WO2015091734A1 - Novel malaria vaccines - Google Patents

Abstract

The present invention relates to novel compositions for the immunisation against malaria as well as to uses of such compositions and to methods for producing such compositions. In particular, the invention relates to an immunogenic composition comprising a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction. Optionally the adjuvant further comprises a TLR4 agonist.

Description

NOVEL MALARIA VACCINES

This work was performed under Cooperative Research and Development Agreement W81XWH-14-0042 of 12 November 2013 among GlaxoSmithKline Biologicals S.A., Walter Reed Army Institute of Research (WRAIR) and United States Army Medicals and Materiel Development Activity (USAMMDA)

Technical field

The present invention relates to novel compositions for the immunisation against malaria as well as to uses of such compositions and to methods for producing such compositions. In particular, the invention relates to an immunogenic composition comprising a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction.

Background of the invention

Malaria is one of the world's major health problems. During the 20th century, economic and social development, together with anti-malarial campaigns, has resulted in the eradication of malaria from large areas of the world, reducing the affected area of the world surface from 50% to 27%. Nonetheless, half of the world's population is living in areas where malaria is transmitted. An estimated 3.3 billion people are at risk of contracting malaria. For the year 2010, the World Health Organization reported an estimated 219 million cases of malaria globally. The disease killed approximately 660,000 people, the vast majority of whom were children under the age of five living in sub-Saharan Africa.

One of the most acute forms of the disease is caused by the protozoan parasite Plasmodium falciparum which is responsible for most of the mortality attributable to malaria. Other Plasmodium species that can cause malaria include P. vivax, P. knowlesi, P. ovale and P. malariae. The life cycle of the parasite is complex, requiring two hosts, man and mosquito for completion. The infection of man is initiated by the inoculation of sporozoites through the saliva of an infected mosquito. The sporozoites migrate to the liver and there infect hepatocytes (liver stage) where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells to initiate cyclical replication in the asexual blood stage. The cycle is completed by the differentiation of a number of merozoites in the red blood cells into sexual stage gametocytes which are ingested by the mosquito, where they develop through a series of stages in the midgut to produce sporozoites which migrate to the salivary gland.

The sporozoite stage has been identified as one potential target of a malaria vaccine. The major surface protein of the sporozoite is known as circumsporozoite protein (CS protein). The RTS,S malaria vaccine based on CS protein has been under development since 1987 and is currently the most advanced malaria vaccine candidate being studied. This vaccine specifically targets the pre-erythrocytic stage of P. falciparum. Recent data from a large-scale Phase III clinical trial, wherein RTS,S was administered in three doses, one month apart, showed that over 18 months of follow-up, RTS,S was shown to almost halve the number of malaria cases in young children (aged 5-17 months at first vaccination) and to reduce by around a quarter the malaria cases in infants (aged 6-12 weeks at first vaccination) (Otieno et al. (2013) Results were presented at the 6th Multilateral Initiative on Malaria (MIM) Pan-African Conference, Durban). In spite of this success of the RTS,S vaccine, malaria vaccines with an efficacy closer to 100% are still needed.

In the search for alternative antigens for use in a malaria vaccine, the protein CelTOS, for cell- traversal protein for ookinetes and sporozoites, has been identified. CelTOS from P. berghei has been shown to mediate malarial invasion of both vertebrate and insect host cells and is required for establishing their successful infection. WO2010/062859 is directed to the use of CelTOS as a target antigen for a pre-erythrocytic malaria vaccine. It was shown that immunisation of mice with P. falciparum CelTOS (PfCelTOS) combined with water-in-oil adjuvant MONTANIDE ISA-720™ induced protection against challenge with P. berghei (see also Bergmann-Leitner et al. (2010) PLoS One 5(8) el2294). Further work with a P. berghei CelTOS combined with water-in-oil adjuvant MONTANIDE ISA-720™ indicated that both humoral and cellular immune responses were required to mediate complete sterile protection against sporozoite challenge (Bergmann-Leitner et al. (2011) Vaccine 29:5940). While using an in vivo imaging system (MS) and quantification of the absolute bioluminescence on anatomical sites in infected mice, PfCelTOS combined with water-in-oil adjuvant MONTANIDE ISA-720™ indicated a role for both humoral and cellular immune effector mechanisms (Bergmann-Leitner et al (2014) Trials in Vaccinology 3:6-10). Moreover, PfCelTOS compositions adjuvanted with GLA-SE (a stable oil-in-water emulsion combined with a Toll-like receptor 4 (TLR4) agonist) were found to elicit strong Thl-type immune responses in mice (Fox et al. (2012) Clin. Vaccine Immunol 19: 1633). However, in a small human trial in healthy malaria-naive adults, no protection was observed (Cowden et al. (2012), Presentation at the 2012 ASTMH meeting, unpublished).

Many different adjuvants have been described and tested; see e.g. O'Hagan (2000) Vaccine Adjuvants: preparation methods and research protocols. Homana Press, Totowa, New Jersey. WO 96/33739 and WO2007/068907 describe adjuvants comprising an immunologically active saponin fraction, such as QS21, and optionally a TLR4 agonist, such as 3-O-deacylated monophosphoryl lipid A (3D-MPL). The RTS,S vaccine described above is adjuvanted with AS01, a liposomal formulation containing QS21 and 3D-MPL. However, results obtained with adjuvants are often inconsistent in that a ranking of a group of adjuvants for efficacy with a particular antigen is often specific for that antigen and a change in antigen will frequently result in a different ranking. Regulatory authorities require rigorous efficacy, safety and stability testing of new antigen-adjuvant combinations.

While significant progress has been made in the field of malaria vaccine research and development, there is still a need for novel malaria vaccines which are highly efficacious, safe and induce a broad spectrum of cross-reactive immune responses. Summary of the invention

In a first aspect of the invention, there is provided an immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin fraction.

In another aspect, the invention relates to an immunogenic composition comprising a CelTOS antigen and an adjuvant comprising an immunologically active saponin adjuvant and a TLR4 agonist. In another aspect, the invention relates to an immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises QS21.

In a further aspect, the invention relates to the use of said composition in medicine, in particular in the prevention of malaria. The invention also relates to a method for the immunisation against malaria comprising administration of a composition of the invention to a human subject.

In a further aspect, the invention relates to a kit comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically-active saponin fraction. In a another aspect, the invention relates to a kit comprising a first container comprising a CelTOS antigen and a second container comprising an adjuvant, wherein the adjuvant comprises an immunologically-active saponin fraction.

In an even further aspect, there is provided a process for making an immunogenic composition according to the invention comprising the step of admixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction.

Brief description of the figures

Figure 1: Immunisation and sampling schedule.

Figure 2: PfCelTOS specific antibody concentrations in μg/ml as determined by ELISA.

Figure 3: IFN-y producing splenocytes following ex vivo stimulation (number of spot-forming cells (SFC) per 10⁶ splenocytes) GST = glutathione-S-transferase, Pf30, PflO and Pf3 indicate 30, 10 and 3 μg/ml of PfCelTOS, respectively. PblO and Pb3 indicate 10 and 3 μg/ml of PbCelTOS, respectively. sc=subcutaneous; im=intramuscular

Figure 4: IL-4 producing splenocytes following ex vivo stimulation (number of spot-forming cells per 10⁶ splenocytes) GST = glutathione-S-transferase tested at 10 μg/ml, Pf30, PflO and Pf3 indicate 30, 10 and 3 μg/ml of PfCelTOS, respectively. PblO and Pb3 indicate 10 and 3 μg/ml of PbCelTOS, respectively; sc=subcutaneous; im=intramuscular

Figure 5: Capacity of immunogenic compositions to induce sterile protection in mice against a heterologous challenge with P. berghei sporozoites. Day 6, 8 and 14 refer to the number of days after sporozoite challenge. These days correspond to Day 76, 78 and 84 after start of the experiment.

Figure 6: (Cross-) reactivity of pooled antisera with E. coli expressed recombinant PfCelTOS, PbCelTOS and P. knowlesi CelTOS (PkCelTOS) as determined by Western blotting.

Figure 7: Immunofluorescence assay against fixed P. falciparum sporozoites.

Figure 8: Inhibition of sporozoite gliding motility by mouse antisera.

Figure 9: Antibody responses following two immunizations with PfCelTOS/ASOl vaccine in individual subjects Detailed description

As explained above, in one aspect, the invention relates to an immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin fraction. CelTOS antigens

The term "CelTOS antigen" when used herein indicates an immunogenic CelTOS polypeptide or a polynucleotide encoding an immunogenic CelTOS polypeptide. CelTOS is also known as Ag2. A "CelTOS polypeptide" when used herein is a polypeptide comprising the sequence of SEQ ID NO: l or SEQ ID NO:2 or a polypeptide comprising a fragment of the sequence of SEQ ID NO:l or SEQ ID NO:2 or a polypeptide comprising a variant of the sequence of SEQ ID NO: l or SEQ ID NO:2.

In some embodiments, the CelTOS antigen in the immunogenic composition is a polypeptide. In other embodiments, however, the CelTOS antigen is a polynucleotide encoding CelTOS, for example a DNA sequence encoding CelTOS. The polynucleotide may e.g. be incorporated into an adenoviral carrier. Vaccines based on replication-defective adenoviral vectors have been described in the art, see e.g. Tatsis et al. (2004) Mol Ther. 10:616 or Tatsis et al. (2006) Gene Therapy 13:421 for chimpanzee-origin adenovirus vectors. Use of adenoviral vectors for malaria antigens has e.g. been described in WO2004055187 and WO2009071613.

Immunogenic compositions wherein the CelTOS antigen is a CelTOS polypeptide are preferred. In one embodiment, the CelTOS polypeptide comprises or consists of a polypeptide sequence naturally occurring in nature, e.g. a polypeptide sequence corresponding to CelTOS from a species selected from the group consisting of: P. falciparum, P. vivax, P. knowlesi, P. ovale and P. malariae.

In a preferred embodiment, the CelTOS polypeptide comprises or consists of a Plasmodium falciparum CelTOS, e.g. a 182 amino acid polypeptide as defined in SEQ ID NO: l.

SEQ ID NO:l (Accession number Q8I5P1: Plasmodium falciparum 3D7 CelTOS; also GenBank: AAN36249).

MNALRRLPVICSFLVFLVFSNVLCFRGNNGHNSSSSLYNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNELVSVL QKNSPTFLESSFDIKSEVKKHAKSMLKELIKVGLPSFENLVAENVKPPKVDPATYGIIVPVLTSLFNKVETAVGAKVSDEI WNYNSPDVSESEESLSDDFFD

In another embodiment, the CelTOS polypeptide comprises or consists of Plasmodium vivax CelTOS, e.g. a 196 amino acid polypeptide as defined in SEQ ID NO:2.

SEQ ID NO:2 (Accession number Q53UB7: Plasmodium vivax CelTOS; also NCBI:XP_001617263) MHLFNKPPKGKMNKVNRVSIICAFLALFCFVNVLSLRGKSGSTASSSLEGGSEFSERIGNSLSSFLSESASLEVIGNELAD NIANEIVSSLQKDSASFLQSGFDVKTQLKATAKKVLVEALKAALEPTEKIVASTIKPPRVSEDAYFLLGPWKTLFNKVED VLHKPIPDTIWEYESKGSLEEEEAEDEFSDELLD

In another embodiment, the CelTOS polypeptide comprises or consists of Plasmodium knowlesi CelTOS, e.g. a 185 amino acid polypeptide as defined in SEQ ID NO:3. SEQ ID NO:3 (Accession number B3LCG1; Plasmodium knowlesi CelTOS; also NCBI: XM_002262206) MNKVNRVSIICAFLALFCFVNVLSLRGKSGLTASSSLEGGSEFSERIGNTLSSFLSESASLEVIGNELADNIANEIVGSLQ NDSASFLQSEFDVKAQLKATAKKVLTEALKAALEPTEKIVASTIKPPRIKEDIYFLLSPWRSLFNKVEDVLHKPVSDDIW NYESRGSSSEEEDEVDSDEDFLD

SEQ ID NO: 1 is approximately 45% identical to SEQ ID NO:2 over 182 residues, SEQ ID NO: 1 is approximately 44% identical to SEQ ID NO:3 over 178 residues, and SEQ ID NO:2 is approximately 84% identical to SEQ ID NO:3 over 185 residues. In addition, these related CelTOS sequences, and the amino acid sequences conserved amongst them, provide guidance for producing variant polypeptides, including one or more conservative and/or one or more nonconservative amino acid substitutions, as detailed below.

In a further embodiment, the CelTOS polypeptide comprises or consists of a fragment of the sequence of SEQ ID NO: l, 2 or 3. Such a fragment can be of any length provided that it retains immunogenic properties. For example, the fragment can comprise 5 or more consecutive amino acids of the sequence of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3, such as 6 or more consecutive amino acids, e.g. 7 or more consecutive amino acids, such as 8 or more consecutive amino acids, e.g. 9 or more, 10 or more, 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 120 or more, 140 or more, 160 or more, or 180 or more , up to 182 consecutive amino acids of the sequence of SEQ ID NO: l, or up to 196 consecutive amino acids of SEQ ID NO:2, or up to 185 consecutive amino acids of SEQ ID NO:3. Epitopes of PfCelTOS have been described in Bergmann-Leitner, et al. (2013) PloS 8:e71610. Suitable fragments of PfCelTOS for use in the present invention include, but are not limited to:

• Fragments comprising residues 25-83 of PfCelTOS, such as:

o WLCFRGNNGHNSSSSL YNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNEL L SVZ.<?/rA5PTFLES (SEQ ID NO:6)

o NVLCFRGNNGHNSSSSL YNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNEL VSVLQKN (SEQ ID

NO: 7)

o FRGNNGHNSSSSL YNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNEL VSVLQKNSPTFLES (SEQ ID NO: 8)

• Fragments comprising residues 125-182 of PfCelTOS, such as:

o GLPSFENLVAEN VKPPKVDPA TYGIIVPVL TSLFNKVETA VGAKVSDEIWNYNSPD VSESEESLSDDFFD

(SEQ ID NO:9)

o LVAEN VKPPKVDPA TYGIIVPVLTSLFNKVETA VGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 10)

o AEN VKPPKVDPA TYGIIVPVL TSLFNKVETA VGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO : 11) · Fragments comprising residues 125-143 of PfCelTOS, such as:

o LVAEN VKPPKVDPA TYGIIVPVL 7SLFN K (SEQ ID NO: 12)

o LVAEN VKPPKVDPA TYGIIVPVL (SEQ ID NO: 13)

o VKPPKVDPA TYGIIVPVL 7SLFNK (SEQ ID NO: 14)

• Fragments comprising residues 149-182 of PfCelTOS, such as: o VPVLTSLFNK VETA VGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO : 15) o SLFNK VETA VGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 16)

o FNK VETA VGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 17)

In further embodiments, the CelTOS polypeptide comprises or consists of two or more fragments of the sequence of SEQ ID NO: l or SEQ ID NO:2 or SEQ ID NO:3, wherein the two or more fragments are not consecutive in SEQ ID NO: l or SEQ ID NO:2, or SEQ ID NO:3, but e.g. form a conformational epitope.

In a further embodiment, the CelTOS polypeptide comprises or consists of a variant of the sequence of SEQ ID NO: l or SEQ ID NO:2.

A variant polypeptide may contain a number of substitutions, preferably conservative substitutions, (for example, 1-50, such as 1-25, in particular 1-10, and especially 1 amino acid residue(s) may be altered) when compared to the reference sequence. In general, conservative substitutions will fall within one of the amino-acid groupings specified below, though in some circumstances other substitutions may be possible without substantially affecting the immunogenic properties of the antigen as determined by methods well known in the art, and as described in the Examples herein. The following eight groups each contain amino acids that are typically conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

Suitably such substitutions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.

Protein variants may also include those wherein additional amino acids are inserted compared to the reference sequence, for example, such insertions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the addition of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such insertions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen. One example of insertions includes a short stretch of histidine residues (e.g. 2-6 residues) to aid expression and/or purification of the antigen in question.

Variants also include those wherein amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the deletion of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such deletions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen. The skilled person will recognise that a particular protein variant may comprise substitutions, deletions and additions (or any combination thereof).

Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity (such as at least about 95%, at least about 98% or at least about 99%) to the associated reference sequence.

The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Optionally, the percentage identity exists over a region that is e.g. at least 25, such as at least 50, e.g. at least 75 amino acids, such as at least about 100, e.g. at least about 150 amino acids in length. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence.

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov). Unless stated otherwise, the percent identity of a sequence to a reference sequence over a particular fragment length or over the entire length of the reference sequence is determined using the Align Sequences Protein BLAST function (available at http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Variants of a polypeptide sequence will typically have essentially the same activity as the reference sequence. By essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood with specific antigens (e.g. restimulation for a period of between several hours to up to two weeks, such as up to one day, 1 day to 1 week or 1 to 2 weeks) that measures the activation of the cells via lymphoproliferation, production of cytokines in the supernatant of culture (measured by ELISA, CBA etc) or characterisation of T and B cell responses by intra and extracellular staining (e.g. using antibodies specific to immune markers, such as CD3, CD4, CD8, IL2, TNFa, IFN-y, CD40L, CD69 etc) followed by analysis with a flowcytometer. Suitably, by essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in a T cell proliferation and/or IFN-gamma production assay.

CelTOS polypeptides for use in the invention can e.g. be produced in E. coli as described in WO2010/062859. Suitably, the immunogenic composition of the invention comprises between 2 and 200 μg of CelTOS polypeptide, such as between 5 and 100 , e.g. between 10 and 50 μg micrograms of CelTOS polypeptide, such as between 10 and 30 μg of CelTOS polypeptide. In a further embodiment, the composition of the invention comprises one or more further antigens, preferably further Plasmodium antigens. In one preferred embodiment, the composition of the invention comprises:

1. a polypeptide comprising the sequence of SEQ ID NO:l or comprising a fragment of the sequence of SEQ ID NO:l or comprising a variant of the sequence of SEQ ID NO: l, and

2. a polypeptide comprising the sequence of SEQ ID NO:2 or comprising a fragment of the sequence of SEQ ID NO:2 or comprising a variant of the sequence of SEQ ID NO:2.

For example, the composition of the invention may comprise

• a polypeptide comprising a fragment of SEQ ID NO: l, e.g. a fragment of SEQ ID NO: l comprising residues 25-83 and/or residues 125-182, and

· a polypeptide comprising a fragment of SEQ ID NO:2, e.g. a fragment of SEQ ID NO:2 comprising residues 36-94 and/or residues 136-196.

In another preferred embodiment, the further antigen is an antigen derived from the circumsporozoite (CS) protein of Plasmodium. A suitable variant of the CS protein may be a variant wherein parts of the CS protein are in the form of a hybrid protein with the surface antigen S from hepatitis B virus (HBsAg). The hybrid protein may e.g. comprise:

1) a sequence of at least 160 amino acids which is at least 70% identical to the C-terminal portion of the CS protein, optionally lacking a functional hydrophobic anchor sequence,

2) four or more tandem repeats of the CS protein immunodominant region, such as NANP repeats, and

3) HBsAg

The hybrid protein may be a protein which comprises a fragment of the CS protein of P. falciparum corresponding to amino acids 207-395 of the CS protein of P. falciparum clone 3D7 fused in frame via a linear linker to the N-terminus of HBsAg. The linker may comprise a portion of preS2 from HBsAg. CS constructs suitable for use in the present invention are outlined in WO 93/10152, which granted in the US as US Pat. Nos. 5,928,902 and 6,169,171, both of which are incorporated by reference for the purpose of describing suitable proteins for use in the present invention.

A particular hybrid protein for use in the invention is the hybrid protein known as RTS (figure 4) (described in WO93/10152 wherein it is denoted RTS* and in WO98/05355) which consists of:

- a methionine residue

- three amino acid residues, Met Ala Pro

- a stretch of 189 amino acids representing amino acids 207 to 395 of the CS protein of P. falciparum strain 3D7 - a glycine residue

- four amino acid residues, Pro Val Thr Asn, representing the four carboxy terminal residues of the hepatitis B virus (adw serotype) preS2 protein, and

- a stretch of 226 amino acids, encoded by nucleotides 1653 to 2330, and specifying the S protein of hepatitis B virus (adw serotype).

Most preferably, the further antigen is RTS,S. RTS,S is a particle comprised of a mixture of native hepatitis B virus surface antigen (HBsAg) and hybrid HBsAg protein containing parts of the CS protein. RTS,S has been reviewed in e.g. Vekemans et al. (2009) Vaccine 275:G67 and Regules et al. (2011) Expert Rev. Vaccines 10:589. RTS,S is also described in WO93/10152. Further suitable CS derived antigens have been described in WO2014111733 (herein incorporated by reference).

In other embodiments, the immunogenic composition of the invention comprises in addition to a CelTOS antigen one or more further P. falciparium (Pf) and/or P. vivax (Pv) antigens selected from the group consisting of: PfDBP, (Duffy binding protein) PvDBP, PfTRAP (thrombospondin-related adhesive protein), PvTRAP, PfMSPl (merozoite surface protein), PfMSP2, PfMSP3, PfMSP4, PfMSP5, PfMSP6, PfMSP7, PfMSP8, PfMSP9, PvMSPl, PvMSP2, PvMSP3, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PfAMAl (apical membrane antigen), PvAMAl, PfRBP (reticulocyte binding protein), PvRBP, PfEMPl (erythrocyte membrane protein), PvEMPl, Pfsl6, Pf332, Pfs25, Pfs28, PfLSAl (liver stage antigen), PfLSA3, PvLSAl, PvLSA3, PfEBA (erythrocyte binding antigen), PvEBA, PfGLURP (glutamate-rich protein), PvGLURP, PfRAPl (rhoptry-associated protein), PvRAPl, PfRAP2, PvRAP2, PfSequestrin, PvSequestrin, PfSALSA (sporozoite and liver stage antigen), PvSALSA, PfEXPl (export protein), PvEXPl, PfSTARP (sporozoite threonine-asparagine-rich protein), PvSTARP, Pv25, Pv28, Pfs27/25, Pfs48/45, Pfs230 and Pf332, or a fragment or variant of any of these.

Adjuvants

As explained above, the immunogenic composition of the invention comprises an adjuvant, which comprises an immunologically active saponin fraction. In one embodiment, the adjuvant comprises an immunologically active saponin fraction and a TLR4 agonist.

Adjuvants comprising saponins have been described in the art. Saponins are described in: Lacaille-Dubois and Wagner (1996) A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2:363. Saponins are known as adjuvants in vaccines. For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), was described by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, 243) to have adjuvant activity. Purified fractions of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (Kensil et al. (1991) J. Immunol. 146: 431. Quil A fractions are also described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R. , Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55).

Two such fractions, suitable for use in the present invention, are QS7 and QS21 (also known as QA-7 and QA-21). QS21 is a preferred immunologically active saponin fraction for use in the present invention. QS21 has been reviewed in Kensil (2000) In O'Hagan: Vaccine Adjuvants: preparation methods and research protocols. Homana Press, Totowa, New Jersey, Chapter 15. Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7, are e.g. described in WO 96/33739, WO 96/11711 and WO2007/068907.

In addition to the other components, the adjuvant preferably comprises a sterol. The presence of a sterol may further reduce reactogenicity of compositions comprising saponins, see e.g. EP0822831. Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. Cholesterol is particularly suitable. Suitably, the immunologically active saponin fraction is QS21 and the ratio of QS21:sterol is from 1: 100 to 1: 1 w/w, such as from 1: 10 to 1: 1 w/w, e.g. from 1:5 to 1: 1 w/w.

Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3D-MPL. US patent 4,436,727 discloses MPL and its manufacture. US patent 4,912,094 and reexamination certificate Bl 4,912,094 discloses 3D-MPL and a method for its manufacture. Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a synthetic lipid A-like molecule (see, e.g. Fox et al. (2012) Clin. Vaccine Immunol 19: 1633). In a further embodiment, the TLR4 agonist may be a synthetic TLR4 agonist such as a synthetic disaccharide molecule, similar in structure to MPL and 3D-MPL or may be synthetic monosaccharide molecules, such as the aminoalkyl glucosaminide phosphate (AGP) compounds disclosed in, for example, WO9850399, WO0134617, WO0212258, W03065806, WO04062599, WO06016997, WO0612425, WO03066065, and WO0190129. Such molecules have also been described in the scientific and patent literature as lipid A mimetics. Lipid A mimetics suitably share some functional and/or structural activity with lipid A, and in one aspect are recognised by TLR4 receptors. AGPs as described herein are sometimes referred to as lipid A mimetics in the art. In a preferred embodiment, the TLR4 agonist is 3D-MPL.

In a preferred embodiment of the composition of the invention, the immunologically active saponin fraction is QS21 and the TLR4 agonist is 3D-MPL.

In a suitable form of the present invention, the compositions of the invention may comprise QS21 in substantially pure form, that is to say, the QS21 is at least 80%, at least 85%, at least 90% pure, for example at least 95% pure, or at least 98% pure. Compositions of the invention may comprise QS21 in an amount of between about 1 μg to about 100 μg per human dose, for example between about 1 μg and about 60 μg or between about 10 μg and about 100 μg, for example, about 10 μg, about 12 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg or about 50 μg. QS21 may e.g. be present in an amount between about 40 μg and 60 μg or between about 45 and about 55 μg or about 50 μg. Alternatively, QS21 may be present in an amount between 21 μg and 29 μg or between about 23 μg and about 27 μg or about 25 μg. In a further embodiment, compositions of the invention may comprise QS21 in an amount of about 10 μg, for example between about 6 μg and about 14 μg, about 8 μg and about 12 μg. In a further embodiment, compositions of the invention may comprise QS21 in an amount of around about 5 μg, for example between about 3 μg and about 7 μg or between about 4 μg and about 6 μς.

Compositions of the invention may comprise 3D-MPL in an amount of between about 1 μg to about 100 μg per human dose, for example between about 1 μg and about 60 μg or between about 10 μg and about 100 μg, for example, about 10 μg, about 12 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μς, about 40 μς or about 50 μς. 3D-MPL may e.g. be present in an amount between about 40 μς and 60 μς or between about 45 and about 55 μς or about 50 μς. Alternatively, 3D-MPL may be present in an amount between 21 μς and 29 μς or between about 23 μς and about 27 μς or about 25 μς. In a further embodiment, compositions of the invention may comprise 3D-MPL in an amount of about 10 μς, for example between about 6 μς and about 14 μς, about 8 μς and about 12 μς. In a further embodiment, compositions of the invention may comprise 3D-MPL in an amount of around about 5 μς, for example between about 3 μς and about 7 μς or between about 4 μς and about 6 μς.

In a preferred embodiment, composition of the invention comprises between about 10 μς and about 60 μς of QS21 and between about 10 μς and about 60 μς of 3D-MPL, suitably about 50 μς of QS21 and about 50 μς of 3D-MPL or about 25 μς of QS21 and about 25 μς of 3D-MPL per human dose.

In some embodiments, the adjuvant is presented in the form of an oil-in-water emulsion, e.g. comprising squalene, alpha-tocopherol and a surfactant (see e.g. W095/17210) or in the form of a liposome. A liposomal presentation is preferred. The term "liposome" when used herein refers to uni- or multilamellar (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10 lamellar depending on the number of lipid membranes formed) lipid structures enclosing an aqueous interior. Liposomes and liposome formulations are well known in the art. Liposomal presentations are e.g. described in WO 96/33739 and WO2007/068907. Lipids which are capable of forming liposomes include all substances having fatty or fat-like properties. Lipids which can make up the lipids in the liposomes may be selected from the group comprising glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, sterols, archeolipids, synthetic cationic lipids and carbohydrate containing lipids. In a particular embodiment of the invention the liposomes comprise a phospholipid. Suitable phospholipids include (but are not limited to): phosphocholine (PC) which is an intermediate in the synthesis of phosphatidylcholine; natural phospholipid derivates: egg phosphocholine, egg phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipids; and synthetic phospholipid derivates: phosphocholine (didecanoyl-L-a-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoyl phosphatidylcholine [DPPC], Distearoyl phosphatidylcholine [DSPC], Dioleoyl phosphatidylcholine, [DOPC], 1-palmitoyl, 2- oleoylphosphatidylcholine [POPC], Dielaidoyl phosphatidylcholine [DEPC]), phosphoglycerol (1,2- Dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], l,2-dipalmitoyl-sn-glycero-3-phosphoglycerol [DPPG], l,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], l-palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol [POPG]), phosphatidic acid (l,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoyl phosphatidic acid [DPPA], distearoyl-phosphatidic acid [DSPA]), phosphoethanolamine (1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine [DMPE], l,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], l,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], l,2-Dioleoyl-sn-Glycero-3-

Phosphoethanolamine [DOPE]), phoshoserine, and polyethylene glycol [PEG] phospholipid.

Liposome size may vary from 30 nm to several μηη depending on the phospholipid composition and the method used for their preparation. In particular embodiments of the invention, the liposome size will be in the range of 50 nm to 500 nm and in further embodiments 50 nm to 200 nm. Dynamic laser light scattering is a method used to measure the size of liposomes well known to those skilled in the art.

In a particularly suitable embodiment, liposomes used in the invention comprise DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, compositions of the invention comprise QS21 in any amount described herein in the form of a liposome, wherein said liposome comprises DOPC and a sterol, in particular cholesterol.

In a preferred embodiment, composition of the invention comprises between about 10 μg and about 100 μg of QS21 and between about 10 μg and about 100 μg of 3D-MPL, suitably about 50 μg of QS21 and about 50 μg of 3D-MPL in a liposome formulation or about 25 μg of QS21 and about 25 μg of 3D-MPL per human dose in a liposome formulation.

In a further aspect, the invention relates to a process for making an immunogenic composition as defined herein comprising the step of admixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction. The CelTOS antigen may be provided in an lyophilized form or in a liquid formulation. Similarly, in embodiments wherein the CelTOS antigen is combined with a further antigen, such as RTS,S, the antigens may be co-lyophilized together in one vial.

In another aspect, a kit comprising a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction is provided. In a further aspect, a kit is provided comprising a first container comprising a CelTOS antigen and a second container comprising an adjuvant, wherein the adjuvant comprises an immunologically-active saponin fraction.

It is well known that for parenteral administration solutions should be physiologically isotonic {i.e. have a pharmaceutically acceptable osmolality) to avoid cell distortion or lysis. An "isotonicity agent" is a compound that is physiologically tolerated and imparts a suitable tonicity to a formulation {e.g. immunogenic compositions of the invention) to prevent the net flow of water across cell membranes that are in contact with the formulation. Aqueous adjuvant compositions are known which contain 100 mM sodium chloride or more, for example adjuvant system A (ASA) in WO 2005/112991 and WO2008/142133 or the liposomal adjuvants disclosed in WO2007/068907.

In some embodiments, the isotonicity agent used for the composition is a salt. In other embodiments, however, the composition comprises a non-ionic isotonicity agent and the concentration of sodium chloride or the ionic strength in the composition is less than 100 mM, such as less than 80 mM, e.g. less than 30 mM, such as less 10 mM or less than 5 mM. In a preferred embodiment, the non-ionic isotonicity agent is a polyol, such as sorbitol. The concentration of sorbitol may e.g. between about 3% and about 15% (w/v), such as between about 4% and about 10% (w/v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist wherein the isotonicity agent is salt or a polyol have been described in WO2010142685, see e.g. Examples 1 and 2 in WO2010142685.

Uses, doses and immunisation regimes

As mentioned above, in one aspect, the invention relates to the use of the immunogenic compositions, as defined herein, in medicine. In particular, the immunogenic compositions of the invention can be used as a vaccine, i.e. for the prevention or prophylaxis of malaria in human subjects, including prevention of malaria infection and malaria disease and/or reducing the severity of malarial disease.

Thus, in one aspect, the invention relates to an immunogenic composition as defined herein for use in the prevention of malaria. Similarly, the invention relates to use of an immunogenic composition as defined herein for the manufacture of a medicament for the prevention of malaria. Also, the invention relates to a method for the immunisation against malaria comprising administration of an immunogenic composition as defined herein to a human subject.

The immunogenic compositions of the invention may be used for the prevention of malaria caused by P. falciparum, P. vivax, P. knowlesi, P. ovale and P. malariae, or for raising an immune response in a subject against one or more antigens (e.g., CelTOS) of any of these species. The immunogenic compositions of the invention are particularly suitable for use in the prevention of malaria caused by P. falciparum or P. knowlesi.

The immunogenic compositions of the invention may be used for any patient group, including the pediatric as well as the adult population. A particularly suitable target patient group for immunisation with the compositions as defined herein comprises the pediatric population, including children aged 6-12 weeks and children aged 5-17 months. A further particularly suitable target population includes travellers to regions where malaria is endemic.

In some embodiments, the immunogenic composition of the invention is administered only once to a subject. In other embodiment, the immunogenic composition of the invention is administered more than once, such as 2, 3, 4 or 5 times. If the composition is administered more than once, typically, there is a time interval between the administrations, such as 1-4 weeks or more, e.g. of 1-3 weeks or more, to allow for the first immunisation to produce its immunogenic effect.

In even further embodiments, the immunogenic composition of the invention is administered one or more times followed by one or more further administrations (boosters) with a different immunogenic composition, e.g. an unadjuvanted composition comprising CelTOS or a composition comprising CelTOS with a different adjuvant. In a further embodiment, the first (priming) immunisation is carried out with a composition according to the invention comprising a polynucleotide encoding CelTOS and one or more of further (booster) immunisations are carried out with a composition comprising a CelTOS polypeptide. An example of a sequence encoding PfCelTOS is the sequence set forth in NCBI Reference Sequence XM_001350533.1 (herein incorporated by reference) (SEQ ID NO:4):

1 atgaatgcct taagaagatt accagttatt tgctctttct tagtatttct tgtcttttcc

61 aatgttttat gtttcagagg aaacaacgga cacaattctt catcatctct ctataatgga

121 agccaattta ttgaacaatt aaataacagt tttacttcag cttttcttga atcacaatca

181 atgaataaga ttggtgatga tttagcagag accatatcaa atgaacttgt cagtgtttta

241 caaaaaaatt caccaacctt tttagaatca agctttgata tcaaatcaga agtaaaaaaa

301 cacgcaaaat ctatgttaaa ggaattaatc aaagtaggat tgccatcatt cgaaaatctc

361 gtagctgaaa atgttaaacc accaaaagtc gacccagcaa catatggtat aatagtacca

421 gtattaacat ctttatttaa taaggtagaa acagctgtag gtgcgaaagt ttctgatgag

481 atatggaatt acaattcacc agacgtctca gaaagtgaag aaagtttatc agatgatttt 541 ttcgattaa

An example of a sequence encoding PvCelTOS is the sequence set forth in GenBank: AB194053.1 (herein incorporated by reference) (SEQ ID NO:5):

1 atgcatttat ttaacaaacc ccccaaaggc aaaatgaaca aagtaaaccg agtctcgatt

61 atttgtgctt tcttggcact tttttgcttc gtaaatgtgt tgtccttgcg gggaaagagc

121 ggctcgactg cctcgtcttc tcttgaagga ggaagcgaat tttccgagcg catagggaac

181 agcttatcgt cattcctttc cgaatcagca tctttggaag ttattggaaa tgaactggcc

241 gacaacatcg ccaacgaaat tgttagctcc ctgcaaaagg attcagcatc ctttttacaa

301 agtgggtttg acgtaaaaac ccagttaaag gctactgcca agaaggtctt agtggaagcg

361 ttaaaagcag cattagagcc aacggaaaaa attgttgcct ccacgattaa gccaccacgt

421 gtcagcgaag atgcctactt cttattggga ccggtcgtca agactctctt taacaaagtt

481 gaggacgttt tacacaagcc aatacctgat accatttggg aatacgaatc caagggttcc

541 ctcgaagagg aagaagctga agatgagttc tctgatgagt tgttagatta g

In one embodiment, the use according to the invention comprises combining the immunogenic composition of the invention with the antigen RTS,S either in the same composition or in a treatment regimen comprising separate administration of the composition according to the invention and a composition comprising RTS,S. For example, the composition of the invention could be co-administered with a composition comprising RTS,S at separate anatomical sites (e.g. right and left arms) or at the same anatomical site. Alternatively, the administrations could be sequential, alternating one or more administrations of the composition of the invention with one or more administrations of compositions comprising RTS,S, starting with either of them and e.g. alternating at each immunization time point. Immunizations could be given at a given anatomical site or could also alternate between two anatomical sites (the RTS,S-containing composition at one site and the CelTOS-containing composition at a distant site). Preferably, the composition comprising RTS,S would also be adjuvanted with an adjuvant comprising an immunologically active saponin fraction, e.g. QS21, and optionally a TLR4 agonist, such as 3D-MPL.

The immunogenic compositions of the invention may be administered in various ways, including oral, parental and mucosal administration, such as intramuscular, subcutaneous, intradermal, intravenous or intranasal administration. However, parental administration is preferred. Most preferably, the use comprises intramuscular or subcutaneous administration of the composition.

Suitably, the immunogenic compositions according to the present invention have a human dose volume of between 0.25 ml and 1 ml, in particular a dose volume of about 0.5 ml, or 0.7 ml. This may depend on the delivery route with smaller doses being given by the intranasal or intradermal route. The teaching of all references in the present application, including patent applications and granted patents, are herein fully incorporated by reference. The terms 'comprising', 'comprise' and 'comprises' herein are optionally substitutable with the terms 'consisting of, 'consist of, and 'consists of, respectively. The invention will be further described by reference to the following, non-limiting, examples: EXAMPLES

Example 1: Production of antigen and adjuvant

P. falciparum CelTOS antigen was produced in E. coli essentially as described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294. The protein was purified to homogeneity using a two-step purification process, a) affinity purification using Ni⁺²-NTA Sepharose (QIAGEN) and b) a Q Sepharose anion exchanger (GE). The purified protein was buffer exchanged by ultrafiltration (UF; GE Healthcare, Piscataway, NJ) to the final buffer composition 10 mM sodium phosphate (monobasic), 150 mM sodium chloride, pH 7.2.

Adjuvant AS01 premix was made as described in WO 96/33739, incorporated herein by reference. In particular the AS01 adjuvant was prepared essentially as Example 1.1 of WO 96/33739. The AS01 adjuvant comprises: liposomes, which in turn comprise dioleoyl phosphatidylcholine (DOPC), cholesterol and 3D MPL (in an amount of 500 μg DOPC, 125 μg cholesterol and 25 μg 3D-MPL), QS21 (25 μg), phosphate NaCI buffer and water to a volume of 0.5ml.

MPL emulsion (MPL-E) contained 50 micrograms of 3D-MPL per ml of 2.5% v/v squalene, 2.5% v/v alpha-tocopherol and 0.91% v/v Tween® 80 in PBS at pH 7.4

Montanide™ ISA 720 VG (VG=veggie, no animal products contained within) (see also Miles et al. 2005 Vaccine 23:2530) was purchased from Seppic Inc., NJ, USA. It is a water-in-oil emulsion with a ratio 70:30 (volume/volume).

Example 2: Immunisation

Ten groups of BalbC mice were immunised three times (Day 0, Day 21, Day 42) according to the schedule set forth in Figure 1. The compositions and modes of administration tested were as follows: Group 1: 10 g of PfCelTOS antigen adjuvanted with ISA-720™, subcutaneous

Group 2: 10 μg of PfCelTOS antigen adjuvanted with MPL-E, subcutaneous

Group 3: 1 μg of PfCelTOS antigen adjuvanted with AS01, subcutaneous

Group 4: 10 μg of PfCelTOS antigen adjuvanted with AS01, subcutaneous

Group 5: 1 μg of PfCelTOS antigen adjuvanted with AS01, intramuscular

Group 6: 10 μg of PfCelTOS antigen adjuvanted with AS01, intramuscular

Group 7: ISA-720™ adjuvant alone, subcutaneous

Group 8: MPL-E adjuvant alone, subcutaneous

Group 9: AS01 adjuvant alone, subcutaneous

Group 10: AS01 adjuvant alone, intramuscular

Thus, Groups 3 to 6 received compositions comprising CelTOS antigen and a saponin (QS21) containing adjuvant (AS01).

Compositions for immunisation were prepared as follows: AS01 or MPL-

Group Total volume PBS ISA 720 Ag PfCelTOS (l.lmg/ml)

E adjuvant

number (μΙ) (μΙ) (μΙ) (μΙ)

premix (μΙ)

1 2000 418 1400 - 182

2 2000 - - 1982 182

3 2000 - - 1998 18

4 2000 - - 1982 182

5 2000 - - 1998 18

6 2000 - - 1982 182

7 2000 600 1400

8, 9, 10 2000 - - Ready to Use formulation

The total volume delivered for all mouse groups was kept at 100 μΙ, except that for the intramuscular injections, the dose was split between the large muscles in the hind legs of each mouse, therefore 2 x 50 μΙ for each injection. Subcutaneous injections were in the inguinal region.

There were 15 mice in each group. The 15 mice sera per group were used to measure PfCelTOS specific antibody concentrations (Example 3), to test for recognition of fixed sporozoites as determined by IFA (Example 7) and to test for inhibition of sporozoite gliding motility (Example 8). Ten mice per group were used for the challenge experiment (Example 5) and five mice per group were used for the ELISpot experiment (Example 4).

Example 3: Antibody responses

PfCelTOS specific antibody concentrations were determined in serum samples collected on Day - 5, 17, 37 and 58 (only Groups 1 to 8 were tested individually, while the adjuvant control groups were tested as pooled sera). Sera obtained from adjuvant controls did not react on PfCelTOS specific ELISAs (data not shown). Quantitative ELISA was carried out on the samples using standard procedures. Plates were coated with 25 ng PfCelTOS protein per well.

Results

The ELISA results are presented in Figure 2. As can be seen from this figure, all antigen-containing compositions induced PfCelTOS-specific antibodies. Antibody responses were higher in the groups that had been immunised with 10 μg of PfCelTOS than in the groups that had been immunised with only 1 μg of the same antigen. The PfCelTOS-specific antibody levels induced by intramuscular administration of 10 μg of PfCelTOS/ASOl (Group 6) were about two-fold higher than when the antigen was adjuvanted with Montanide ISA-720™ and administered subcutaneously (Group 1) (35.52 versus 14.82 μg/mL, respectively).

Example 4: Cellular responses Profiles of the cellular immune responses were investigated by quantifying the number of interferon gamma (IFN-y) and interleukin 4 (IL-4) producing splenocytes, using the methods and assays described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294. As a negative control, samples were stimulated ex vivo with glutathione-S-transferase (GST).

P. berghei (Pb) CelTOS used for ex vivo stimulations was produced in E. coli and purified, essentially as described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294. The protein included a 16 amino acid N-terminal linker sequence containing 6 histidines.

Results

Figure 3 shows the number of IFN-y producing splenocytes following ex vivo stimulation (Groups 1 and 7 were not tested in this study). Upon ex vivo stimulation with P. falciparum CelTOS, large numbers of PfCelTOS-specific splenocytes producing IFN-y could be detected in all groups that had received PfCelTOS antigen (Groups 2-6). However, the responses were higher in the groups that had received compositions that were adjuvanted with the saponin (QS21)-containing adjuvant AS01 as compared to the group wherein the composition was adjuvanted with MPL-E, regardless of the dose and the route of administration (Compare Groups 3-6 with Group 2).

Figure 4 shows the number of IL-4 producing splenocytes following ex vivo stimulation. Upon ex vivo stimulation with PfCelTOS, clear responses were obtained in all groups that had received 10 μg PfCelTOS antigen (Groups 2, 4 and 6). The highest response was obtained in the groups that had received subcutaneous administration of 10 μg PfCelTOS adjuvanted with AS01 (Group 4). When the results (IFN- Y and IL-4) were compared with historical data from immunizations with PfCelTOS adjuvanted in Montanide ISA-720, the magnitude of the responses obtained with the AS01 formulations exceeded those obtained with Montanide ISA-720 (data not shown). The cellular analysis did not reveal a dose response in regards to the numbers of IFN-γ or IL-4 producing PfCelTOS specific splenocytes.

When ex vivo stimulation was performed with the heterologous antigen P. berghei CelTOS (PbCelTOS), a cross-reactive IL-4 response was seen in the groups that had received high dose PfCelTOS adjuvanted with AS01 (Groups 4 and 6). Such a response was not seen in the group wherein the composition was adjuvanted with MPL-E (Group 2).

Example 5: Sporozoite challenge

The capacity of immunogenic compositions to induce sterile protection in mice against a heterologous challenge with P. berghei sporozoites was tested. Sporozoite challenge was performed as described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294. On Day 76, Day 78 and Day 84 (final day), blood samples were taken, spotted onto a microscope slide, smeared, fixed with Methanol and stained with Giemsa stain and air dried. Parasitemia was analysed microscopically on these samples to determine if the kinetics of infection differed by vaccination group. If by the final blood smear on Day 84 (Day 14 since the challenge), mice remained aparasitemic, the mice were considered 'sterilely protected'. Efficacy was calculated by the equation shown here:

Efficacy = [l-[(number of infected animals (I) vaccine/total number of animals (n) vaccine) ÷ (number of infected animals control (I)/total number of animals (n) control)]]* 100.

Groups 7, 8, 9 and 10 were used as the control groups for Group 1, Group 2, Groups 3+4 and

Groups 5+6, respectively.

Results

All three combinations of 10 μg PfCelTOS antigen with adjuvant (ISA-720™, MPL-E or AS01) induced protection against sporozoite challenge. Protection was also observed at the lower dose of 1 μg PfCelTOS/ASOl when given intramuscularly (Figure 5).

Example 6: Cross-reactivity with other species (Western blot)

Western blotting was performed to test for cross-reactivity of antisera with non-fa lci pa rum species. SDS-PAGE and western blotting procedures were according to standard operating procedures. For each group, pooled, post 3rd bleed, serum was tested at the dilution required to achieve an OD = 1 (as determined by ELISA using PfCelTOS, essentially as described in Example 3). Equal amounts of recombinant proteins (0.5 μg per lane) were loaded into multiple lanes on 4-20% Tris-glycine SDS-PAGE gels.

P. knowlesi CelTOS was produced in E. coli as follows. The gene was subcloned into a modified pETK vector and expressed in BL21 DE3 E. coli. The purification process used to isolate this protein to homogeneity is the same process used for the PbCelTOS. In all cases, Ni-NTA affinity chromatography is used first followed by polishing on Q sepharose anion exchanger. The proteins were buffer exchanged into final buffer composition of 1 x PBS pH 7.4 for storage.

Results

Serum from all four 10 μg PfCelTOS dose groups tested (Groups 1, 2, 4 and 6) reacted with CelTOS from P. falciparum and CelTOS from P. berghei (see lanes 2 and 3 in each of the panels). However, only sera from Group 6 (mice immunised intramuscularly with PfCelTOS/ AS01) reacted with CelTOS from P. knowlesi, suggesting that intramuscular immunisation with PfCelTOS/ASOl elicited an immune-response with broader cross-reactivity (Figure 6).

Example 7: Immunofluorescence assay on sporozoites

Immunofluorescence assays were performed as essential described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294 to determine whether antibodies induced by PfCelTOS/ASOl could recognize native antigen on or inside dissected salivary gland sporozoites on fixed parasites. Immunostaining using the polyclonal anti-PfCelTOS antisera from these Balb/c mice showed reactivity on fixed homologous P. falciparum and the heterologous P. berghei sporozoites. In addition, for PfCelTOS/ASOl 10 μg dose delivered both intramuscularly and subcutaneously, the antibodies reacted on 'live' P. falciparum sporozoites verifying the extracellular (surface) localization of the PfCelTOS and validating the potential of antibodies to act as effectors in protection (data not shown). The data in Figure 7 are graphed as the median antibody endpoint titer (dilution) and the first and third quartiles. Outliers and extreme outliers appear outside of the 1^st and 3^rd quartiles.

Example 8: Inhibition of sporozoite gliding motility by antisera

The inhibition of sporozoite gliding motility assay is an in iz/f/u test to characterize and semi-quantify antiparasitic activity of polyclonal or monoclonal antibodies targeting the motile stages of the malaria parasite during the pre-erythrocytic stage. Live sporozoites deposit a trail of CS protein on glass (Stewart and Vanderberg JP (1988) J Protozool 35: 389). As sporozoite motility is an indirect measure of the viability and quality of the parasites, a motility assay can be used to test whether antibodies have an effect on viability and health of the parasites. Such a motility assay was carried out on the mice antisera, using essentially the same method as described in Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294. Sporozoites of P. berghei as well as sporozoites of P. falciparum were tested. The percentage inhibition of sporozoite gliding motility is calculated by counting the number of sporozoites and number of CSP trails per field (from at least 10 fields/slide) and is calculated as: % inhibition = [l-[(n trails exp/n spz exp)/ (n trails con/n spz con)]]*100; where exp is experimental and con is control-naive serum.

Results

Results of the motility assay are shown in Figure 8. The highest degree of inhibition of sporozoite motility was induced by the sera obtained from the mice immunised intramuscularly with PfCelTOS/ASOl against both parasite species tested, P. falciparum and P. berghei (Group 6).

Example 9: Immunogenicity (antibody responses) following two immunizations with the malaria vaccine, FMP012 (PfCelTOS)/ ASOl, in U.S. naive subjects.

Serum samples were obtained from a Phase 1 study with controlled human malaria infection (CHMI), a non-randomized, open label, dose escalation study in healthy, malaria-naive adults aged 18 to 50 years (inclusive) clinical trial design, to assess safety, immunogenicity, and protection. To date, subjects in 2 groups have received vaccinations at weeks 0, 4, 8 and have been monitored for clinical adverse events and laboratory abnormalities. The PfCelTOS antigen is administered with adjuvant ASOl.

A total of 30 subjects, were divided into low and high dose groups (15 per group), and received 3 doses of the PfCelTOS /ASOl vaccine to date. Group 1 received 10 μg of PfCelTOS formulated with ASOl adjuvant, Group 2 received 30 μg of PfCelTOS formulated with ASOl adjuvant. The adjuvant composition is constant and 500 μί. was delivered by the intramuscular route to both groups. The study design included a staggered start for Group 1 and Group 2 with immunizations separated by 14 days. Five subjects from each group were immunized in a pilot group, 1 day prior to the remainder of the group, for the first vaccination only (day -1 for Group 1, day 13 for Group 2). For this report, only the antibody titer for the PfCelTOS/ASOl Group 1 (10 μg) will be presented. Briefly, total IgG responses to the PfCelTOS antigen were measured using standard ELISA methodologies by the Malaria Serology Lab (MSL) at WRAIR. Plates were coated with 100 μί^εΙΙ of CelTOS antigen (Bergmann-Leitner et al. (2010) PLoS ONE 5(8) el2294) at a concentration of 0.25 μg/mL, placed inside a humidity chamber and incubated overnight (16-20 hrs) at 4°C. Plates were washed four times with IX PBS (pH 7.4) containing 1% Tween-20 and blocked with 0.5% boiled casein (Sigma, St. Louis, MO, USA). Plates were washed four times with IX PBS solution between all subsequent steps except the development reaction. Serum samples from study subjects were serially diluted from 1:50 on each plate and incubated at 22°C for 2 hrs. Peroxidase-labelled goat anti-human IgG (KPL, Gaithersburg, MD, USA) was added to each well at a 1:4,000 dilution and incubated for 1 hr at 22°C. ABTS Peroxidase substrate (KPL, Gaithersburg, MD, USA) was added to induce reaction development. At the end of 1-hr incubation at 22°C, a stop solution (20% sodium dodecyl sulphate) was added and the plates were read using a Spectromax340PC plate reader. The absorbance at 414 nm was determined for each well and these data were applied to a four parameter logistic curve using SoftMax GxP software (Molecular Devices, Sunnyvale, CA, USA). The serum titer was defined as the serum dilution to achieve an optical density (OD) equal to 1.0.

The results are graphically reported in Figure 9 using Minitab V16 for Individual Values for Group 1 on a linear scale for the Mean antibody titer. The 95% Confidence Interval is shown. In addition, the Geometric Mean titer and 95% Confidence Intervals and Median, with first and third quartiles were determined:

· Geometric Mean and 95% Confidence Intervals = 23,158 (8,276 - 38,981)

• Mean and 95% Confidence Interval = 36,986 (15,739 - 58,233)

• Median = 20,100 (First quartile, 9,505 and Third quartile, 50,474)

The antibody titers were higher than previously observed with three doses of PfCelTOS combined with a non-saponin-containing adjuvant (Cowden et al. (2012) Presentation at the 2012 ASTMH meeting, unpublished).

EQUIVALENTS

The present invention provides among other things immunogenic compositions comprising a CelTOS antigen and an adjuvant. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov).

Claims

Claims

1. An immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin fraction.

2. The composition of claim 1, wherein the adjuvant comprises an immunologically active saponin fraction and a TLR4 agonist.

3. The composition of any one of the preceding claims, wherein the immunologically active saponin fraction is QS21.

4. The composition of any one of the preceding claims, wherein the adjuvant further comprises a sterol.

5. The composition of claim 4, wherein the sterol is cholesterol.

6. The composition of any one of the preceding claims, wherein the immunologically active saponin fraction is QS21 and wherein the ratio of QS21:sterol is from 1: 100 to 1:1 w/w.

7. The composition of any one of the preceding claims, wherein the adjuvant comprises a TLR4 agonist and wherein the TLR4 agonist is a lipopolysaccharide.

8. The composition of claim 7, wherein the lipopolysaccharide is 3D-MPL.

9. The composition of any one of the preceding claims, wherein the immunologically active saponin fraction is QS21 and wherein the TLR4 agonist is 3D-MPL and wherein both QS21 and 3D-MPL are present in an amount of between 10 and 100 μg per human dose.

10. The composition of any one of the preceding claims, wherein the adjuvant is presented in the form of a liposome.

11. The composition of any one of the preceding claims, wherein the CelTOS antigen is a CelTOS polypeptide.

12. The composition of claim 11, wherein the polypeptide is Plasmodium falciparum CelTOS.

13. The composition of claim 11 or 12, wherein the polypeptide comprises the sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or an immunogenic fragment of said polypeptide, or a variant of said polypeptide.

14. The composition of any one of the preceding claims, wherein the composition further comprises one or more other Plasmodium antigens.

15. The composition of any one of the preceding claims, wherein the composition further comprises a circumsporozoite protein antigen, such as RTS,S.

16. The composition of any one of the preceding claims for use in medicine.

17. The composition of any one of the preceding claims for use in the prevention of malaria.

18. The composition of any one of the preceding claims for use in the prevention of malaria disease caused by P. falciparum, P. vivax or P. knowlesi.

19. The composition of any one of claims 16 to 18, wherein the use comprises intramuscular administration of the composition.

20. The composition of any one of claims 16 to 18, wherein the use comprises subcutaneous administration of the composition.

21. The composition of any one of claims 16 to 18, wherein the use comprises 2 or 3 administrations of the composition.

22. Use of a composition of any one of claims 1 to 15 for the manufacture of a medicament for the prevention of malaria.

23. A method for the immunisation against malaria comprising administration the composition of any of claims 1 to 15 to a human subject.

24. A kit comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically-active saponin fraction.

25. The kit of claim 24, wherein the kit comprises one or more of the additional features of claims 2-15.

26. A process for making an immunogenic composition of any one of claims 1-15 comprising the step of admixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction.

27. The process of claim 26, wherein the composition comprises one or more of the additional features of claims 2-15.