BE1022373B1 - NEW ANTIMALARIAL VACCINES - Google Patents

Abstract

The present invention relates to novel compositions for immunization against malaria as well as uses of such compositions and methods of making 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

NEW ANTIMALARIAL VACCINES

This work was carried out within the framework of the Cooperative Research and Development Agreement W81XWH-14-0042 of November 12, 2013 at GlaxoSmithKline Biologicals SA, Walter Reed Army Institute of Research (WRAIR) and United States Army Medical and Materials Development Activity (USAMMDA).

Technical area

The present invention relates to novel compositions for the immunization against malaria as well as uses of such compositions and 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 moiety.

Context of the invention

Malaria is one of the major health problems in the world. During the 20th century, economic and social development, carried out jointly with anti-malaria campaigns, led to the eradication of malaria from large areas of the world, reducing the affected area of the world's surface from 50% to 27%. Nevertheless, half of the world's population lives in areas where malaria is transmitted. It is estimated that 3.3 billion people are at risk of contracting malaria. During 2010, the World Health Organization reported a global estimate of 219 million malaria cases. The disease killed an estimated 660,000 people, the vast majority of whom were children under 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, the man and the mosquito for its accomplishment. 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 the hepatocytes (hepatic stage) where they differentiate, through the exoerythrocytic intracellular stage, at the merozoite stage they infect the erythrocytes to initiate cyclic replication in the asexual blood stage. The cycle is complemented by the differentiation of a number of merozoites in erythrocytes into sexually mature gametocytes that are ingested by the mosquito, where they develop through a series of steps in the midgut to produce sporozoites that migrate to the salivary gland.

The sporozoite stage has been identified as a potential target for a malaria vaccine. The main surface protein of the sporozoite is known as the circumsporozoite protein (CS protein). The RTS, S-based malaria vaccine based on CS protein has been in development since 1987 and is currently the most advanced candidate malaria vaccine in the study. This vaccine specifically targets the pre-erythrocytic stage of P. falciparum. Recent data from a large-scale phase III clinical trial, in which RTS, S was administered in three doses, one month apart, showed that over 18 months of follow-up, RTS, S had almost divided by two the number of cases of malaria in young children (aged 5 to 17 months at the first vaccination) and reduced by about a quarter the cases of malaria in infants (aged 6 to 12 weeks at the first vaccination) ) (Otieno et al. (2013) results were presented at the 6th Multilateral Initiative on Malaria (MIM) Pan-African Conference, Durban). Despite this success of the RTS, S vaccine, malaria vaccines with efficiencies closer to 100% are still needed.

In the search for antigen variants for use in an antimalarial vaccine, the CelTOS protein for cell crossing protein for ookinets and sporozoites has been idnetified. CelTOS from P. berghei has been shown to mediate malaria invasion of host cells from both vertebrates and insects and is necessary for the establishment of their successful infection. WO 2010/062859 is directed to the use of CelTOS as the target antigen for a pre-erythrocyte malaria vaccine. Immunization of mice with CelTOS of P. falciparum (PfCelTOS) combined with water-emulsion adjuvant in MONTANIDE IS-720TM oil has been shown to provide protection against challenge with P. berghei (see also Bergmann-Leitner et al (2010) PLoS One 5 (8) e12294). Further work with a CelTOS from P. berghei combined with MONTANIDE ISA-720TM water-in-oil emulsion adjuvant 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). Using an in vivo imaging system (IVIS) and quantification of absolute bioluminescence at anatomical sites in infected mice, PfCelTOS combined with the water-in-oil emulsion adjunct MONTANIDE ISA-720 ™ indicated a role for effector mechanisms

both humoral and cellular immune (Bergmann-Leitner et al (2014) Trials in Vaccinology 3: 6-10). In addition, it has been found that compositions of PfCelTOS adjuvated with GLA-SE (a stable oil-in-water emulsion combined with a Toll-4 receptor agonist (TLR4)) elicit strong Th1-like immune responses in mice (Fox et al (2012) Clin Immunol Vaccine 19: 1633). However, in a small human study in healthy adults who have never been in contact with malaria ("naive for malaria"), no protection has been observed (Cowden et al (2012), Presentation at the 2012 ASTMH meeting, unpublished).

Many different adjuvants have been described and tested; see, for example, O'Hagan (2000) Vaccine Adjuvants: preparation methods and research protocols. Homana Press,

Totowa, New Jersey. WO 96/33739 and WO 2007/068907 disclose 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, the results obtained with adjuvants are often irregular in that the classification of a group of adjuvants for their effectiveness with a particular antigen is often specific for this antigen and a change of antigen will frequently lead to a different classification. Regulatory authorities require rigorous analysis of the efficacy, safety and stability of new antigen-adjuvant combinations.

While significant progress has been made in the area of malaria vaccine research and development, there is still a need for new malaria vaccines that are highly effective, 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 moiety.

In another aspect, the invention provides 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 provides an immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises QS21.

In another aspect, the invention relates to the use of said composition in medicine, particularly in the prevention of malaria. The invention also relates to a method for immunizing against malaria comprising administering a composition of the invention to a human subject.

In another aspect, the invention provides a kit comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin moiety. In 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 yet another aspect, there is provided a method of making an immunogenic composition according to the invention comprising the step of mixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction.

Brief description of the figures

Figure 1 - Schedule of immunization and sampling.

Figure 2 - Concentrations of PfCelTOS specific antibodies in μg / ml determined by the ELISA technique.

Figure 3 - Splenocytes producing IFN-γ after ex vivo stimulation (number of spot-forming cells (SFC) for 106 splenocytes), GST = glutathione-S-transferase,

Pf30, Pf10 and Pf3 indicate, respectively, 30, 10 and 3 μg / ml of PfCelTOS. Pb10 and Pb3 indicate, respectively, 10 and 3 μg / ml of PbCelTOS. sc = subcutaneous; im = intramuscular

Figure 4 - Splenocytes producing IL-4 after ex vivo stimulation (number of spot-forming cells (SFC) for 106 splenocytes), GST = glutathione-S-transferase tested at 10 μg / ml, Pf30, Pf10 and Pf3 indicate respectively, 30, 10 and 3 μg / ml of PfCelTOS. Pb10 and Pb3 indicate, respectively, 10 and 3 μg / ml of PbCelTOS; sc = subcutaneous; im = intramuscular

Figure 5 - Ability of immunogenic compositions to induce sterile protection in mice against a heterologous challenge with P. berghei sporozoites. Days 6, 8 and 14 refer to the number of days after the test with sporozoites. These days are days 76, 78 and 84 after the start of the experiment.

Figure 6 - Reactivity (cross) of E. coli grouped E. coli expressing PfCelTOS, PbCelTOS and CelTOS of recombinant P. knowlesi (PkCelTOS), determined by Western blot analysis.

Figure 7 - Immunofluorescence test on fixed sporozoites of P. falciparum.

Figure 8 - Inhibition of sporozoite motility by mouse antisera.

Figure 9 - Antibody responses after two immunizations with PfCelTOS / AS01 vaccine in individual subjects.

detailed description

As has been 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 moiety.

CelTOS Antigens

The term "CelTOS antigen", when used herein, refers to an immunogenic CelTOS polypeptide or 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: 1 or SEQ ID NO: 2 or a polypeptide comprising a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a polypeptide comprising a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the CelTOS antigen in the immunogenic composition is a polypeptide. However, in other embodiments, the CelTOS antigen is a polynucleotide encoding CelTOS, for example a DNA sequence encoding CelTOS. The polynucleotide can be, for example, incorporated into an adenoviral support. Adenoviral vector vaccines defective for replication have been described in the state of the art, see, for example, Tatsis et al. (2004) Mol Ther. 10: 616 or Tatsis et al. (2006) Gene Therapy 13: 421 for adenoviral vectors from chimpanzees. The use of adenoviral vectors for malarial antigens has been described, for example, in WO 2004055187 and WO 2009071613.

Immunogenic compositions in which 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, for example, a polypeptide sequence corresponding to CelTOS of 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 by SEQ ID NO: 1. SEQ ID NO: 1 accession Q8I5P1: CelTOS of

Plasmodium falciparum 3D7; also GenBank: AAN36249).

MNALRRLPVICSFLVFLVFSNVLCFRGNNGHNSSSSLYNGSQFIEQLNNSFTSAFLE

SQSMNKIGDDLAETISNELVSVLQKNSPTFLESSFDIKSEVKKHAKSMLKELIKVGLPSFEN

LVAENVKPPKVDPATYGIIVPVLTSLFNKVETAVGAKVSDEIWNYNSPDVSESEESLSDDFF

D

In another embodiment, the CelTOS polypeptide comprises or consists of Plasmodium vivax CelTOS, for example, a 196 amino acid polypeptide as defined by SEQ ID NO: 2. SEQ ID NO: 2 (accession number Q53UB7 : CelTOS of

Plasmodium vivax; also NCBI: XP_001617263)

MHLFNKPPKGKMNKVNRVSIICAFLALFCFVNVLSLRGKSGSTASSSLEGGSEFSER

IGNSLSSFLSESASLEVIGNELADNIANEIVSSLQKDSASFLQSGFDVKTQLKATAKKVLVE

ALKAALEPTEKIVASTIKPPRVSEDAYFLLGPVVKTLFNKVEDVLHKPIPDTIWEYESKGSL

EEEEAEDEFSDELLD

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

MNKVNRVSIICAFLALFCFVNVLSLRGKSGLTASSSLEGGSEFSERIGNTLSSFLSE

SASLEVIGNELADNIANEIVGSLQNDSASFLQSEFDVKAQLKATAKKVLTEALKAALEPTEK

IVASTIKPPRIKEDIYFLLSPVVRSLFNKVEDVLHKPVSDDIWNYESRGSSSEEEDEVDSDE

DFLD SEQ ID NO: 1 is approximately 45% identical with SEQ ID NO: 2 on 182 residues, SEQ ID NO: 1 is approximately the same as 44% with SEQ ID NO: 3 on 178 residues, and SEQ ID NO: 2 is approximately 84% identical with SEQ ID NO: 3 over 185 residues. In addition, these cognate sequences of CelTOS, and the amino acid sequences conserved among them, serve as a guide for producing variant polypeptides, including one or more conservative amino acid substitutions and / or one or more amino acid substitutions. non-conservative, as detailed below.

In another embodiment, the CelTOS polypeptide comprises or consists of a fragment of the sequence of SEQ ID NO: 1, 2 or 3. Such a fragment may be of any length provided that it retains the properties immunogenic. For example, the fragment may comprise 5 or more consecutive amino acids of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, such as 6 consecutive amino acids or more, for example, 7 acids. consecutive amino acids or more, such as 8 or more consecutive amino acids, for example, 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 plus, 160 or more, or 180 or more, up to 182 consecutive amino acids of the sequence of SEQ ID NO: 1, or up to 196 consecutive amino acids of SEQ ID NO: 2, or up to 185 amino acids The 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 to 83 of PfCelTOS, such as: o NVLCFRGNNGHNSSSSLYNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNEL VSVLQKNSPTFLES (SEQ ID NO: 6) o NVLCFRGNNGHNSSSSLYNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNEL VSVLQKN ( SEQ ID NO: 7) o FRGNNGHNSSSSLYNGSQFIEQLNNSFTSAFLESQSMNKIGDDLAETISNELVSVL QKNSPTFLES (SEQ ID NO: 8) • fragments comprising residues 125-182 of PfCelTOS, such as: o GLPSFENLVAENVKPPKVDPATYGIIVPVLTSLFNKVETAVGAKVSDEIWNYNSPD VSESEESLSDDFFD (SEQ ID NO: 9) o LVAENVKPPKVDPATYGIIVPVLTSLFNKVETAVGAKVSDEIWNYNSPDVSESEES LSDDFFD (SEQ ID NO: 10) o AENVKPPKVDPATYGIIVPVLTSLFNKVETAVGAKVSDEIWNYNSPDVSESEESLS DDFFD (SEQ ID NO: 11) • fragments comprising residues 125-143 of PfCelTOS as: LVAENVKPPKVDPATYGIIVPVLTSLFNK (SEQ ID NO: 12) LVAENVKPPKVDPATYGIIVPVLT (SEQ ID NO: 13) VKPPKVDPATYGIIVPVLTSLFNK (SEQ ID NO: 14) • Fragments comprising residues 149 to 182 of PfCelTOS, such as: o VPVLTSLFNKVETAVGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 15) o SLFNKVETAVGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 16) o FNKVETAVGAKVSDEIWNYNSPDVSESEESLSDDFFD (SEQ ID NO: 17)

In other embodiments, the CelTOS polypeptide comprises or consists of two or more fragments of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, where the two or more fragments are not consecutive in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, but, for example, form a conformational epitope.

In another embodiment, the CelTOS polypeptide comprises or consists of a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

A variant polypeptide may contain a number of substitutions, preferably conservative substitutions (for example, 1 to 50, such as 1 to 25, especially 1 to 10, and especially 1 amino acid may be modified) when compared. to the reference sequence. In general, the conservative substitutions will fall within one of the amino acid groups specified below, although under certain circumstances other substitutions may be possible without substantially affecting the immunogenic properties of the antigen as it may be. is determined by methods well known in the state of the art, and as described in the examples herein. The following eight groups each contain amino acids that are generally conservative substitutions for each other: 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 therefore do not have a significant impact on the immunogenic properties of the antigen.

Protein variants may also include those in which additional amino acids are inserted relative to the reference sequence, for example, such insertions may occur at 1-10 locations (such as 1-5 locations, suitably 1 to 2 locations, particularly 1 location) and may, for example, involve the addition of 50 amino acids or less at each location (such as 20 or less, especially 10 or less, especially 5 or less). Conveniently, such insertions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. An exemplary insertion includes a short sequence of histidine residues (eg, 2 to 6 residues) to assist the expression and / or purification of the antigen in question.

Variants also include those in which amino acids have been deleted as 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, particularly 1 location) and may, for example, involve deletion of 50 amino acids or less at each location (such as 20 or less, especially 10 or less, especially 5 or less). Suitably such deletions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. It will be understood by those skilled in the art that a particular protein variant may include substitutions, deletions, and additions (or all combinations thereof).

The variants preferably have 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%) with the associated reference sequence.

The terms "identical" or "identity" in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of residues of amino acids or nucleotides that are the same (that is, 70% identity, possibly 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity on a specified region), when compared and aligned for maximum match on a comparison window, or a designated region, measured using one of the following sequence comparison algorithms or by manual alignment and visual examination. Such sequences are then said to be "substantially identical". This definition also refers to the complement of a sequence to be tested. Optionally, the percent identity exists over a region that has a length, for example, of at least 25, such as at least 50, for example at least 75 amino acids, such as at least about 100, for example at least about 150 amino acids. Suitably, the comparison is made on 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. The BLAST analysis software is available to the public through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov). Unless otherwise indicated, the percent identity of a sequence against a reference sequence over a particular fragment length or over the entire length of the reference sequence is determined using the BLAST Protein Align Sequence function (available at http://blast.ncbi.nlm.nih.gov/Blast.cgi).

The variants of a polypeptide sequence will generally have essentially the same activity as the reference sequence. By substantially the same activity, at least 50%, suitably at least 75% and especially at least 90% of reference sequence activity is included in an in vitro restimulation test of peripheral blood mononuclear cells (PBMCs). or whole blood with specific antigens (for example, a restimulation for a period between several hours and up to two weeks, such as up to a day, 1 day to 1 week or 1 to 2 weeks) that measures the cell activation via lymphoproliferation, cytokine production in culture supernatant (measured by ELISA, CBA, etc.) or characterization of T and B cell responses by intracellular and extracellular staining (eg, using specific antibodies against immune markers, such as CD3, CD4, CD8, IL2, TNFa, IFN-gamma, CD40L, CD69, etc.) followed by cytomic analysis. to be flow. Suitably, by essentially the same activity, at least 50%, suitably at least 75% and especially at least 90% of reference sequence activity in a T cell proliferation assay and / or production of IFN gamma.

CelTOS polypeptides for use in the invention may be produced, for example, in E. coli as described in WO 2010/062859.

Suitably, the immunogenic composition of the invention comprises between 2 and 200 μg of CelTOS polypeptide, as between 5 and 100 μg, for example, between 10 and 50 μg (micrograms) of CelTOS polypeptide, as between 10 and 30 μg of CelTOS polypeptide.

In another embodiment, the composition of the invention comprises one or more other antigens, preferably other Plasmodium antigens. In a preferred embodiment, the composition of the invention comprises: 1. a polypeptide comprising the sequence of SEQ ID NO: 1 or comprising a fragment of the sequence of SEQ ID NO: 1 or comprising a variant of the SEQ sequence ID NO: 1, 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: 1, for example a fragment of SEQ ID NO: 1 comprising residues 25 to 83 and / or residues 125 to 182, and A polypeptide comprising a fragment of SEQ ID NO: 2, for example a fragment of SEQ ID NO: 2 comprising residues 36 to 94 and / or residues 136 to 196.

In another preferred embodiment, the other antigen is an antigen derived from Plasmodium circumsporozoite (CS) protein. An appropriate variant of the CS protein may be a variant in which portions of the CS protein are in the form of a hybrid protein with the surface antigen S from the hepatitis B virus (HBs Ag). The hybrid protein may comprise, for example: 1) a sequence of at least 160 amino acids which is at least 70% identical with the C-terminal portion of the CS protein, to which there is possibly a hydrophobic anchoring sequence missing functional, 2) four or more tandem repeats of the immunodominant region of the CS protein, such as NANP repeats, and 3) HBsAg.

The hybrid protein may be a protein which comprises a fragment of the P. falciparum CS protein corresponding to amino acids 207 to 395 of the cloned P.falciparum CS protein CS3 fused via a linker linearly at the N-terminus of HBsAg. The linker may include a portion of preS2 from HBsAg. CS constructs suitable for use in the present invention are disclosed in WO 93/10152, which has been issued in the US under US Patent Numbers 5,928,902 and 6,169,171, both incorporated herein by reference. 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 WO 93/10152 in which it is indicated by RTS * and in WO 98/05355 ) which consists of: - a methionine residue - three amino acid residues, Met Ala Pro - a 189 amino acid sequence representing amino acids 207 to 395 of the CS protein of the P. falciparum strain 3D7 - a glycine residue - four amino acid residues, Pro Val Thr Asn, representing the four carboxy-terminal residues of the preS2 protein of hepatitis B virus (serotype adw), and - a sequence of 226 amino acids, encoded by nucleotides 1653 to 2330, and specifying the hepatitis B virus S protein (serotype adw).

Most preferably, the other antigen is RTS, S. RTS, S is a particle composed of a mixture of the native hepatitis B virus surface antigen (HBsAg) and a hybrid HBsAg protein containing portions of the CS protein. RTS, S has been described, for example, in Vekemans et al. (2009) Vaccine 275: G67 and Regules et al. (2011) Expert

Rev. Vaccines 10: 589. RTS, S is also described in WO 93/10152. Other suitable CS-derived antigens have been described in WO 2014111733 (incorporated herein by reference).

In other embodiments, the immunogenic composition of the invention comprises in addition to a CelTOS antigen, one or more other antigens of P. falciparum (Pf) and / or P. vivax (Pv) selected from the group consisting of: PfDBP, (Duffy binding protein) PvDBP, PfTRAP (thrombospondin-associated adhesive protein), PvTRAP, PfMSP1 (merozoite surface protein), PfMSP2, PfMSP3, PfMSP4, PfMSP5, PfMSP6, PfMSP7, PfMSP8, PfMSP9, PvMSP1, PvMSP2,

PvMSP3, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PfAMAl (Apical Membrane Antigen), PvAMAl, PfRBP (Reticulocyte Binding Protein), PvRBP, PfEMP1 (Erythrocyte Membrane Protein), PvEMP1, Pfs16, Pf332, Pfs25, Pfs28 , PfLSA1 (hepatic stage antigen), PfLSA3, PvLSA1, PvLSA3, PfEBA (erythrocyte binding antigen), PvEBA, PfGLURP (glutamate-rich protein), PvGLURP, PfRAP1 (rhoptry-associated protein), PvRAP1, PfRAP2, PvRAP2, PfSequestrine, PvSequestrine, PfSALSA (sporozoite and hepatic 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.

admixtures

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

Adjuvants comprising saponins have been described in the state of 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), has been described by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv für die gesamte Virusforschung, Vol 44, Springer Verlag, Berlin, 243) as possessing adjuvant activity The purified fractions of Quil A were isolated by HPLC, which retain activity adjuvant without Quil A-associated toxicity (Kensil et al (1991) J. Immunol 146: 431) Quil A fractions were also described in US 5,057,540 and "Saponins as vaccinia adjuvants", Kensil, CR, Crit Rev. Ther Drug Carrier Syst, 1996, 12 (12): 1-55).

Two of these moieties, 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 moiety for use in the present invention. QS21 has been described by Kensil (2000) in O'Hagan: Vaccine Adjuvants: preparation methods and research protocols. Homana Press, Totowa, New Jersey, Chapter 15. Particular adjuvant systems comprising QuilA fractions, such as QS21 and QS7, are described, for example, in WO 96/33739, WO 96/11711 and WO 2007 / 068,907.

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

Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3D-MPL. U.S. Patent 4,436,727 describes MPL and its manufacture. U.S. Patent 4,912,094 and Reexamination Certificate B1,4912,094 discloses 3D-MPL and a method for its manufacture. Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a lipid A synthetic molecule (see, for example, Fox et al (2012) Clin Immunol Vaccine 19: 1633). In another 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 it may be synthetic monosaccharide molecules, such as compounds aminoalkyl glucosaminide phosphate (AGP) described, for example, in WO 9850399, WO 0134617, WO 0212258, WO 3065806, WO 04062599, WO 06016997, WO 0612425, WO 03066065, and WO 0190129. Such molecules have also described in the scientific and patent literature as lipid A mimetics. The lipid A mimetics appropriately share some functional and / or structural activity with lipid A, and in one aspect they are recognized by the receptors. of TLR4. AGPs as described herein are sometimes referred to as lipid A mimetics in the state of 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 an appropriate form of the present invention, the compositions of the invention may comprise QS21 in a substantially pure form, i.e., the QS21 is at least 80% pure, at least 85% pure, at least 90%, for example at least 95% pure, or at least 98% pure. The compositions of the invention may comprise QS21 in an amount of between about 1 μg and 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 be present, for example, in an amount between about 40 μg and 60 μg or between about 45 and about 55 μg or about 50 μg. Alternatively, the 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 another embodiment, the 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 another embodiment, the compositions of the invention may comprise QS21 in an amount of about 5 μg, for example between about 3 μg and about 7 μg or between about 4 μg and about 6 μg.

The compositions of the invention may comprise 3D-MPL in an amount of between about 1 μg and 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. 3D-MPL may be present, for example, in an amount between about 40 μg and 60 μg or between about 45 and about 55 μg or about 50 μg. Alternatively, the 3D-MPL 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 another embodiment, the compositions of the invention may comprise 3D-MPL 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 another embodiment, the compositions of the invention may comprise 3D-MPL in an amount around about 5 μg, for example between about 3 μg and about 7 μg or between about 4 μg and about 6 μg.

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

In some embodiments, the adjuvant is in the form of an oil-in-water emulsion, for example, comprising squalene, alpha-tocopherol and a surfactant (see, for example, WO 95 / 17210) or in the form of a liposome. A liposomal presentation is preferred. The term "liposome", when used herein, refers to single- or multilamellar lipid structures (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10-lamellar depending on the number of membranes formed lipids) containing an aqueous interior. Liposomes and liposomal formulations are well known in the state of the art. Liposomal presentations are described, for example, in WO 96/33739 and WO 2007/068907. Lipids that are capable of forming liposomes include all substances having fat or grease properties. The lipids that may constitute the lipids in the liposomes may be chosen from the group comprising glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulpholipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, and sterols. archaolipids, synthetic cationic lipids and lipids containing carbohydrates. 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 derivatives: egg phosphocholine, egg phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipids; and synthetic phospholipid derivatives: phosphocholine (didecanoyl-La-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoylphosphatidylcholine [DPPC], distearoylphosphatidylcholine [DSPC], dioleoylphosphatidylcholine, [DOPC] ], 1-palmitoyl, 2-oleoylphosphatidylcholine [POPC], diallydoylphosphatidylcholine [DEPC]), phosphoglycerol (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], 1,2-dipalmitoyl-sn-glycerol 3-phosphoglycerol [DPPG], 1,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG]), phosphatidic acid ( 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoylphosphatidic acid [DPPA], distearoylphosphatidic acid [DSPA]), phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine [DMPE], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-distearoyl-sn-gly cero-3-phosphoethanolamine [DSPE], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]), phosphoserine, and polyethylene glycol [PEG] phospholipid.

The size of the liposomes can vary from 30 nm to several μm depending on the composition of phospholipids and the process used for their preparation. In particular embodiments of the invention, the size of the liposomes will be in the range of 50 nm to 500 nm and in other 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, the liposomes used in the invention comprise DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, the compositions of the invention comprise QS21 in any amount herein described in the form of a liposome, wherein said liposome comprises DOPC and a sterol, particularly cholesterol.

In a preferred embodiment, the 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 liposomal formulation or about 25 μg of QS21 and about 25 μg of 3D-MPL per human dose in a liposomal formulation.

In another aspect, the invention relates to a method of making an immunogenic composition as defined herein comprising the step of mixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin moiety. An antigen is proposed which may be in lyophilized form or in a liquid formulation. Similarly, in embodiments where the CelTOS antigen is combined with another antigen, such as RTS, S, the antigens can be colyophilized together in a vial.

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

It is well known that for parenteral administration the solutions will have to be physiologically isotonic (i.e. they will have a pharmaceutically acceptable osmolality) to avoid cell distortion or lysis. An "isotonicity agent" is a compound that is physiologically tolerated and imparts appropriate tonicity to a formulation (eg, the immunogenic compositions of the invention) to prevent the net flow of water through 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 WO 2008/142133 or liposomal adjuvants described in US Pat. WO 2007/068907.

In some embodiments, the isotonicity agent used for the composition is a salt. However, in other embodiments, the composition comprises a nonionic 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, for example, less than at 30 mM, as less than 10 mM or less than 5 mM. In a preferred embodiment, the nonionic isotonicity agent is a polyol, such as sorbitol. The concentration of sorbitol may range, for example, from about 3% to about 15% (w / v), such as from about 4% to about 10% (w / v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist, where the isotonicity agent and a salt or a polyol, have been described in WO 2010142685, see for example Examples 1 and 2 in the WO document 2010142685.

Uses, doses and immunization 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 vaccines, i.e. for the prevention or prophylaxis of malaria in humans, including the prevention of malaria infection and malarial disease and / or the reduction of the severity of a malarial disease.

Thus, in one aspect, the invention relates to an immunogenic composition as defined herein for use in the prevention of malaria. In a similar manner, the invention relates to the use of an immunogenic composition as defined herein for the manufacture of a medicament for the prevention of malaria. In addition, the invention provides a method of immunizing against malaria comprising administering an immunogenic composition as defined herein to a human subject.

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

The immunogenic compositions of the invention can be used for any group of patients, including the pediatric as well as adult population. A group of target patients particularly suitable for immunization with the compositions as defined herein includes the pediatric population, including children aged 6 to 12 weeks and children aged 5 to 17 months. Another particularly appropriate target population includes travelers to areas where malaria is endemic.

In some embodiments, the immunogenic composition of the invention is administered only once to a subject. In another 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, generally, there is a time interval between administrations, such as 1 to 4 weeks or more, for example 1 to 3 weeks or more, to allow the first immunization to produce its immunogenic effect.

In still other embodiments, the immunogenic composition of the invention is administered one or more times, this followed by one or more other administrations (recalls) with a different immunogenic composition, for example a non-adjuvanted composition comprising CelTOS or a composition comprising CelTOS with a different adjuvant. In another embodiment, the first immunization (sensitization) is performed with a composition according to the invention comprising a polynucleotide encoding CelTOS and one or more other immunizations (recalls) are made with a composition comprising a CelTOS polypeptide. An example of a coding sequence for PfCelTOS is the sequence represented by the NCBI reference sequence XM_001350533.1 (hereby incorporated by reference) (SEQ ID NO: 4): ## EQU1 ## where: 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 coding sequence for PvCelTOS is the sequence shown in GenBank: AB194053.1 (hereby incorporated by reference) (SEQ ID NO: 5): Atacctatat 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 RTS antigen, S is in the same composition or in a therapeutic regime comprising a separate administration of the composition according to the invention. invention and a composition comprising RTS, S. For example, the composition of the invention may be coadministered with a composition comprising RTS, S at discrete anatomical sites (e.g. right and left arm) or at the same anatomical site. Alternatively, the administrations may 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 one or the other of them and, by example, alternating at each point of immunization time. The immunizations may be given at a given anatomical site or they may also be alternated between two anatomical sites (the composition containing RTS, S at a site and the composition containing CelTOS at a remote site). Preferably, the composition comprising RTS, S will also be adjuvanted with an adjuvant comprising an immunologically active saponin fraction, for example QS21, and optionally a TLR4 agonist, such as 3D-MPL.

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

Suitably, the immunogenic compositions of the present invention have a human dose volume of between 0.25 ml and 1 ml, particularly a dose volume of about 0.5 ml, or 0.7 ml. This may depend on the route of administration of smaller doses that are given intranasally or intradermally. The teaching of all references in this application, including patent applications and issued patents, is hereby incorporated in its entirety by reference. The terms "comprising", "include" and "includes" here are optionally substitutable by the terms "consisting of", "consist of" and "consists of", respectively. The invention will be further described with reference to the following non-limiting examples.

Examples

Example 1 - Production of antigen and adjuvant The CelTOS antigen of P. falciparum was produced in E. coli essentially as described in Bergmann-Leitner et al. (2010) PLoS ONE 5 (8) e12294. The protein was purified to homogeneity, using a two-step purification method, a) affinity purification using Ni + 2-NTA Sepharose (QIAGEN) and b) an anion exchanger Q Sepharose ( GE). The purified protein was exchanged for buffer by ultrafiltration (UF, GE Healthcare, Piscataway, NJ) to the final buffer composition of 10 mM sodium phosphate (monobasic), 150 mM sodium chloride, pH 7, 2.

The premix of AS01 adjuvant was manufactured as described in WO 96/33739, incorporated herein by reference. In particular, AS01 adjuvant was prepared essentially as in Example 1.1 of WO 96/33739. Adjuvant AS01 comprises: liposomes, which in turn comprise dioleoylphosphatidylcholine (DOPC), cholesterol and 3D-MPL (in an amount of 500 μg of DOPC, 125 μg of cholesterol and 25 μg of 3D-MPL) , QS21 (25 μg), NaCl phosphate buffer and water for a volume of 0.5 ml. The MPL emulsion (MPL-E) contained 50 micrograms of 3D-MPL per ml of 2.5% v / v of squalene, 2.5% v / v of alpha-tocopherol and 0.91% v / v Tween® 80 in PBS pH 7.4.

Montanide ™ ISA 720 VG (VG = veggie (vegetables), no animal products contained therein) (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 70/30 ratio (volume / volume).

Example 2 - Immunization

Ten groups of BalbC mice were immunized three times (Day 0, Day 21, Day 42) according to the schedule presented in Figure 1. The compositions and modes of administration tested were as follows:

Group 1: 10 μg 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: adjuvant ISA-720 ™ alone, subcutaneous

Group 8: MPL-E adjuvant alone, subcutaneous

Group 9: Adjuvant AS01 alone, subcutaneous

Group 10: adjuvant AS01 alone, intramuscular

Thus, groups 3-6 received compositions comprising CelTOS antigen and saponin-containing adjuvant (QS21) (AS01).

The compositions for immunization were prepared as follows:

The total volume administered for all groups of mice was kept at 100 μΐ, except that for intramuscular injections, the dose was divided between the large muscles in the hind paws of each mouse, therefore 2 x 50 μ! for each injection. Subcutaneous injections were performed in the inguinal region.

There were 15 mice in each group. Sera from mice per group were used to measure PfCelTOS specific antibody concentrations (Example 3), to test IFA fixed sporozoite recognition (Example 7) and to test sporozoite motility inhibition. (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 sera samples collected at days 5, 17, 37 and 58 (only groups 1-8 were individually tested, while adjuvant control groups were tested under form of grouped sera). The sera obtained from the adjuvant controls did not react on the PfCelTOS-specific ELISA assays (data not shown). Quantitative ELISA was performed on the samples using standard procedures. The plates were covered with 25 ng of PfCelTOS protein per well. Results

The ELISA results are shown in FIG. 2. As can be seen from this figure, all compositions containing the antigen induced antibodies specific for

PfCelTOS. Antibody responses were higher in groups that had been immunized with 10 μg of PfCelTOS compared to the group that had been immunized with only 1 μg of the same antigen. PfCelTOS-specific antibody levels induced by intramuscular administration of 10 μg of PfCelTOS / AS01 (group 6) were approximately two-fold higher compared to the time when the antigen was adjuvanted with Montanide ISA-720 ™ and administered by subcutaneous (group 1) (35.52 versus 14.82 μg / ml, respectively).

Example 4 - Cellular Responses

The patterns of cellular immune responses were investigated by quantifying the number of interferon-gamma-producing (IFN-gamma) and interleukin-4 (IL-4) splenocytes using the methods and tests described in Bergmann-Leitner. et al. (2010) PLoS ONE 5 (8) e12294. As a negative control, the samples were stimulated ex vivo with glutathione-S-transferase (GST).

CelTOS of P. berghei (Pb) 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) e12294. The protein included an N-terminal 16-amino acid sequence containing 6 histidines. Results

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

Figure 4 shows the number of splenocytes producing IL-4 following ex vivo stimulation. After ex vivo stimulation with PfCelTOS, clear responses were obtained in all groups that received 10 μg of PfCelTOS antigen (groups 2, 4 and 6). The highest response was obtained in groups that received subcutaneous administration of 10 μg PfCelTOS adjuvanted with AS01 (group 4). When the results (IFN-γ 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). Cell analysis did not reveal a dose response with respect to PfCelTOS-specific splenocyte counts producing IFN-γ or IL-4.

When ex vivo stimulation was performed with the heterocytic antigen CelTOS from P. berghei (PbCelTOS), a cross-reactivity response for IL-4 was observed in the groups that had received a high dose of PfCelTOS adjuvanted with AS01 (groups 4 and 6). Such a response was not observed in the group in which the composition was adjuvated with MPL-E (group 2).

Example 5 - Sporozoite Test

The ability of the immunogenic compositions to induce sterile protection in mice against a heterologous challenge with P. berghei sporozoites was tested. The sporozoite challenge was performed as described in Bergmann-Leitner et al. (2010) PLoS ONE 5 (8) e12294. At day 76, day 78 and day 84 (final day), blood samples were taken, deposited on a microscope slide, spread, fixed with methanol and stained with Giemsa stain and air dried. Parasitaemia was analyzed microscopically on these samples to determine if the kinetics of infection differed by vaccination group. If, according to the final blood smear at day 84 (day 14 since challenge), the mice remained aparasitaemic, the mice were considered "sterile protected". Efficiency was calculated by the equation presented here:

Efficacy = [1 - [(number of infected animals (I) vaccine / total number of animals (n) vaccine) v (number of infected animals controls (I) / total number of animals (n) controls)] ] * 100.

Groups 7, 8, 9 and 10 were used as a control group for group 1, group 2, 3 + 4 groups and 5 + 6 groups, respectively. Results

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

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

Western blot analysis was performed to test cross-reactivity of antisera with non-falciparum species. The SDS-PAGE and Western blot procedures were in accordance with standard operating procedures. For each group, the pooled serum, after the 3rd sample, was tested at the required dilution to obtain OD = 1 (determined by ELISA using PfCelTOS, essentially as described in Example 3). Equal amounts of recombinant proteins (0.5 μg per lane) were loaded in several lanes on 4 to 20% SDS-PAGE Tris-glycine gels.

CelTOS of P. knowlesi was produced in E. coli as follows. The gene was subcloned into a modified pETK vector and expressed in E. coli BL21 DE3. The purification process used to isolate this protein until it becomes homogeneous is the same method as that used for PbCelTOS. In all cases, affinity chromatography with Ni-NTA is used first, followed by passage over a Q Sepharose anion exchanger. Proteins were exchanged with the buffer in a final buffer composition of 1X PBS pH 7.4 for storage. Results

Serum from the four groups at the 10 μg dose of PfCelTOS tested (Groups 1, 2, 4 and 6) reacted with CelTOS of P. falciparum and CelTOS of P. berghei (see Corridors 2 and 3 on each panel) . However, only sera from group 6 (mice immunized intramuscularly with PfCelTOS / AS01) reacted with CelTOS from P. knowlesi, suggesting that intramuscular immunization with PfCelTOS / AS01 triggered an immune response with broader cross-reactivity ( Figure 6).

Example 7 - Immunofluorescence test on sporozoites

Immunofluorescence assays were performed as essentially described in Bergmann-Leitner et al. (2010) PLoS ONE 5 (8) e12294 to determine whether PfCelTOS / AS01-induced antibodies could recognize native antigen on or within sporozoites of salivary glands dissected on fixed parasites. Immunostaining using anti-PfCelTOS polyclonal antisera from these Balb / c mice showed reactivity on P. falciparum homologous and heterologous P. berghei-attached sporozoites. In addition, for the 10 μg dose of PfCelTOS / AS01 administered both intramuscularly and subcutaneously, the antibodies reacted on "live" sporozoites of P. falciparum to verify the extracellular localization (surface) of PfCelTOS. and validating the potential of antibodies to act as effectors in protection (data not shown). The data of Figure 7 is plotted as the median titer of antibodies, as well as the first and third quartiles, obtained at the end of the reaction. Outliers and extreme outliers appear outside the 1st and 3rd quartiles.

EXAMPLE 8 Inhibition of Sporozoite Motility by Antisera The Inhibition of the Sporozoite Motility Test is an in vitro test to characterize and semi-quantify the antiparasitic activity of polyclonal or monoclonal antibodies targeting the motile stages of the malaria parasite. during the pre-erythrocytic stage. Live sporozoites deposit a CS protein trail on the glass (Stewart and Vanderberg JP (1988) J Protozol 35: 389). Because sporozoite motility is an indirect measure of the viability and quality of parasites, a motility test can be used to test whether antibodies have an effect on the viability and health of the parasites. Such a motility test was performed on the mouse antisera, using essentially the same method as that described in Bergmann-Leitner et al. (2010) PLoS ONE 5 (8) e12294. Sporozoites of P. berghei and sporozoites of P. falciparum were tested. The percentage inhibition of sporozoite motility is calculated by counting the number of sporozoites and the number of CSP streaks per field (from at least 10 fields / slide) and is calculated as:% inhibition = [ 1 - [(n strands exp / n spz exp) / (n strikes control / n spz control)]] * 100; where exp means experimental and control means control serum that has never been in contact with the malaria parasite ("naive" control serum). Results

The results of the motility test are shown in FIG. 8. The highest degree of inhibition of sporozoite motility was induced by the sera obtained from mice immunized intramuscularly with PfCelTOS / AS01 against the two parasite species tested. P. falciparum and P. berghei (group 6).

Example 9 Immunogenicity (Antibody Responses) Following Two Immunizations with the Malaria Vaccine, FMP012 (PfCelTOS) / AS01, in US Subjects Never in Contact with Malaria (American "Naïve" Subjects)

Serum samples were obtained from a Phase I study with a controlled human malaria infection (CHMI), a non-random, open-label, escalating study in a clinical trial design on healthy adults never having have been in contact with malaria ("naive" for malaria) aged 18 to 50 years (including), to estimate the absence of risk, immunogenicity, and protection. To date, subjects in 2 groups received vaccinations at weeks 0, 4, 8 and were followed for adverse clinical effects and laboratory abnormalities. The PfCelTOS antigen is administered with AS01 adjuvant.

A total of 30 subjects were divided into low and high dose groups (15 per group), and received 3 doses of PfCelTOS / AS01 vaccine to date. Group 1 received 10 μg of PfCelTOS formulated with adjuvant AS01, group 2 received 30 μg of PfCelTOS formulated with adjuvant AS01. The composition of the adjuvant is constant and 500 μ! were administered intramuscularly to both groups. The design of the study included an off-label start for group 1 and group 2 with immunizations separated by 14 days. Five subjects in each group were immunized in a pilot group, 1 day before the rest of the group, for the first vaccination only (day -1 for group 1, day 13 for group 2). For this report, only the antibody titre for group 1 of PfCelTOS / AS01 (10 μg) will be presented.

Briefly, total IgG responses to PfCelTOS antigen were measured using standard ELISA methodologies by the Malaria Serology Lab (MSL) at WRAIR. The plates were covered with 100 μl / well of CelTOS antigen (Bergmann-Leitner et al (2010) PLoS ONE 5 (8) e12294) at a concentration of 0.25 μg / ml, placed in a humid chamber and incubated. overnight (16 to 20 h) at 4 ° C. The plates were washed four times with 1X 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 1X PBS solution between all subsequent steps except for the development reaction. Serum samples from the study subjects were serially diluted from 1/50 on each plate and incubated at 22 ° C for 2 h. Peroxidase labeled goat anti-human IgG antibody (KPL, Gaithersburg, MD, USA) was added to each well at a 1/4000 dilution and incubated for 1 h at 22 ° C. The ABTS peroxidase substrate (KPL, Gaithersburg, MD, USA) was added to induce the development of the reaction. At the end of the 1 hour incubation at 22 ° C, stop solution (20% sodium dodecyl sulfate) was added and the plates were read using a Spectromax® 0PC plate reader. 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). Serum titer was defined as serum dilution reaching an optical density (OD) of 1.0.

The results are reported graphically in Figure 9 using Minitab V16 for the individual values for Group 1 on a linear scale for the average antibody titre. The 95% confidence interval is represented. In addition, the geometric mean of the headings and the 95% confidence intervals and the median, with the 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)

Antibody titers were higher than those observed previously with three doses of PfCelTOS combined with an adjunct containing no saponin (Cowden et al (2012) Presentation at the 2012 ASTMH meeting, unpublished).

equivalent

The present invention provides inter alia immunogenic compositions comprising a CelTOS antigen and an adjuvant. While specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art after reading this description. The full scope of the invention will have to be determined with reference to the claims accompanied by their full scope of equivalents, and description, accompanied by such variations.

Incorporation by reference

All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated as incorporated by reference. In the event of a conflict, this application, including all definitions here, will prevail.

Also included by reference in their entirety are all polynucleotide and polypeptide sequences, which refer to an accession number correlating an entry into a public database such as those maintained by The Institute for Genomics Research (TIGR) (www.tigr .org) and / or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov).

Claims (27)

An immunogenic composition comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin moiety. The composition of claim 1, wherein the adjuvant comprises an immunologically active saponin moiety and a TLR4 agonist. 3. The composition according to any one of the preceding claims, wherein the immunologically active saponin fraction is QS21. 4. The composition according to any one of the preceding claims, wherein the adjuvant further comprises a sterol. The composition of claim 4 wherein the sterol is cholesterol. A composition according to any one of the preceding claims, wherein the immunologically active saponin fraction is QS21 and wherein the ratio of QS21 / sterol is 1/100 to 1/1 w / w. The composition of any one of the preceding claims, wherein the adjuvant comprises a TLR4 agonist and wherein the TLR4 agonist is a lipopolysaccharide. The composition of claim 7 wherein the lipopolysaccharide is 3D-MPL. A composition according to 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 between 10 and 100 μg per human dose. A composition according to any one of the preceding claims wherein the adjuvant is presented as a liposome. 11. A composition according to any one of the preceding claims, wherein the CelTOS antigen is a CelTOS polypeptide. The composition of claim 11, wherein the polypeptide is CelTOS of Plasmodium falciparum. The composition of claim 11 or 12, wherein the polypeptide comprises the sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or an immunogenic fragment of said polypeptide, or a variant of said polypeptide. A composition according to any one of the preceding claims, wherein the composition further comprises one or more other Plasmodium antigens. A composition according to any one of the preceding claims, wherein the composition further comprises an antigen of the circumsporozoite protein, such as RTS, S. 16. Composition according to any one of the preceding claims, for use in medicine. 17. A composition according to any one of the preceding claims for use in the prevention of malaria. 18. A composition according to any one of the preceding claims for use in the prevention of P. falciparum, P. vivax or P. knowlesi malarial disease. The composition of any one of claims 16 to 18, wherein the use comprises intramuscular administration of the composition. 20. A composition according to any one of claims 16 to 18, wherein the use comprises subcutaneous administration of the composition. 21. A composition according to any one of claims 16 to 18, wherein the use comprises 2 or 3 administrations of the composition. 22. Use of a composition according to any one of claims 1 to 15 for the manufacture of a medicament for the prevention of malaria. 23. A method of immunizing against malaria comprising administering the composition of any one of claims 1 to 15 to a human subject. 24. Kit comprising a CelTOS antigen and an adjuvant, wherein the adjuvant comprises an immunologically active saponin fraction. Kit according to claim 24, wherein the kit comprises one or more of the additional features of claims 2 to 15. 26. A method of manufacturing an immunogenic composition according to any one of claims 1 to 15 comprising the step of mixing a CelTOS antigen and an adjuvant comprising an immunologically active saponin fraction. The method of claim 26, wherein the composition comprises one or more of the additional features of claims 2 to 15.