose. In an embodiment, the method comprises administering the third dose about 168 days after the first dose. In an embodiment, the method comprises administering the third dose about 6 months after the first dose. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition 12 months to about 16 months after administration of the first dose. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 15 months after administration of the first dose. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 421 days after administration of the first dose. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 1 year after administration of the third dose.In an embodiment, the method comprises administering (i) a first dose of an immunogenic composition, the first dose comprising about 10 μg of antigen; (ii) a second dose of an immunogenic composition, the second dose comprising about 10 μg of antigen; and (iii) a third dose of an immunogenic composition, the third dose comprising about 2 μg of antigen. In an embodiment, the method comprises administering (i) a first dose of an immunogenic composition, the first dose comprising about 50 μg of antigen; (ii) a second dose of an immunogenic composition, the second dose comprising about 50 μg of antigen; and (iii) a third dose of an immunogenic composition, the third dose comprising about 10 μg of antigen. In an embodiment, the method includes administering (i) a first dose of the immunogenic composition, the first dose comprising about 10 μg of antigen; (ii) a second dose of the immunogenic composition, the second dose comprising about 10 μg of antigen; and (iii) a third dose of the immunogenic composition, the third dose comprising about 10 μg of antigen. In an embodiment, the method includes administering (i) a first dose of the immunogenic composition, the first dose comprising about 5 μg of antigen; (ii) a second dose of the immunogenic composition, the second dose comprising about 5 μg of antigen; and (iii) a third dose of the immunogenic composition, the third dose comprising about 5 μg of antigen. In an embodiment, the method includes administering a fourth dose, a fifth dose, or both doses of the immunogenic composition. In an embodiment, the method comprises administering a second dose about one month after the first dose and a third dose about two months after the second dose. In an embodiment, the method comprises administering a second dose about one month after the first dose and a third dose about six months after the second dose. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about nine months to about two years after administration of the first dose of the immunogenic composition.In an embodiment, the method comprises administering a fourth dose of the immunogenic composition 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 after administration of the first dose of the immunogenic composition. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 10 months after administration of the first dose of the immunogenic composition. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 12 months after administration of the first dose of the immunogenic composition. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 14 months after administration of the first dose of the immunogenic composition. In an embodiment, the method comprises administering a fourth dose of the immunogenic composition about 15 months after administration of the first dose of the immunogenic composition. In an embodiment, the method comprises administering a fifth dose of the immunogenic composition 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 after administration of the fourth dose of the immunogenic composition. In an embodiment, the parasite of the Plasmodium genus is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. In an embodiment, the CS protein is derived from Plasmodium falciparum.In embodiments, the method comprises administering an immunogenic composition to a subject comprising: 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 at 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 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, up to 22 months, up to 23 months, or up to 24 months malaria. In embodiments, the method prevents infection by a parasite of the genus Plasmodium. In embodiments, the method provides for an immunogenic composition to provide an immunogenic effect of 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%. and 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% efficacy for at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 15 months, at least about 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 ... or for 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 22 months, at least 23 months, or at least 24 months. In embodiments, the method prevents malaria with about 69% effectiveness for up to about 12 months. In embodiments, the method prevents malaria with about 69% effectiveness for at least about 12 months. In embodiments, the method prevents malaria with about 77% effectiveness for up to about 12 months. In embodiments, the method prevents malaria with about 77% effectiveness for at least about 12 months. In embodiments, the method prevents malaria with about 77% effectiveness for up to about 24 months.In embodiments, the method prevents malaria with about 77% effectiveness for at least about 24 months. In embodiments, the method prevents malaria with about 81% effectiveness for up to about 12 months. In embodiments, the method prevents malaria with about 81% effectiveness for at least about 12 months. In embodiments, the method prevents malaria with about 78% effectiveness for up to about 12 months. In embodiments, the method prevents malaria with about 78% effectiveness for at least about 12 months. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows an image of particles containing the R21 fusion protein.

[0012] [Diagram 2] Figure 2 is a graph showing the percentage of subjects over time who are not diagnosed with malaria after being intentionally infected with human malaria (CHMI) and receiving the immunogenic composition of the present disclosure. Group 2 received an immunogenic composition comprising 10 μg of R21 protein and 50 μg of saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 6 months. Group 3 received an immunogenic composition comprising 10 μg of R21 protein and 50 μg of saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 2 months. Group 6 was a control group that did not receive the immunogenic composition. The efficacy of each group was calculated on day 21 using the following formula: 100 x (percentage of subjects diagnosed with malaria who did not receive the immunogenic composition - percentage of subjects diagnosed with malaria who received the immunogenic composition) / (percentage of subjects diagnosed with malaria who did not receive the immunogenic composition). The efficacy of group 2 was 75%. The efficacy of group 3 was 62.5%. The percentage of controls diagnosed with malaria was 100%.

[0013] [Diagram 3] Figure 3 is a graph showing the percentage of subjects free of a diagnosis of malaria over time after the subjects were intentionally infected with human malaria (CHMI). Group 2 received 10 μg of R21 protein and 50 μg of saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, 6, and 14 months. Group 3 received 10 μg of R21 protein and 50 μg of saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, 2, and 10 months. Group 4 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 6 months. Group 7 was the control group that did not receive vaccination. Efficacy was calculated at day 14 as in Figure 2. Group 2 showed 60% efficacy. Group 3 showed 100% efficacy. Group 4 showed 40% efficacy. The percentage of controls diagnosed with malaria was 83.3%.

[0014] [Figure 4] Figure 4 shows malaria protective immunoglobulin levels (reflected by ELISA units) in UK and Kenyan adults. UK and Kenyan adults were administered 10 μg R21 protein and 50 μg saponin adjuvant (i.e. saponin adjuvant containing 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix).

[0015] [Diagram 5]Figure 5 shows malaria protective immunoglobulin levels (reflected by ELISA units) in adults, infants, and young children. Adults, infants, and young children were administered 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant containing 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix).

[0016] [Figure 6A] FIG. 6A shows the dosing regimen of R21 protein and saponin adjuvant ("MM") evaluated in Example 1.

[0017] [Figure 6B] Figure 6B shows a graph of the incidence of malaria in patients administered an immunogenic composition comprising 5 μg R21 protein and 25 μg saponin adjuvant (Group 1), an immunogenic composition comprising 5 μg R21 protein and 50 μg saponin adjuvant (Group 2), or a control rabies vaccine (i.e., RABIVAX-S). The immunogenic composition or control rabies vaccine was administered on days 0, 28, and 56. The saponin adjuvant comprised 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix.

[0018] [Figure 7A]FIG. 7A is a graph showing anti-NANP antibody titers over time in patient serum following administration of one of three immunogenic compositions: (i) an immunogenic composition comprising 5 μg R21 protein and 25 μg saponin adjuvant (Group 1); (ii) an immunogenic composition comprising 5 μg R21 protein and 50 μg saponin adjuvant (Group 2); (iii) or a control rabies vaccine (i.e., RABIVAX-S). The saponin adjuvant comprises 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. The immunogenic composition or the control rabies vaccine was administered on days 0 ("1st dose"), 28 ("2nd dose"), 56 ("3rd dose"), and 421 ("4th dose"). Patients in group 2 had higher anti-NANP antibody titers after three doses of vaccine than patients in group 1 (p<0.0001), and patients in both groups 1 and 2 had increased anti-NANP antibody titers after the boost dose.

[0019] [Figure 7B] Figure 7B is a graph showing anti-NANP antibody titers over time in patient serum following administration of one of the three immunogenic compositions of Figure 7A on days 0 ("1st dose"), 28 ("2nd dose"), 56 ("3rd dose"), 421 ("Boost 1"), and 786 ("Boost 2").

[0020] [Figure 7C] FIG. 7C is a graph showing titers of IgG specific to the C-terminus of the CS protein over time in patient sera over time following administration of one of the three immunogenic compositions of FIG. 7A on days 0 ("1st dose"), 28 ("2nd dose"), 56 ("3rd dose"), 421 ("Boost 1"), and 786 ("Boost 2").

[0021] [Figure 7D]Figure 7D is a graph showing the avidity of CS protein to anti-NANP antibodies from patient sera. Patients were administered an immunogenic composition comprising 5 μg of R21 protein and 25 μg of saponin adjuvant (Group 1) or an immunogenic composition comprising 5 μg of R21 protein and 50 μg of saponin adjuvant (Group 2). The immunogenic composition was administered on days 0, 28, 56, 421, and 786. Avidity was measured 28 days after administration of the dose on day 56 ("D84"), 30 days after administration of the boost dose on day 421 ("1B+30"), and 30 days after administration of the boost dose on day 786 ("2B+30"). Avidity was also measured on day 816 in patients who received the immunogenic composition on days 0, 28, and 56, but not on days 421 or 786 ("no boost").

[0022] [Figure 7E] Figure 7E is a graph showing the avidity of CS protein to IgG specific for the C-terminus of CS protein from patient serum. Patients were administered an immunogenic composition comprising 5 μg of R21 protein and 25 μg of saponin adjuvant (Group 1) or an immunogenic composition comprising 5 μg of R21 protein and 50 μg of saponin adjuvant (Group 2). The immunogenic composition was administered on days 0, 28, 56, 421, and 786. Avidity was measured 28 days after administration of the dose on day 56 ("D84"), 30 days after administration of the boost dose on day 421 ("1B+30"), and 30 days after administration of the boost dose on day 786 ("2B+30"). Avidity was also measured on day 816 in patients who received the immunogenic composition on days 0, 28, and 56, but not on days 421 or 786 ("no boost").

[0023] [Figure 8] FIG. 8 is a schematic representation of the primary structure of the R21 protein used in the immunogenic compositions described herein.

[0024] [Figure 9A]Figures 9A and 9B show the antibody responses induced over time in patients receiving different dosing regimens of R21 and saponin adjuvant. Figure 9A shows the antibody responses of groups 1 and 3 (see Table 2 for regimen description). Figure 9B shows the antibody responses of groups 2, 4, and 5 (see Table 2 for regimen description). The saponin adjuvant contains 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. [Figure 9B] Figures 9A and 9B show the antibody responses induced over time in patients receiving different dosing regimens of R21 and saponin adjuvant. Figure 9A shows the antibody responses of groups 1 and 3 (see Table 2 for regimen description). Figure 9B shows the antibody responses of groups 2, 4, and 5 (see Table 2 for regimen description). The saponin adjuvant contains 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix.

[0025] [Figure 10A] FIG. 10A shows the antibody responses (anti-NANP-specific IgG) induced by administration of the different dosing regimens of Table 2. [Figure 10B] FIG. 10B shows the antibody responses (IgG specific to the C-terminus of the CS protein of the R21 protein) induced by administration of the different dosing regimens of Table 2. [Figure 10C] FIG. 10C shows the avidity of the anti-NANP-specific IgG of FIG. 10A for the CS protein. [Figure 10D] FIG. 10D shows the avidity of IgG specific to the C-terminus of the CS protein of FIG. 10B for the CS protein.

[0026] [Figure 11A]Figure 11A shows the correlation of antibody titers from patient sera with protection from malaria. Patients were administered Group 2 or Group 3 dosing regimens and stratified by protection and no protection from malaria. The dosing regimens for Groups 2 and 3 are shown in Table 2. [Figure 11B] 11B shows the correlation of protection in patients in groups 2 and 3 with antibody responses above or below EU1100. The dosing regimens for groups 2 and 3 are shown in Table 2.

[0027] [Figure 12] FIG. 12 shows the dosing regimen of R21 protein and saponin adjuvant ("MM") evaluated in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" can refer to one protein or a mixture of such proteins, and a reference to "the method" includes reference to equivalent steps and / or methods known to those of skill in the art and / or the like.

[0029] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced 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.

[0030] As used herein, the terms "about" or "approximately," when placed before a numerical value, indicate a value within a range of plus or minus 10%. For example, "about 100" includes 90 and 110.

[0031] 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.

[0032] As used herein, an "immunogenic composition" is a composition that contains an antigen that generates a humoral and / or cellular immune response in a subject against the antigen when the composition is administered to a subject.

[0033] As used herein, a "subunit" composition (e.g., a vaccine) comprises one or more selected antigens, but not all antigens from a pathogen. Such compositions are substantially free of intact virus or lysates of such cells or particles, and are typically prepared from at least partially purified, and often substantially purified, immunogenic polypeptides from a pathogen. Antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0034] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; ameliorating or alleviating the onset, symptoms of an infection or disease; or a combination thereof.

[0035] "Prevention," as used herein, is used interchangeably with "prophylaxis" and can mean the complete prevention of an infection or disease, or the prevention of the onset of symptoms of that infection or disease; the delay in the onset of an infection or disease or symptoms thereof; or the reduction in the severity of an infection or disease or symptoms thereof that has subsequently developed.

[0036] As used herein, an "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that alleviates 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 (e.g., plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays).

[0037] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against the pathogen. The immune response may include the formation of antibodies and / or a cellular response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen administered to a subject to obtain an immune response. Preferably, the vaccine induces an immune response effective in preventing infection from a parasite of the genus Plasmodium.

[0038] 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 (birth to 2 years old), or a newborn (up to 2 months old). In certain embodiments, the subject is up to 4 months old or up to 6 months old. In some embodiments, the adult is about 65 years old or older, or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who is hoping to become pregnant. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject can be a pet animal (such as a dog or cat).

[0039] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the U.S. Federal or state government for use in mammals, more specifically humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0040] As used herein, the terms "co-formulation mix", "co-formulation", "co-formulated vaccine composition", "pre-filled syringe", "premix" refer to a vaccine formulation that is prepared for short- to long-term storage prior to the time of administration to a subject. Such a vaccine formulation includes a combination of antigen and adjuvant in the same container and is prepared prior to administration. In an embodiment, the pre-filled syringe contains a vaccine formulation that includes the R21 antigen and an adjuvant (e.g., a saponin adjuvant such as a matrix adjuvant).

[0041] 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 certain percentage of identical nucleotides or amino acid residues when compared. Percentage identity can be calculated using the online available tool CLUSTALW2. The following parameters can be used for CLUSTALW2 pairwise alignment: protein weight matrix=Gonnet; gap open=10; gap extension=0.1.

[0042] The patients described herein may be diagnosed with primary, secondary, or tertiary clinical malaria. Patients diagnosed with primary clinical malaria exhibit: (a) an axillary temperature of 37.5°C or higher and / or a history of fever within the last 24 hours, and (b) asexual Plasmodium falciparum (P. falciparum) parasitemia with a parasite count of more than 5000 / μL. Patients diagnosed with secondary clinical malaria exhibit: (a) an axillary temperature of 37.5°C or higher and / or a history of fever within the last 24 hours, and (b) asexual P. falciparum parasitemia with a parasite count of more than 0 / μL or a parasite count of more than 2500 / μL. Patients diagnosed with tertiary clinical malaria are those who show either (i): (a) an axillary temperature of 37.5°C or higher and / or a history of fever within the last 24 hours and (b) P. falciparum asexual parasitemia with a parasite count of more than 500 / μL; or (ii) (a) an axillary temperature of 37.5°C or higher and / or a history of fever within the last 24 hours and (b) P. falciparum asexual parasitemia with a parasite count of more than 20,000 / μL or a positive rapid detection test for malaria antigen.

[0043] The term "efficacy" of the immunogenic compositions described herein (also referred to herein as "vaccine efficacy") refers to the percentage reduction in disease (e.g., malaria) in a group administered the immunogenic composition compared to a group not administered the immunogenic composition. In embodiments, efficacy (E) is calculated using the following formula: 100 x (percentage of subjects not administered the immunogenic composition diagnosed with disease - percentage of subjects diagnosed with malaria administered the immunogenic composition) / (percentage of subjects not administered the immunogenic composition diagnosed with disease). Immunogenic compositions comprising circumsporozoite (CS) protein, hepatitis B surface antigen, saponin adjuvant, and combinations thereof - Patent Application 20090233633

[0044] Disclosed herein is an immunogenic composition comprising an antigen of a parasite of the genus Plasmodium. In an embodiment, the parasite of the genus Plasmodium is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. In an embodiment, the antigen comprises a circumsporozoite (CS) protein or a fragment thereof.

[0045] The CS protein is composed of an N-terminus that binds to heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C-terminus that includes a thrombospondin-like type I repeat (TSR) domain. In embodiments, the antigen comprises the N-terminus of the CS protein, the NANP, the TSR domain, or a combination thereof. In embodiments, the antigen comprises the C-terminus of the TSR or a fragment thereof and the NANP or a fragment thereof (e.g., FIG. 8).

[0046] In an embodiment, the antigen comprises Hepatitis B surface antigen (HBsAg) or a fragment thereof. In an embodiment, the HBsAg fragment is about 226 amino acids. In an embodiment, the HBsAg fragment comprises from about 180 amino acids to about 220 amino acids.

[0047] In an embodiment, the antigen is a fusion protein comprising a CS protein or a fragment thereof and HBsAg or a fragment thereof. In an embodiment, the antigen has the amino acid sequence of SEQ ID NO:1: [ka]

[0048] The antigen having the amino acid sequence of SEQ ID NO:1 comprises the C-terminus of the CS protein (underlined, SEQ ID NO:8), the NANP repeat region (bold, SEQ ID NO:7), and the HBsAg antigen (italics, SEQ ID NO:9). In embodiments, the antigen comprises a NANP repeat region 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:7. In embodiments, the antigen comprises a C-terminus of the CS protein 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:8. In embodiments, the antigen comprises an HBsAg 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:9.

[0049] In an embodiment, the antigen comprises the polypeptide of SEQ ID NO:1, or a fragment thereof.

[0050] In embodiments, the amino acid sequence of the antigen has 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 protein having the amino acid sequence of SEQ ID NO: 1. In embodiments, the protein having the amino acid sequence of SEQ ID NO: 1 is referred to herein as "R21". Alternative fusion proteins that may be utilized in the immunogenic compositions herein are described in U.S. Patent Application Publication No. 2020 / 0,207,811, which is incorporated herein by reference in its entirety for all purposes.

[0051] In embodiments, a "fragment portion thereof" of a protein is between 10 and 1500 amino acids in length (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 amino acids in length). In embodiments, the fragment comprises at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the amino acids of the wild-type version of the protein.

[0052] In embodiments, the antigen has the amino acid sequence of SEQ ID NO:2: MDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVPVTNMENITSGFLGPLLVLQAGFFLLT RILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIEPEA

[0053] In embodiments, the amino acid sequence of the antigen has 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 protein having the amino acid sequence of SEQ ID NO:2.

[0054] In an embodiment, the nucleic acid encoding an antigen of a parasite of the genus Plasmodium is extended at the 5' end, the 3' end, or both. In an embodiment, the extension encodes a protein tag used for purification or detection. In an embodiment, the protein tag is a polyglutamic acid tag, a FLAG-tag, an HA-tag, a polyHis-tag (having about 5-10 histidines) (SEQ ID NO: 3), a hexahistidine tag (SEQ ID NO: 4), an 8X-His-tag (having 8 histidines) (SEQ ID NO: 5), 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 an embodiment, the extension comprises a C-tag. The C-tag comprises the sequence EPEA (SEQ ID NO: 6).

[0055] In an embodiment, the nucleic acid encoding the antigen is cloned into a Hansenula polymorpha (H. polymorpha) expression plasmid. In an embodiment, the H. polymorpha plasmid is pFPMT121. In an embodiment, the pFPMT121 plasmid carries the LEU2 gene for selection in yeast. In an embodiment, the auxotrophic H. polymorpha yeast strain ALU3 (relevant genotypes: ade1, leu2, ura3) is used as the expression host. In an embodiment, yeast cells carrying the plasmid containing the antigen are produced by electroporation. In an embodiment, gene expression of the antigen is induced by introduction of methanol (e.g., 1% v / v) into the yeast cells. In an embodiment, the antigen is isolated by ultracentrifugation.

[0056] In an embodiment, the antigen is expressed in Pichia pastoris cells. In an embodiment, after expression in P. pastoris cells, the yeast cell membrane is lysed. In an embodiment, the cells are lysed with a lysis reagent selected from Tris buffer (e.g., 10 mM, pH 7.8), Triton® X-100 (e.g., 0.1%), EDTA (e.g., 1 mM), benzonase (e.g., 250 U / mL), or a combination thereof. In an embodiment, the mixture containing the lysis reagent and yeast cells is vortexed. In an embodiment, the cell debris is removed from the lysed cells by centrifugation. In an embodiment, the yeast lysate is layered on a discontinuous CsCl gradient and ultracentrifuged. In an embodiment, the antigen is purified using. In an embodiment, the antigen is eluted with Tris buffer (e.g., 10 mM). In an embodiment, the antigen is desalted.

[0057] In an embodiment, the antigen is purified by affinity chromatography. In an embodiment, the purification involves exposing an antigen containing a C-tag (e.g., an antigen of SEQ ID NO: 2) to a C-tag affinity chromatography column; 2 (wherein the buffer is at about pH 7) to remove impurities; 20 mM Tris and 2 M MgCl 2 (wherein the buffer is at about pH 7).

[0058] The following publications describe methods of expressing and purifying the disclosed antigens: Kurtovic et al. Front Immunol. 2021;12:641421; Degelmann et al. FEMS Yeast Rest (2002)2(3):349-361; Lahtchev et al. Arch Microbiol (2002)177(2):150-158; Faber et al. Curr Genet (1994)25(4):305-310; Guengerich et al. Genet Biotechnol: Springer (2004)2:273-87; Mukhopadhyay et al. Biotechnol Bioeng. (2022)119(10):2784-2793; Datoo et al. Lancet. (2021);397:1809-1818; and Datoo et al. al. Lancet Infect Dis (September 7, 2022), doi.org / 10.1016 / S1473-3099(22)00442-X. Each of the foregoing references is incorporated by reference herein in its entirety for all purposes.

[0059] In embodiments, the antigen is produced in a yeast, hi embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha (also referred to as "Ogataea polymorpha"), Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipites, Yarrowia lipolytica, Ogataea minuta, Candida boidinii, and Kluyveromyces lactis.

[0060] Adjuvants In embodiments, the immunogenic composition comprises an adjuvant.

[0061] In certain embodiments, the compositions disclosed herein may be combined with one or more adjuvants to enhance immune response.In other embodiments, the compositions are prepared without the use of adjuvants and are therefore available for administration as adjuvant-free compositions.Advantageously, the adjuvant-free compositions disclosed herein may 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 and above.

[0062] Aluminum-based adjuvants In embodiments, the adjuvant is alum (e.g., AlPO 4 or Al(OH) 3 ). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may be at least 80% bound to the alum, at least 85% bound, at least 90% bound, or at least 95% bound. Often, the nanoparticles are 92%-97% bound to the alum in the composition. The amount of alum is typically present in a range between 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 for a 120 μg dose of protein nanoparticles.

[0063] In an embodiment, the adjuvant is a saponin adjuvant.

[0064] Saponin adjuvants Also, adjuvants containing saponin can be combined with the immunogens disclosed herein.Saponin is a glycoside derived from the bark of Quillaja saponaria Molina tree.Typically, saponin is prepared by obtaining multiple fractions using a multi-step purification process.As used herein, the term "saponin fraction from Quillaja saponaria Molina" is generally used to describe the semi-purified or limited saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0065] Saponin fraction Several approaches to generate saponin fractions are suitable. Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and may be prepared as follows: A fat-soluble fraction from Quil A (crude aqueous Quillaja saponaria Molina extract) is separated by chromatography and eluted with 70% aqueous acetonitrile to recover the fat-soluble fraction. This fat-soluble fraction is then separated by semi-preparative HPLC with elution using 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 purification of the fractions can be found in U.S. Pat. No. 5,057,540. When prepared as described herein, each of Quillaja saponaria Molina fractions A, B, and C represents a group or family of closely chemically related molecules with definable properties. The chromatographic conditions under which these fractions are obtained are such that there is a high degree of consistency in batch-to-batch reproducibility in terms of elution profile and biological activity.

[0066] Other saponin fractions have been described: fractions B3, B4, and B4b are described in EP 0436620; fractions QA1 to QA22 are described in EP 03632279B2, 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 EP03632279B2 may be used, in particular QA-7, QA-17, QA-18 and QA-21. These fractions are obtained as described in EP03632279B2, in particular in Example 1 on pages 6 and 8 and 9.

[0067] 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 embodiments, a substantially pure saponin fraction may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0068] ISCOM Structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (immunostimulating complexes). ISCOMs may be prepared as described in EP0109942B1, EP0242380B1, and EP0180546B1. In certain embodiments, transport and / or passenger antigens may be used as described in EP9600647-3 (PCT / SE97 / 00289).

[0069] Matrix adjuvants In an embodiment, 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 one sterol, such as cholesterol. In certain aspects, the ISCOM matrix complex also comprises a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant-active) substances that are not necessarily glycosides, and may be produced as described in EP0436620B1 (incorporated herein in its entirety by reference).

[0070] In another embodiment, the ISCOM is an ISCOM complex. The ISCOM complex comprises at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex comprises an antigen that is associated with the particle by detergent treatment, such that a portion of the antigen is incorporated into the particle. In contrast, the ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix particle and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0071] According to one embodiment, the ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one additional adjuvant similarly 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 purified subfraction (e.g., QA1-21).

[0072] In certain embodiments, 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 particles may each contain any weight percent of fraction A and any weight percent of another saponin fraction (such as a crude saponin fraction or fraction C). Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may 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 one saponin fraction (e.g., fraction A), and the remainder being each another saponin (e.g., any coarse fraction or any other faction (e.g., fraction C)) up to 100%. Weights are calculated as the total weight of saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in US Patent Application Publication No. 2013 / 0129770, which is incorporated by reference in its entirety.

[0073] In certain embodiments, an ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction (e.g., fraction A) and the remainder up to 100% by weight of another fraction (e.g., crude saponin fraction or fraction C). Weights are calculated as the total weight of the saponin fractions.

[0074] 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). Weights are calculated as the total weight of the saponin fractions.

[0075] 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). Weights are calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA1-21.

[0076] 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 may contain a plurality of particles, where each particle contains only one saponin fraction. That is, a particular composition may 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 the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[0077] In certain embodiments, 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.

[0078] A mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used in the composition, where one saponin fraction Quillaja saponaria Molina and another saponin fraction Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0079] ISCOM matrices or ISCOM complex particles each having one saponin fraction may be present in the composition in any combination of weight percents. In certain embodiments, the composition may comprise 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 an ISCOM matrix or complex comprising a first saponin fraction, with the remainder being made up of ISCOM matrices or complexes containing different saponin fractions. In some embodiments, the remainder is one or more ISCOM matrices or complexes, where each matrix or complex particle comprises only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles may comprise more than one saponin fraction.

[0080] In certain compositions, the only saponin fraction in a first ISCOM matrix or ISCOM complex particle is fraction A and the only saponin fraction in a second ISCOM matrix or ISCOM complex particle is fraction C.

[0081] A preferred composition comprises a first ISCOM matrix comprising fraction A and a second ISCOM matrix comprising 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 is ​​present in the range of about 70% to about 85% and the fraction C ISCOM matrix is ​​present in the range of about 15% to about 30% by weight of the total saponin adjuvant in the composition. In an embodiment, the fraction A ISCOM matrix comprises 50% to 96% by weight of the combined weight of the fraction A ISCOM matrix and the fraction C ISCOM matrix in the adjuvant, respectively, and the fraction C ISCOM matrix comprises the remainder. In an embodiment, the Fraction A ISCOM matrix comprises at least 75% by weight, and the Fraction C ISCOM matrix comprises the remainder, based on the combined weight of the Fraction A ISCOM matrix and Fraction C ISCOM in the adjuvant. In an embodiment, the Fraction A ISCOM matrix is ​​present at about 92% and the Fraction C ISCOM matrix is ​​present at about 8% based on the total weight of the saponin adjuvant in the composition. In a particularly preferred composition, referred to herein as MATRIX-M™, the Fraction A ISCOM matrix is ​​present at about 85% and the Fraction C ISCOM matrix is ​​present at about 15% based on the total weight of the saponin adjuvant in the composition. MATRIX-M™ may be interchangeably referred to as Matrix-M1.

[0082] Exemplary QS-7 and QS-21 fractions, their production and 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, herein incorporated by reference.

[0083] Other adjuvants may additionally or alternatively be used in some compositions. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" (herein incorporated 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 that contains killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in 2% squalene / TWEEN® polysorbate 80 emulsion. In an embodiment, the adjuvant can be paucilamellar lipid vesicles; for example, NOVASOMES®. NOVASOMES® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. These include BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928).

[0084] Excipients In embodiments, the immunogenic compositions described herein include various excipients and 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, sodium chloride may be present at higher concentrations (e.g., from about 200 mM to about 500 mM). In embodiments, 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).

[0085] Administration and Dosage Provided herein is a novel method of administering the immunogenic composition. In an embodiment, the immunogenic composition induces an immune response against malaria in a subject in need thereof. In an embodiment, the immunogenic composition induces an immune response against a parasite selected from Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi.

[0086] 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, one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or ten 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.

[0087] In embodiments, the amount of antigen administered per dose ranges from about 0.1 μg to about 100 μg, including all ranges and subranges therebetween. For example, about 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, or about 10 μg per dose of the immunogenic composition. g, about 10μg, about 11μg, about 12μg, about 13μg, about 14μg, about 15μg, about 16μg, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 26μg, 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 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, 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 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 of antigen may be administered. In embodiments, the antigen is an antigen that has at least 70%, at least 80%, 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 R21 or the polypeptide of SEQ ID NO:1.In embodiments, the antigen has at least 70%, at least 80%, 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 the polypeptide of SEQ ID NO:2.

[0088] In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the amount of adjuvant in each dose of the immunogenic composition ranges from about 1 μg to about 100 μg. For example, in an embodiment, the amount of adjuvant in each dose of the immunogenic composition 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 40 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 50 μg, about Approximately 15μg, approximately 16μg, approximately 17μg, approximately 18μg, approximately 19μg, approximately 20μg, approximately 21μg, approximately 22μg, approximately 23μg, approximately 24μg, approximately 25μg, approximately 26μg, approximately 27μg, approximately 28μg, approximately 29 μg, approximately 30 μg, approximately 31 μg, approximately 32 μg, approximately 33 μg, approximately 34 μg, approximately 35 μg, approximately 36 μg, approximately 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 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 μg, The adjuvant is 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. In an embodiment, the adjuvant is a saponin adjuvant. In an embodiment, the saponin adjuvant comprises 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. In an embodiment, the saponin adjuvant comprises about 25 μg of 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix.In an embodiment, the saponin adjuvant comprises about 50 μg of 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix.

[0089] In embodiments, the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition is administered about 1 week, about 2 weeks, about 3 weeks, about 4 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, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 53 weeks, about 54 weeks, about 55 weeks, about 56 weeks, about 57 weeks, about 58 weeks, about 59 weeks, about 60 weeks, about 61 weeks, about 62 weeks, about 63 weeks, about 64 weeks, about 65 weeks, about 66 weeks, about 67 weeks, about 68 weeks, about 69 weeks, about 70 weeks, about 71 weeks, about 72 weeks, about 73 weeks, about 74 weeks, about 75 weeks, about 76 weeks, about 77 weeks, about 78 weeks, about 79 weeks, about 80 weeks, about 81 weeks Administration is performed 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, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, or about 52 weeks later. 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 after administration of the first dose. After, approximately 22 days later, approximately 23 days later, approximately 24 days later, approximately 25 days later, approximately 26 days later, approximately 27 days later, approximately 28 days later, approximately 29 days later, approximately 30 days later, approximately 31 days later, approximately 32 days later, approximately 33 days later. After, approximately 34 days later, approximately 35 days later, approximately 36 days later, approximately 37 days later, approximately 38 days later, approximately 39 days later, approximately 40 days later, approximately 41 days later, approximately 42 days later, approximately 43 days later, approximately 44 days later, approximately 45 days later , about 46 days later, about 47 days later, about 48 days later, about 49 days later, about 50 days later, about 51 days later, about 52 days later, about 53 days later, about 54 days later, about 55 days later, about 56 days later, about 57 days later , approximately 58 days later, approximately 59 days later, approximately 60 days later, approximately 61 days later, approximately 62 days later, approximately 63 days later, approximately 64 days later, approximately 65 days later, approximately 66 days later, approximately 67 days later, approximately 68 days later, approximately 69 days later , about 70 days later, about 71 days later, about 72 days later, about 73 days later, about 74 days later, about 75 days later, about 76 days later, about 77 days later, about 78 days later, about 79 days later, about 80 days later, about 81 days later , after about 82 days, after about 83 days, after about 84 days, after about 85 days, after about 86 days, after about 87 days, after about 88 days, after about 89 days, after about 90 days, after about 91 days, after about 92 days, after about 93 days,After about 94 days, after about 95 days, after about 96 days, after about 97 days, after about 98 days, after about 99 days, after about 100 days, after about 101 days, after about 102 days, after about 103 days, after about 104 days, after about 105 days, after about 106 days, after about 107 days, after about 108 days, after about 109 days, after about 110 days, after about 111 days, after about 112 days, after about 113 days, after about 114 days, after about 115 days, after about 116 days, after about 117 days, after about 118 days, after about 119 days, after about 120 days, after about 121 days, after about 122 days, after about 123 days, after about 114 days, after about 115 days, after about 116 days, after about 117 days, after about 118 days, after about 119 days, after about 120 days, after about 121 days, after about 122 days, after about 123 days, after about 124 days, after about 125 days, after about 126 days, after about 127 days, after about 128 days, after about 129 days, after about 130 days, after about 131 days, after about 132 days, after about 133 days, after about 134 days, after about 135 days, after about 136 days, after about 137 days, after about 138 days, after about 139 days, after about 140 days, after about 141 days, after about 142 days, after about 143 days, after about 144 days, after about 145 days, after about 146 days, after about 147 days, after about 148 days, after about 149 days, after about 150 days, after about 151 days, after about 152 days, after about 153 days, after about 154 days, after about 155 days, after about 156 days, after about 157 days, after about 158 days, after about 159 days, after about 160 days, after about 161 days, after about 162 days, after about 163 days, after about 164 days, after about 165 days, after about 166 days, after about 167 days, after about 168 days, after about 169 days, after about 170 days, after about 171 days, after about 172 days, after about 173 days, after about 174 days, after about 175 days, after about 176 days, after about 177 days, after about 178 days, after about 179 days, after about 180 days, after about 181 days, after about 182 days, after about 183 days, after about 184 days, after about 185 days, after about 186 days, after about 187 days, after about 188 days, after about 189 days, after about 190 days, after about 191 days, after about 192 days, after about 193 days, after about 194 days, after about 195 days, after about 196 days, after about 197 days, after about 198 days, after about 199 days, after about 200 days, after about 201 days, after about 202 days, after about 203 days, after about 204 days, after about 205 days, after about 206 days, after about 207 days, after about 208 days, after about 209 days, after about 210 days, after about 211 days, after about 212 days, after about 213 days, after about 214 days, after about 215 days, after about 216 days, after about 217 days, after about 218 days, after about 219 days, after about 220 days, after about 221 days, after about 222 days, after about 223 days, after about 224 days, after about 225 days, after about 226 daysApproximately 227 days later, approximately 228 days later, approximately 229 days later, approximately 230 days later, approximately 231 days later, approximately 232 days later, approximately 233 days later, approximately 234 days later, approximately 235 days later , approximately 236 days later, approximately 237 days later, approximately 238 days later, approximately 239 days later, approximately 240 days later, approximately 241 days later, approximately 242 days later, approximately 243 days later, approximately 244 days later , approximately 245 days later, approximately 246 days later, approximately 247 days later, approximately 248 days later, approximately 249 days later, approximately 250 days later, approximately 251 days later, approximately 252 days later, approximately 253 days later After, approximately 254 days, approximately 255 days, approximately 256 days, approximately 257 days, approximately 258 days, approximately 259 days, approximately 260 days, approximately 261 days, approximately 262 days. After, approximately 263 days later, approximately 264 days later, approximately 265 days later, approximately 266 days later, approximately 267 days later, approximately 268 days later, approximately 269 days later, approximately 270 days later, approximately 271 272 days later, 273 days later, 274 days later, 275 days later, 276 days later, 277 days later, 278 days later, 279 days later, 280 days later 281 days later, 282 days later, 283 days later, 284 days later, 285 days later, 286 days later, 287 days later, 288 days later, 28 days later 9 days later, approximately 290 days later, approximately 291 days later, approximately 292 days later, approximately 293 days later, approximately 294 days later, approximately 295 days later, approximately 296 days later, approximately 297 days later, approximately 29 8 days later, approximately 299 days later, approximately 300 days later, approximately 301 days later, approximately 302 days later, approximately 303 days later, approximately 304 days later, approximately 305 days later, approximately 306 days later, approximately 3 07 days later, approximately 308 days later, approximately 309 days later, approximately 310 days later, approximately 311 days later, approximately 312 days later, approximately 313 days later, approximately 314 days later, approximately 315 days later, approximately 3 16 days later, approximately 317 days later, approximately 318 days later, approximately 319 days later, approximately 320 days later, approximately 321 days later, approximately 322 days later, approximately 323 days later, approximately 324 days later, approximately 325 days later, approximately 326 days later, approximately 327 days later, approximately 328 days later, approximately 329 days later, approximately 330 days later, approximately 331 days later, approximately 332 days later, approximately 333 days later, approximately 334 days later, approximately 335 days later, approximately 336 days later, approximately 337 days later, approximately 338 days later, approximately 339 days later, approximately 340 days later, approximately 341 days later, approximately 342 days later, Approximately 343 days later, approximately 344 days later, approximately 345 days later, approximately 346 days later, approximately 347 days later, approximately 348 days later, approximately 349 days later, approximately 350 days later, approximately 351 days later, After about 352 days, after about 353 days, after about 354 days, after about 355 days, after about 356 days, after about 357 days, after about 358 days, after about 359 days, after about 360 days, after about 361 days, after about 362 days, after about 363 days, after about 364 days, after about 365 days, after about 13 months, after about 14 months, after about 15 months, after about 16 months,The second dose is administered about 28 days after administration of the first dose. In an embodiment, the third dose is administered about 56 days after administration of the first dose. In an embodiment, the third dose is administered about 168 days after administration of the first dose. In an embodiment, the third dose is administered about 168 days after administration of the first dose. In an embodiment, the third dose is administered about 168 days after administration of the first dose. In an embodiment, the second dose is administered about 28 days after administration of the first dose, and the third dose is administered about 56 days after administration of the first dose. In an embodiment, the second dose is administered about 28 days after administration of the first dose, and the third dose is administered about 168 days after administration of the first dose.

[0090] In an embodiment, the second dose of the immunogenic composition is administered about one month (e.g., 28 days, 29 days, 30 days, or 31 days) after the first dose of the immunogenic composition. In an embodiment, the third dose of the immunogenic composition is administered about 2 months to about 24 months, about 2 months to about 6 months, about 10 months to about 15 months, about 12 months to about 18 months, or about 12 months to about 16 months after the first dose of the immunogenic composition. In an embodiment, the fourth dose of the immunogenic composition is administered about 2 months to about 24 months, about 2 months to about 6 months, about 10 months to about 15 months, about 12 months to about 18 months, or about 12 months to about 16 months after the first dose of the immunogenic composition. In embodiments, the third dose of the immunogenic composition is administered at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 13 months, at least 14 months, or at least 15 months after administration of the first dose of the immunogenic composition. In embodiments, the third dose of the immunogenic composition is administered up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, up to 13 months, up to 14 months, or up to 15 months after administration of the first dose of the immunogenic composition. In embodiments, the fourth dose of the immunogenic composition is administered at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 13 months, at least 14 months, or at least 15 months after administration of the first dose of the immunogenic composition.In embodiments, the fourth dose of the immunogenic composition is administered up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, 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, up to 22 months, up to 23 months, or up to 24 months after administration of the first dose of the immunogenic composition.

[0091] In embodiments, when a dose is administered after the first dose, the subsequent dose may contain an amount of antigen less than the amount of antigen in the first dose. For example, if the first dose contains 5 μg of antigen, the third dose may contain 1 μg of antigen. In embodiments, the subsequent dose may contain about 1%, about 2%, about 3% less antigen than the first dose. About 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% lower. For example, if the first dose contains 5 μg of antigen, a subsequent dose that is 50% lower than the first dose contains 2.5 μg of antigen. In an embodiment, the third dose contains 1 / 5 of the antigen of the first dose. In an embodiment, the first dose contains 10 μg of antigen and the third dose contains 2 μg of antigen. In an embodiment, the first dose contains 50 μg of antigen and the third dose contains 10 μg of antigen.

[0092] In an embodiment, three doses (a first dose, a second dose, and a third dose) of the immunogenic composition described herein are administered. In an embodiment, the first dose comprises 10 μg of antigen, the second dose comprises 10 μg of antigen, and the third dose comprises 10 μg of antigen. In an embodiment, the first dose comprises 5 μg of antigen, the second dose comprises 5 μg of antigen, and the third dose comprises 5 μg of antigen. In an embodiment, the first dose comprises 50 μg of antigen, the second dose comprises 50 μg of antigen, and the third dose comprises 10 μg of antigen. In an embodiment, the first dose comprises 10 μg of antigen, the second dose comprises 10 μg of antigen, and the third dose comprises 2 μg of antigen.

[0093] In an embodiment, four doses (a first dose, a second dose, a third dose, and a fourth dose) of the immunogenic composition described herein are administered. In an embodiment, the first dose comprises 10 μg of antigen, the second dose comprises 10 μg of antigen, the third dose comprises 10 μg of antigen, and the fourth dose comprises 10 μg of antigen. In an embodiment, the first dose comprises 5 μg of antigen, the second dose comprises 5 μg of antigen, the third dose comprises 5 μg of antigen, and the fourth dose comprises 5 μg of antigen.

[0094] In an embodiment, five doses (first dose, second dose, third dose, fourth dose, and fifth dose) of the immunogenic composition described herein are administered. In an embodiment, the first dose comprises 10 μg of antigen, the second dose comprises 10 μg of antigen, the third dose comprises 10 μg of antigen, the fourth dose comprises 10 μg of antigen, and the fifth dose comprises 10 μg of antigen. In an embodiment, the first dose comprises 5 μg of antigen, the second dose comprises 5 μg of antigen, the third dose comprises 5 μg of antigen, the fourth dose comprises 5 μg of antigen, and the fifth dose comprises 5 μg of antigen.

[0095] In an embodiment, the method comprises administering an immunogenic composition according to the dosing regimen of Table A, where the amount refers to the dose of antigen and the number of days or months for the second, third, fourth, or fifth dose are the number of days or months after the first dose. [Table A-1] [Table A-2]

[0096] The compositions disclosed herein may be administered via systemic or mucosal or transdermal routes, or directly to specific tissues.As used herein, the term "systemic administration" includes parenteral administration routes.In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, or kidney dialysis infusion techniques.Typically, 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.

[0097] In an embodiment, 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 an embodiment, the dose is administered in a volume of 0.25 mL. In an embodiment, the dose is administered in a volume of 0.5 mL. In an embodiment, the dose is administered in a volume of 0.6 mL.

[0098] In embodiments, the immunogenic composition may contain an 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.

[0099] In an embodiment, the immunogenic composition described herein has efficacy in preventing malaria infection.Efficacy can be determined by comparing the number of subjects who are diagnosed with malaria and are not administered the malaria immunogenic composition (controls diagnosed with malaria) with the number of subjects who are subsequently diagnosed with malaria and are administered the malaria immunogenic composition.The following formula is used to calculate efficacy: 100 x (percentage of controls diagnosed with malaria-percentage of subjects who are administered the immunogenic composition and are diagnosed with malaria) / (percentage of controls diagnosed with malaria).

[0100] 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%.

[0101] In some embodiments, the present disclosure provides a co-formulation (i.e., pre-filled syringe or pre-mix) strategy for immunogenic compositions comprising antigens of Plasmodium genus parasites and adjuvants (e.g., saponin adjuvants). The typical vaccine administration strategy currently utilized is bedside mixed formulation. That is, the vaccine composition and 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 antigens (e.g., R21 antigens) and their subsequent immunogenic potential. The present disclosure provides immunogenic compositions that can be pre-mixed and stored. The disclosed vaccination strategies and formulations can improve vaccination efficiency and reduce the risk of bedside mismixing while maintaining overall safety and immunogenicity.

[0102] Various containers, including syringes for single doses and plastic ampoules, can be used to store and transport the premix formulation. In some examples, plastic ampoules can be manufactured using mold-fill-synthetic manufacturing techniques or methods. Generally, mold-fill-synthetic (BFS) manufacturing methods include extruding plastic materials (e.g., resins) to form parisons, which are then poured into molds and cut to size. A filling needle or mandrel is then used to inflate the plastic, resulting 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 carried out in a sterile environment without direct human intervention.

[0103] In some instances, ampoules produced by BFS may be particularly suitable for the pharmaceutical industry, as they can aseptically produce sterile ampoules containing the desired liquid. However, BFS technology is not compatible with all pharmaceutical liquids, products, etc. For example, some known BFS manufacturing methods involve delivering the liquid or product to the ampule, at which point the plastic is still relatively hot, which may have deleterious effects on temperature-sensitive liquids and / or products, such as, for example, vaccines, biologicals, etc. However, advances in low-temperature BFS technology have increased the variety of suitable products, liquids, etc., allowing some vaccines, biologicals, and / or temperature-sensitive pharmaceuticals to be included in BFS ampoules.

[0104] In some examples, a BFS ampoule can have a size, shape, and / or configuration based at least in part on a desired application and / or a desired pharmaceutical liquid or dosage that the ampoule is configured to contain. For example, some known BFS ampoules can include a pierce through top, a screw-off cap, and / or a cap that includes a male or female luer, and the like. Some known BFS ampoules can have a size and / or shape based on a volume or dosage of liquid that is configured to be placed therein. Additionally, some known BFS ampoules can be manufactured in a series of multiple temporarily connected ampoules, which can improve manufacturing, packaging, and / or storage efficiencies, and the like.

[0105] In an embodiment, the immunogenic composition described herein is provided in a pre-filled syringe.When preparing the immunogenic composition in a pre-filled syringe, antigen and adjuvant are combined before administration.In an embodiment, the pre-filled syringe comprises an antigen of the Plasmodium genus parasite (e.g., R21) and an adjuvant (e.g., saponin adjuvant). In an embodiment, the pre-filled syringe comprises R21 and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, a first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; a second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not comprise fraction A of Quillaja Saponaria Molina; and 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 and fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively. In an embodiment, the pre-filled syringe comprises R21 and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; a second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not comprise fraction A of Quillaja Saponaria Molina; and 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 and fraction C of Quillaja Saponaria Molina account for about 92% by weight and about 8% by weight, respectively.In an embodiment, the pre-filled syringe comprises R21 and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; a second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not comprise fraction A of Quillaja Saponaria Molina; and, based on 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 about 75% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

[0106] In an embodiment, the subject is administered the immunogenic composition from a pre-filled syringe. In an embodiment, the subject is administered the immunogenic composition comprising both hemagglutinin and CoV S polypeptide in a single pre-filled syringe. In an embodiment, the subject is administered the immunogenic composition comprising CoV S polypeptide but not hemagglutinin from a pre-filled syringe. In an embodiment, the subject is administered the immunogenic composition comprising hemagglutinin but not CoV S polypeptide from a pre-filled syringe. EXAMPLES

[0107] Example 1: A Phase I / IIb Randomized Controlled Study of the Safety and Immunogenicity of an Immunogenic Composition Comprising R21 Protein and Saponin Adjuvant in Mice Ageing 5-17 Months Objective: The efficacy of an immunogenic composition containing R21 protein and a saponin adjuvant was evaluated in children aged 5-17 months.

[0108] Methods: A Phase I / IIb randomized controlled trial of the safety and immunogenicity of a candidate immunogenic composition was conducted in children aged 5-17 months in Nanoro, Burkina Faso. Children were randomized into three groups: Group 1, Group 2, and Group 3. Children in Group 1 received 5 μg of R21 protein with 25 μg of saponin adjuvant on days 0 (1st dose), 28 (2nd dose), 56 (3rd dose), 1 year after the 3rd dose or 421 days after the 1st dose (4th dose), and 1 year after the 4th dose or 786 days after the 1st dose (5th dose). Group 2 children received 5 μg of R21 protein with 50 μg of saponin adjuvant on days 0 (1st dose), 28 (2nd dose), 56 (3rd dose), 1 year after the 3rd dose or 421 days after the 1st dose (4th dose), and 1 year after the 4th dose or 786 days after the 1st dose (5th dose). Group 3 children received a placebo (control). In each composition, the saponin adjuvant contained 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix.

[0109] Production of R21 protein: R21 protein was produced in Hansenula polymorpha yeast cells. Nucleic acid encoding R21 protein was subcloned into a derivative of H. polymorpha expression plasmid pFPMT121 carrying the LEU2 gene instead of the URA3 gene for selection in yeast. Auxotrophic H. polymorpha strain ALU3 (relevant genotypes: ade1, leu2, ura3) derived from wild-type strain ATCC® 34438™ (CBS4732, IFO1476, JCM3621, NBRC1476, NCYC1457, NRRL Y-5445) was used as the expression host. Recombinant yeast cell lines were generated by electroporation and subsequent strain generation and isolation protocols, whereby the expression plasmid was stably integrated into the yeast genome.

[0110] Heterologous yeast strains were stored as glycerol stocks at −80° C. Production cell lines were grown using 20 g L -1 Glycerol (AppliChem, Germany) and 0.1 g L -1 Cultures were performed in 2 L baffled shake flasks filled with 200 mL of animal component-free YPG medium containing adenine (AppliChem). A preculture grown to stationary phase in YPD medium was used as inoculum. Cultures were incubated at 130 rpm at 37°C and a stroke of 5 cm. After 56 h of batch growth and derepression of the promoter system by consumption of glycerol, 1% (v / v) methanol was added to the cultures for induction of target gene expression. After a total culture period of 72 h, cells were harvested by centrifugation (6,000 g, 15 min, 4°C), washed once with wash buffer (50 mM sodium phosphate buffer, 2 mM EDTA, pH 8.0) and stored at -20°C. R21 protein was isolated by ultracentrifugation.

[0111] Results: No suspected unknown serious adverse events (SUSARs) or serious adverse events (SAEs) associated with vaccination were observed. No febrile convulsions were observed. The number of patients experiencing fever was low across all vaccine doses. 9-15% of patients in group 1 experienced fever. 10-30% of patients in group 2 experienced fever. 8-10% of patients in group 3 experienced fever. Table 1 shows the protective efficacy by group. [Table 1]

[0112] FIG. 6B shows a graph of malaria incidence in patients in groups 1, 2, and 3.

[0113] FIG. 7A is a graph showing immunoglobulins protecting against malaria in patients in groups 1, 2, and 3. Patients in group 2 had higher immunoglobulins after three doses of the vaccine than patients in group 1 (p<0.0001). Patients in both groups 1 and 2 also had increased antibody concentrations after booster doses. FIG. 7B is a graph showing anti-NANP antibody titers over time in patients after administration of the dosing regimen. FIG. 7C is a graph showing IgG titers specific to the C-terminus of the CS protein over time in patients after administration of the dosing regimen. FIG. 7D is a graph showing the avidity of CS protein to anti-NANP antibodies one month after administration of the dosing regimen. FIG. 7E is a graph showing the avidity of CS protein to IgG specific to the C-terminus of the CS protein over time in patients after administration of the dosing regimen.

[0114] Assessing time to first clinical malaria episode, efficacy in group 2 patients was 81% [95% Cl: 74-87%] over 12 months after the booster (p<0.0001). Efficacy in group 2 patients against multiple episodes of clinical malaria over 12 months after the booster was 78% [95% Cl: 71-83%] (p<0.001). Efficacy in group 2 patients against multiple episodes of clinical malaria over 24 hours after the first vaccination series was 77% [95% Cl: 71-83%] (p<0.001).

[0115] Example 2A: A Phase I / IIa Open-Label Safety and Immunogenicity Study of an Immunogenic Composition Comprising R21 Protein and a Saponin Adjuvant in Adults Objective: To evaluate the efficacy of an immunogenic composition containing R21 protein and a saponin adjuvant in adults.

[0116] Methods: A phase I / IIa non-randomized open-label study of the safety and immunogenicity of the candidate immunogenic composition was conducted in adults in Oxford, London, and Southampton, England. Group 2 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 6 months. Group 3 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 2 months. Group 6 was a control group that did not receive the vaccine. The efficacy of each group was calculated on day 21 using the following formula: 100 x (percentage of subjects diagnosed with malaria who did not receive the immunogenic composition - percentage of subjects diagnosed with malaria who received the immunogenic composition) / (percentage of subjects diagnosed with malaria who did not receive the immunogenic composition). Subjects were intentionally infected with malaria parasites (CHMI). The percentage of patients not diagnosed with malaria after CHMI was measured.

[0117] Results: Figure 2 is a graph showing the percentage of subjects free of malaria diagnosis over time after intentionally infecting subjects with human malaria (CHMI). Efficacy in group 2 was 75%. Efficacy in group 3 was 62.5%. The percentage of controls diagnosed with malaria was 100%.

[0118] Example 2B: A Phase I / IIa Open-Label Safety and Immunogenicity Study of an Immunogenic Composition Comprising R21 Protein and a Saponin Adjuvant in Adults Objective: The efficacy of an immunogenic composition containing R21 protein and a saponin adjuvant was evaluated in adults.

[0119] Methods: A phase I / IIa non-randomized open-label study of the safety and immunogenicity of the candidate immunogenic composition was conducted in adults in Oxford, London, and Southampton, England. Group 2 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, 6, and 14 months. Group 3 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, 2, and 10 months. Group 4 received 10 μg R21 protein and 50 μg saponin adjuvant (i.e., saponin adjuvant with 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix) at 0, 1, and 6 months. Group 7 was the unvaccinated control group. VE was calculated on day 14 as in Example 2A. Subjects were intentionally infected with malaria parasites (CHMI). The percentage of patients without a diagnosis of malaria after CHMI was measured.

[0120] Results: Figure 3 is a graph showing the percentage of subjects without a diagnosis of malaria over time after subjects were intentionally infected with human malaria (CHMI). Group 2 showed 60% VE. Group 3 showed 100% VE. Group 4 showed 40% VE. The percentage of controls diagnosed with malaria was 83.3%.

[0121] Example 3: A Phase III Randomized Controlled Trial to Evaluate the Efficacy of an Immunogenic Composition Comprising R21 Protein and a Saponin Adjuvant in Children Aged 5 to 36 Months Objective: The efficacy of an immunogenic composition containing R21 protein and a saponin adjuvant is being evaluated in children aged 5-36 months.

[0122] METHODS:A phase III randomized trial is being conducted to evaluate the efficacy of a candidate immunogenic composition against malaria in African children aged 5-36 months. All children received 5 μg of R21 protein with 50 μg saponin adjuvant or a control vaccine on days 0, 28, and 56. Children in Dande, Bagamoyo, and Kirfi will receive a boost on day 421. Children in Nanoro and Buguni will receive a boost dose one year later. A series of primary vaccinations will be administered before or at the start of the malaria season.

[0123] The following outcomes are evaluated: presence of primary, secondary, or tertiary clinical malaria; safety and responsiveness of the immunogenic composition; complaints of adverse events from the composition; and efficacy against clinical malaria. The immunogenicity of the immunogenic composition described herein is compared to a control. Enzyme-linked immunosorbent assay (ELISA) is used to quantify antibodies against CS antigen, the NANP repeat region of CS, and HBsAg.

[0124] Example 4: Immunogenicity of different dosing regimens in malaria naive patients administered an immunogenic composition comprising R21 protein and a saponin adjuvant. Objective: To evaluate the effect of different dosing regimens of an immunogenic composition containing R21 protein and a saponin adjuvant on immunogenicity. The saponin adjuvant contained 85% w / w Fraction A ISCOM matrix and 15% w / w Fraction C ISCOM matrix. After administration of the dosing regimen in Table 2, patients underwent monitored human malaria infection (CHMI).

[0125] The regimens are listed in Table 2: [Table 2-1] [Table 2-2]

[0126] Patients in group 1 did not undergo CHMI and served as a control group. Patients in group 2 received a delayed third dose, administered 5 months after the first dose. Patients in group 3 received each dose approximately 1 month apart. Patients in group 4 received 50 μg of R21 protein in the first and second doses and 1 / 5 the dose of the first and second doses (10 μg of R21 protein) in the third dose. Patients in group 5 received 10 μg of R21 protein in the first and second doses and 1 / 5 the dose of the first and second doses (2 μg of R21 protein) in the third dose.

[0127] Total IgG NANP-specific antibody responses against the central repeat region of the CS protein were assessed by ELISA. Figures 9A and 9B show the induced antibody responses over time in patients in groups 1 and 3 (Figure 9A) and groups 2, 4, and 5. Patients in groups 2, 4, and 5 were administered a delayed third dose approximately 3 months after administration of the first dose. Surprisingly and unexpectedly, patients in group 5 who received 2 μg of R21 in the third dose showed nearly identical antibody induction as patients who received the group 2 dosing regimen (10 μg of R21 in doses 1-3). The antibody responses of patients in group 5 were higher than those of patients who received 50 μg of R21 in the first dose, 50 μg of R21 in the second dose, and 10 μg of R21 in the third dose.

[0128] FIG. 10A shows the antibody response (anti-NANP specific IgG) after administration of the different dosing regimens of Table 2. Patients administered the regimen of group 5 show higher peak antibody responses than patients administered the regimen of group 3. The geometric mean of the ELISA units in FIG. 10A for group 3 is 1389 (95% CI 1031-1871). The geometric mean of the ELISA units in FIG. 10A for group 2 is 2661 (95% CI 1771-3998). The geometric mean of the ELISA units in FIG. 10A for group 4 is 1009 (95% CI 316-3221). The geometric mean of the ELISA units in FIG. 10A for group 5 is 3980 (95% CI 2500-6337). FIG. 10B shows the antibody response (IgG specific to the C-terminus of CS protein) after administration of the different dosing regimens of Table 2. Figure 10C shows the avidity of anti-NANP specific IgG for the CS protein after administration of the different dosing regimens in the table. Figure 10D shows the avidity of IgG specific for the C-terminus of the CS protein after administration of the different dosing regimens in Table 2.

[0129] Figure 11A shows antibody titers from patients receiving dosing regimens of groups 2 and 3 stratified by protection and lack of protection. Figure 11B shows the correlation of protection in patients of groups 2 and 3 with antibody responses above EU1100 or below EU1100.

[0130] Taken together, this data indicates that delaying the third dose (administered approximately 3 months after the first dose) elicits a higher antibody response than a dosing regimen in which the immunogenic composition is administered in three doses spaced approximately one month apart.

[0131] The data also show that a delayed third dose of R21, 1 / 5 of the first dose, induces a superior antibody response in patients compared to a dosing regimen of 10 μg R21 given in three doses spaced approximately one month apart, which has significant economic implications since it reduces the cost of administering immunogenic compositions against Plasmodium parasites (e.g., malaria vaccines).

[0132] Numbered embodiments Embodiment 1. An immunogenic composition comprising an antigen of a parasite of the genus Plasmodium. Embodiment 2. The immunogenic composition of embodiment 1, wherein the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. Embodiment 3. The immunogenic composition of embodiment 1 or 2, wherein the antigen comprises a circumsporozoite (CS) protein or a fragment thereof. Embodiment 4. The immunogenic composition of embodiment 3, wherein the CS protein is derived from Plasmodium falciparum. Embodiment 5. The immunogenic composition of any one of embodiments 1 to 4, wherein the antigen comprises Hepatitis B surface antigen (HBsAg) or a fragment thereof. Embodiment 6. The immunogenic composition of any one of embodiments 1 to 5, wherein the antigen comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1. Embodiment 7. The immunogenic composition of embodiment 1 comprising an adjuvant. Embodiment 8. The adjuvant comprises at least two iscom particles, wherein: The first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; and An immunogenic composition according to embodiment 7, wherein the second iscom particles comprise fraction C of Quillaja Saponaria Molina and do not comprise fraction A of Quillaja Saponaria Molina. Embodiment 9. The immunogenic composition of claim 8, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 10. The immunogenic composition of claim 8, wherein 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 total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 11. The immunogenic composition of any one of embodiments 7 to 10, wherein the adjuvant is administered at a dose of about 50 μg. Embodiment 12. A method for stimulating an immune response against a parasite of the genus Plasmodium in a subject, comprising administering an immunogenic composition according to any one of embodiments 1 to 11. Embodiment 13 The method of embodiment 12, wherein the immunogenic composition comprises an adjuvant. Embodiment 14. The adjuvant comprises at least two iscom particles, wherein: The first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; and 14. The method according to embodiment 13, wherein the second iscom particles comprise Fraction C of Quillaja Saponaria Molina and do not comprise Fraction A of Quillaja Saponaria Molina. Embodiment 15. The method according to embodiment 14, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 16. The method according to embodiment 14, wherein 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 total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 17. The method of any one of embodiments 13 to 16, wherein the adjuvant is administered at a dose of about 50 μg. Embodiment 18. The method of any one of embodiments 13 to 16, wherein the dose of antigen administered is from about 0.1 μg to about 100 μg. Embodiment 19. The method of any one of embodiments 13 to 16, wherein the dose of antigen administered is from about 0.1 μg to about 10 μg. Embodiment 20. The method of any one of embodiments 13 to 16, wherein the dose of antigen administered is from about 0.1 μg to about 5 μg. Embodiment 21. The method of any one of embodiments 13 to 16, wherein the dose of antigen administered is from about 0.1 μg to about 3 μg. Embodiment 22. The method of any one of embodiments 13 to 16, wherein the amount of antigen administered is from about 0.1 μg to about 2 μg. Embodiment 23. The method of any one of embodiments 12-22, comprising administering a first dose and a second dose of the immunogenic composition. Embodiment 24 The method of embodiment 23, comprising administering a third dose of the immunogenic composition. Embodiment 25. The method of embodiment 23, wherein the dose of antigen in the second dose is less than the dose of antigen in the first dose. Embodiment 26 The method of embodiment 24, wherein the dose of antigen in said third dose is less than the dose of antigen in said first dose. Embodiment 27. The method of embodiment 23 or 24, wherein the first dose and the second dose contain the same amount of antigen. Embodiment 28. The method of embodiment 25, wherein the second dose and the third dose comprise the same amount of antigen. Embodiment 29. The method of embodiment 25, wherein the first dose and the third dose contain the same amount of antigen. Embodiment 30. The method of embodiment 26, wherein the third dose comprises 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. Embodiment 31. The method of embodiment 26, 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 antigen in the first dose. Embodiment 32. The method of embodiment 23 or 24, wherein the second dose is administered about 28 days after the first dose. Embodiment 33 The method of embodiment 24, wherein the third dose is administered about 56 days after the first dose. Embodiment 34 The method of embodiment 24, wherein the third dose is administered about 168 days after the first dose. Embodiment 35. A method of stimulating an immune response to a parasite of the genus Plasmodium in a subject, comprising: (i) an immunogenic composition comprising a first dose of an antigen of a parasite of the genus Plasmodium; said first dose comprising about 10 μg of said antigen; (ii) a second dose of the immunogenic composition, the second dose comprising about 10 μg of the antigen; and (iii) a third dose of the immunogenic composition, wherein the third dose comprises about 2 μg of the antigen. The method of claim 1, further comprising administering Embodiment 36 The method of embodiment 35, wherein the second dose is administered about 28 days after the first dose. Embodiment 37. The method of embodiment 35 or 36, wherein the third dose is administered about 168 days after the first dose. Embodiment 38. The method of any one of embodiments 35 to 37, wherein the Plasmodium parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi. Embodiment 39. The method of any one of embodiments 35 to 38, wherein the antigen comprises a circumsporozoite (CS) protein or a fragment thereof. Embodiment 40. The method of any one of embodiments 35 to 39, wherein the CS protein is derived from Plasmodium falciparum. Embodiment 41. The method of any one of 35 to 40, wherein the antigen comprises Hepatitis B surface antigen (HBsAg) or a fragment thereof. Embodiment 42. The method of any one of embodiments 35 to 41, wherein the antigen comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1. Embodiment 43 The method of any one of embodiments 35 to 42, comprising an adjuvant. Embodiment 44. The adjuvant comprises at least two iscom particles, wherein: The first iscom particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina; and 44. The method according to embodiment 43, wherein the second iscom particles comprise Fraction C of Quillaja Saponaria Molina and do not comprise Fraction A of Quillaja Saponaria Molina. Embodiment 45. The method according to embodiment 44, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 46. The method according to embodiment 44, wherein 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 total weight of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant. Embodiment 47. The method of any one of embodiments 35 to 46, wherein the adjuvant is administered at a dose of about 50 μg.

[0133] 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 references, articles, publications, patents, patent publications, and patent applications cited herein is not and shall not be deemed to be any form of admission or suggestion that said documents constitute valid prior art or form part of the common general knowledge in any country throughout the world. This application is hereby incorporated by reference in its entirety for all purposes the disclosures of U.S. Patent Nos. 10,729,764, 9,821,046, and 8,821,881.

Claims

1. 1. An immunogenic composition for stimulating an immune response to a parasite of the genus Plasmodium in a subject, comprising an antigen of a parasite of the genus Plasmodium, wherein the antigen comprises a circumsporozoite (CS) protein or a fragment thereof and a hepatitis B surface antigen (HBsAg) or a fragment thereof; 1. An immunogenic composition, wherein the antigen of the Plasmodium parasite comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence 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 at least 99.5% identity to SEQ ID NO:1 or SEQ ID NO:

2.

2. The immunogenic composition of claim 1 , wherein the immunogenic composition comprises an adjuvant.

3. The immunogenic composition of claim 2, wherein the immunogenic composition comprises from about 1 μg to about 100 μg of adjuvant.

4. The immunogenic composition of claim 2 , wherein the immunogenic composition comprises from about 25 μg to about 75 μg of adjuvant.

5. The adjuvant comprises at least two iscom particles, wherein: the first iscom particles comprising fraction A of Quillaja saponaria Molina and no fraction C of Quillaja saponaria Molina; and 3. The immunogenic composition of claim 2, wherein the second iscom particles comprise fraction C of Quillaja saponaria Molina and do not comprise fraction A of Quillaja saponaria Molina.

6. The immunogenic composition according to claim 5, wherein the fraction A of Quillaja saponaria Molina and the fraction C of Quillaja saponaria Molina account for at least 75% by weight of the total weight of the fraction A of Quillaja saponaria Molina and the fraction C of Quillaja saponaria Molina in the adjuvant, respectively, and the fraction C of Quillaja saponaria Molina accounts for the remainder.

7. The immunogenic composition described in claim 1, wherein the immunogenic composition is contained in a prefilled syringe.

8. The immunogenic composition of claim 1, wherein the amount of antigen is from about 0.1 μg to about 100 μg; from about 0.1 μg to about 10 μg; from about 0.1 μg to about 5 μg; from about 0.1 μg to about 3 μg; or from about 0.1 μg to about 2 μg.

9. 2. The immunogenic composition of claim 1, wherein the parasite of the genus Plasmodium is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi.

10. The immunogenic composition of claim 1 , wherein the CS protein is derived from Plasmodium falciparum. Claim 11: Administration of the immunogenic composition to a human subject results in a reduction in immune response after administration of the immunogenic composition of from about 50% to about 99%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 60% to about 99%, from about 65% to about 95%, from about 65% to about 90%, from about 65% to about 85%, from about 69% to about 81%, from about 60% to about 95% from about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 40% to about 99%, from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 55%, or from about 40% to about 50% effective. By gender, maximum of about 2 months, maximum of about 2.5 months, maximum of about 3 months, maximum of about 3.5 months, maximum of about 4 months, maximum of about 4.5 months, maximum of about 5 months, maximum of about 5.5 months, maximum of about 6 months, maximum of about 6.5 months, maximum of about 7 months, maximum of about 7.5 months, maximum of about 8 months, maximum of about 8.5 months, maximum of about 9 months, maximum of about 9.5 months, maximum of about 10 months, maximum 2. The immunogenic composition of claim 1, which prevents malaria for 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, up to 22 months, up to 23 months, or up to 24 months.

12. The immunogenic composition of claim 1, wherein administration of the immunogenic composition to a human subject prevents malaria for at least about 12 months with an efficacy of about 69%.

13. The immunogenic composition of claim 1, wherein administration of the immunogenic composition to a human subject prevents malaria for at least about 24 months with an efficacy of at least about 77%.

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