Technical field

The present invention relates to methods for inducing an immune response against malaria, in particular methods for immunisation against malaria comprising administration of: i) a circumsporozoite (CS) antigen, which is a CS protein or an immunogenic fragment or variant thereof and ii) a nucleotide vector encoding a thrombospondin-related adhesive protein (TRAP) antigen, which is a TRAP protein or an immunogenic fragment or variant thereof. 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 2012, the World Health Organization reported an estimated 207 million cases of malaria globally. The disease killed approximately 627,000 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. 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 multiply and 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. The RTS,S vaccine is adjuvanted with AS01, a liposomal formulation containing QS21 and 3D-MPL. 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 almost halved the number of malaria cases in young children (aged 5-17 months at first vaccination) and reduced 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, and recently published (The RTS,S Clinical Trial Partnership, July 2014, PLoS Medicine, 7:el001685). In spite of this success of the RTS,S vaccine, a next generation of malaria vaccines with an efficacy closer to 100% are still needed to reduce mortality and address the goals of elimination and eventual eradication of malaria.

A further antigen which is being developed for use in malaria vaccination is thrombospondin- related adhesive protein (TRAP) (also termed thrombospondin-related anonymous protein), an antigen also expressed on sporozoites and at the liver-stage of infection. TRAP has been tested in vaccine clinical trials as both a DNA vaccine and as several viral vectored vaccines (MVA, chimpanzee adenovirus) encoding a fusion protein with a multi-epitope string containing additional B-cell, CD8+ and CD4+ T cell epitopes from several malaria antigens, known as ME-TRAP (Gilbert et al. 1997 Nat Biotechnol. 15:1280; Moorthy et al. 2003 Vaccine 21: 1995; Ewer et al 2013 Nat Commun 4:2836. doi: 10.1038/ncomms3836). WO2008122769 describes recombinant replication-defective simian adenovirus vectors encoding TRAP or variants thereof and the use of these vectors in vaccination. Chimpanzee adenovirus isolate ChAd63 (also known as AdCh63) encoding the ME-TRAP antigen was found to be immunogenic and immunogenicity could be boosted by a subsequent administration of an MVA vector coding for ME-TRAP. More recently, it was found that prime-boost immunisation with ChAd63 ME-TRAP and MVA ME-TRAP in healthy Gambian and Kenyan adults was safe and immunogenic (Ogwang et al. 2013 PLoS 8:e57726).

Investigators have explored combining RTS,S with other protein targets and vaccine platforms. For example, RTS,S has been combined with merozoite surface protein-1 or recombinant TRAP protein in adjuvant (Cummings et al. 2002 Safety, immunogenicity, and efficacy of candidate malaria vaccine containing CSP and MSP-1 antigens. Presented at: 51 ^st Annual Meeting of the ASTMH, Denver, CO, USA; Kester et al. Vaccine 2014, Jun 18). Unfortunately, even though the combination of RTS,S+TRAP was safe and immunogenic (Walsh et al. 2004 Am J Trap Med Hyg 70:499), enhanced protection was not observed (Kester et al. 2014, supra). This suggests that immunological mechanisms, not entirely understood, cannot be relied on when combining separate vaccines that theoretically would provide superior protection compared with each antigen alone (Regules et al. 2011 Expert Rev Vaccines 10:589). In other words it is still not possible to predict whether combining two partially protective malaria vaccines will lead to enhanced efficacy.

In conclusion, while significant progress has been made in the field of malaria vaccine research and development, there is still a need for novel methods of immunisation against malaria which are highly efficacious, broad-spectrum, safe and long-lasting.

Summary of the invention

It has now surprisingly been found that an immunisation regimen comprising administration of both a variant of CS protein and a nucleotide vector encoding a variant of TRAP yielded very high protection rates. Furthermore, when delayed infections were included as a recognized measure of vaccine efficacy, the efficacy became 100%. To our knowledge, this is the first vaccine regimen to show 100% efficacy in any malaria challenge study with a suitable sample size, for example >10 vaccine recipients per group. Most strikingly the efficacy of this combination approach was well maintained over six months post vaccination so that when vaccine recipients who were protected in the first month post vaccination were rechallenged at six months post-vaccination an unprecedented high level of durable efficacy was observed so that seven out of eight such re-challenge individuals were protected again at six months post-vaccination. Thus, in one embodiment, the method of the invention results in an efficacy at six months post vaccination which is higher than the efficacy obtained with an otherwise similar protocol which does not include an immunisation with a vector encoding a TRAP variant, such as a protocol as described in the Examples herein, only including immunisation with RTS,S/AS01B. Specifically the sterile efficacy rate at 6 months with the combination vaccine used here is 72% (82% initial sterile efficacy x 7/8 on re-challenge), whereas the sterile efficacy rate at six month reported by Kester et al ((2009) J Infect Dis 200, 337 346) was only 22% (50% initial sterile efficacy x 4/9 on re-challenge). In another embodiment, the overall efficacy at six month post vaccination (as determined according to the Example herein) is more than 65%, such as more than 70%. In another embodiment, the sterile efficacy at six months post vaccination (as determined according to the Example herein) is more than 50%.

Accordingly, in a first aspect of the invention, there is provided a method for inducing an immune response against malaria in a human subject comprising administration of

i) a circumsporozoite (CS) plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a nucleotide vector encoding a thrombospondin-related adhesive protein (TRAP) plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof. In a further aspect, there is provided an immunogenic composition(s) for use in a method for inducing an immune response against malaria in a human subject, wherein the method comprises administration of i) a CS plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a nucleotide vector encoding a TRAP plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

In an even further aspect, the invention relates to the use of

i) a CS plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a nucleotide vector encoding a TRAP plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

in the manufacture of a medicament for inducing an immune response against malaria in a human subject, wherein i) and ii) are administered sequentially or simultaneously.

In a further aspect, the invention relates to a composition, comprising:

i) a CS plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a nucleotide vector encoding a TRAP plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof. In a further aspect, the invention relates to a kit comprising:

i) a CS plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof, optionally in combination with an adjuvant (e.g., an AS01 adjuvant, such as AS01B) and ii) a nucleotide vector encoding a TRAP plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

Brief description of the figures

Figure la: Kaplan-Meier curves: time to patency. The percentage of subjects remaining aparasitaemic over time subsequent to sporozoite challenge.

Figure lb: Anti-CS antibody levels.

Figure 2a: TRAP T-cell immunogenicity measured by antibody-secreting spot-forming cells (SFC) (Y axis) versus anti-TRAP antibodies (X axis).

Figure 2b: CS T-cell immunogenicity (Y axis) versus anti-CS antibodies (X axis).

Figure 2c: TRAP T-cell immunogenicity (Y axis) versus anti-CS antibodies (X axis).

Figure 2d: Comparison of anti-TRAP antibody levels observed in various studies.

Figure 2e: Anti-CS antibody levels (Y axis) versus days to parasitaemia (blood film).

Figure 2f: Anti-CS antibody levels (Y axis) versus days to parasitaemia (20 parasites per ml by PCR).

Figure 2g: Anti-CS antibody levels (Y axis) versus days to parasitaemia (500 parasites per ml by PCR).

Figure 3: Kaplan-Meier curves: time to patency. The percentage of subjects in Groups 1 and 2 remaining aparasitaemic over time subsequent to re-challenge.

Figure 4: The amino acid sequence of RTS, in which the first 194 amino acids are P. falciparum CS polypeptide sequence and the last 230 amino acids are Hepatitis B S antigen sequence.

Figure 5: The amino acid sequence of ME-TRAP, with the 559 amino acid TRAP sequence, beginning at amino acid 232, underlined.

Figure 6: Nucleotide sequence encoding ME-TRAP

Figure 7: CS antibody responses as measured by ELISA. The VAC59 panel shows data from Example 2.

The VAC55 panel shows data from Example 1.

Detailed description

Definitions

When used herein, the term "immunogenic fragment" includes a fragment of a protein of any length provided that it retains immunogenic properties. For example, the fragment can comprise 5 or more consecutive amino acids, such as 10 or more consecutive amino acids, e.g. 20 or more consecutive amino acids, such as 50 or more consecutive amino acids, e.g. 100 or more consecutive amino acids of a protein.

A "variant" of a polypeptide may contain amino acid 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. 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. The term "conservative amino acid substitution" refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schinner., Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schinner, Principles of Protein Structure, Springer-Verlag). One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of Glu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of Val, Leu and He, (vii) a slightly-polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro, (ix) an aliphatic group consisting of Val, Leu, He, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr. 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 proteins where further epitopes have been added to an antigen. For example, further epitopes from other malaria antigens may have been added to a particular antigen. An example of such a variant is ME-TRAP, which is a variant of TRAP containing further epitopes from other plasmodial antigens, see below. 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. 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.

Further aspects and embodiments of the invention As described above, in a first aspect, the invention relates to a method for inducing an immune response against malaria in a human subject comprising administration of

i) a circumsporozoite (CS) plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a nucleotide vector encoding a thrombospondin-related adhesive protein (TRAP) plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

Typically, the aim of the method of the invention is to induce a protective immune response, i.e. immunise or vaccinate the subject in order to prevent infection and/or malaria disease. In one embodiment, the vaccine efficacy of the method of the invention is improved as compared to a treatment regimen which only comprises RTS,S. For example, the protection rate and/or the vaccine efficacy including delays in time to patency (i.e. the condition of showing detectable parasite infection, as determined according to the Examples herein) may be at least 10%, such as 25% improved over an otherwise existing but partially effective treatment regimen which only comprises RTS,S. In one embodiment, a protection rate and/or vaccine efficacy (including infection delays) of more than 80%, such as more than 90%, as determined according to the Examples herein, is achieved.

The circumsporozoite (CS) protein (see e.g. UniProt No. Q7K740 for the sequence of CS from isolate 3D7) is present on the sporozoites of all Plasmodium and is the most abundant sporozoite surface protein. CS protein is involved in the motility and invasion of the sporozoite during its passage from the site of inoculation into circulation, from where it migrates to the liver and enters hepatocytes.

A number of recombinant and synthetic CS constructs and variants have been tested for utility in immunisation against malaria. In one embodiment, the CS plasmodial antigen is a Plasmodium falciparum or Plasmodium vivax CS protein or an immunogenic fragment or variant thereof.

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 CS variant antigen may e.g. be in the form of a hybrid protein comprising substantially all the C-terminal portion of the CS protein, four or more tandem repeats of the CS protein immunodominant region, and HBsAg. The hybrid protein may comprise a sequence which contains at least 160 amino acids and which is substantially homologous to the C-terminal portion of the CS protein, but devoid of the hydrophobic anchor sequence. The CS protein may be devoid of the last 12 amino-acids from the C terminus. Further, it may contain 4 or more, e.g. 10 or more Asn- Ala-Asn-Pro tetrapeptide (NANP) repeat motifs.

The hybrid protein for use in the invention may be a protein which comprises a portion of the CS protein of P. falciparum substantially as corresponding to amino acids 207-395 of the CS protein derived from P. falciparum clone 3D7, derived from the strain NF54 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.

A particular hybrid protein for use in the invention is the hybrid protein known as RTS (figure 4) (SEQ ID NO: l) (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).

RTS may be in the form of RTS,S mixed particles. RTS,S particles comprise two polypeptides,

RTS and S, that may be synthesized simultaneously and spontaneously form composite particulate structures (RTS,S).

The RTS protein may be expressed in yeast, for example S. cerevisiae. In such a host, RTS will be expressed and spontaneously assembled into lipoprotein particles. The recipient yeast strain may already carry in its genome several integrated copies of a hepatitis B S antigen expression cassette. The resulting strain synthesizes therefore two polypeptides, S and RTS, that spontaneously co-assemble into mixed (RTS,S) particles. These particles may present the CSP sequences of the hybrid at their surface. The RTS and S in these mixed particles may be present at a particular ratio, for example 1:4.

RTS,S has been reviewed in e.g. Vekemans et al. (2009) Vaccine 27:G67 and Regules et al. (2011) Expert Rev. Vaccines 10:589.

In a further embodiment, the CS plasmodial antigen is derived from the CS protein of P. vivax (i.e., CSV). Suitable P. vivax CS protein variants have been described. For example, WO2008009652, which published in the US as US20100150998, describes suitable proteins for use in the present invention, e.g. hybrid fusion proteins comprising: a. at least one repeat unit derived from the repeating region of a type I circumsporozoite protein of P. vivax, b. at least one repeat unit derived from the repeating region of a type II circumsporozoite protein of P. vivax, and c. surface antigen S derived from Hepatitis B virus, or a fragment thereof. SEQ ID NO: 17 of WO2008009652 describes a specific hybrid fusion protein, termed CVS-S. When co-expressed with surface antigen S derived from hepatitis B virus, CSV-S,S vivax particles, analogous to RTS,S falciparum particles, are formed (WO2008009652). Such particles may also be used in the present invention.

In a further embodiment, the CS plasmodial antigen is a mixed particle comprising RTS and CSV- S, and optionally unfused S antigen of Hepatitis B. Such particles have been described in WO2008009650, which published in the US as US20100062028.

Accordingly, in one embodiment of the method or use of the invention, the CS plasmodial antigen is one or more of those selected from the group consisting of:

a. RTS,

b. CSV-S,

c. RTS,S d. CSV-S,S and

e. mixed particles comprising RTS and CSV-S, and optionally unfused S antigen of Hepatitis B virus.

The CS plasmodial antigen is administered in an amount sufficient to generate an immune response in the human subject. In a preferred embodiment, the CS plasmodial antigen is RTS,S and the amount of RTS,S is between 25 and 75, such as 50, micrograms per dose or between 12.5 and 37.5, such as 25, micrograms per dose or between 5 and 20, such as 10 μg per dose.

In a preferred embodiment, the CS plasmodial antigen is administered in combination with an adjuvant.

In one embodiment, the adjuvant comprises a TLR (Toll-like receptor) agonist. The use of TLR agonists in adjuvants is well-known in art and has been reviewed e.g. by Lahiri et al. (2008) Vaccine 26:6777. TLRs that can be stimulated to achieve an adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9. TLR2, TLR4, TLR7 and TLR8 agonists, particularly TLR4 agonists, are preferred.

Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3- O-deacylated monophosphoryl lipid A (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. TLR4 agonists, such as 3-O-deacylated monophosphoryl lipid A (3D-MPL), and their use as adjuvants in vaccines has e.g. been described in WO 96/33739 and WO2007/068907 and reviewed in Alving et al. (2012) Curr Opin in Immunol 24:310.

In a further embodiment, the adjuvant comprises an immunologically active saponin, such as QS21. 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 Quil A, such as QS21 and QS7, are e.g. described in WO 96/33739, WO 96/11711 and WO2007/068907.

In addition to the saponin component, 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.

In a preferred embodiment of the invention, the adjuvant comprises 3D-MPL and QS21.

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. Thus, in a preferred embodiment of the invention, the adjuvant comprises 3D-MPL and QS21 in a liposomal formulation.

The term "liposome" when used herein refers to uni- or multilamellar 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.

Liposome size may vary from 30 nm to several urn 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 one embodiment, the adjuvant comprises between 25 and 75, such as 50 micrograms, of 3D- MPL per dose and between 25 and 75, such as 50 micrograms of QS21 per dose. In another embodiment, the adjuvant comprises between 12.5 and 37.5, such as 25 micrograms, of 3D-MPL per dose and between 12.5 and 37.5, such as 25 micrograms of QS21 per dose. In a further embodiment, the adjuvant comprises between 5 and 20, such as 10 micrograms, of 3D-MPL per dose and between 5 and 20, such as 10 micrograms of QS21 per dose.

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 be 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 a salt or a polyol have been described in WO2010142685, see e.g. Examples 1 and 2 in WO2010142685.

As described above, the methods and uses of the invention include administration of a nucleotide vector encoding a thrombospondin-related adhesive protein (TRAP) antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

In one embodiment of the method or use of the invention, the TRAP plasmodial antigen is a

Plasmodium falciparum or Plasmodium vivax TRAP antigen or an immunogenic fragment or variant thereof (see e.g. Robson et al. (1988) Nature 335:79 for the sequence of TRAP from isolate T9/96 or UniProt ID number Q94727 for P. vivax TRAP). In a further embodiment, the TRAP plasmodial antigen is ME-TRAP, a TRAP fusion protein comprising a multi-epitope string containing additional B-cell, CD8+ and CD4+ T cell epitopes from several malaria antigens (SEQ ID NO:2) (figure 5) (Gilbert et al. 1997 Nat Biotechnol. 15:1280; Moorthy et al. 2003 Vaccine 21: 1995; WO2008122769). Figure 6 shows a vector insert encoding ME-TRAP (SEQ ID NO:3).

In one embodiment, the nucleotide vector encoding the TRAP plasmodial antigen is a recombinant replication-defective simian adenovirus vector. For example, a simian adenovirus vector which comprises a simian adenovirus genome into which is stably integrated a transgene which encodes a TRAP plasmodial antigen operably linked to regulatory sequences which direct expression of the TRAP plasmodial antigen in mammalian cells. In one embodiment, the simian adenoviral genome is the genome of a chimpanzee adenovirus vector. In a further embodiment, the regulatory sequences which direct expression of the transgene comprise a CMV promoter. For example, the regulatory sequences may comprise the promoter of the HCMV IE1 gene and a fragment of the 5' untranslated region of the HCMV IE1 gene including intron A.

In another embodiment, the simian adenovirus vector is ChAd63 which is derived from chimpanzee adenovirus isolate 63 (deposited with the ECACC under accession number 05011210) (SEQ ID NO:115 in US Patent Application 2011/0217332) or ChAd3, which is derived from chimpanzee adenovirus isolate 3 (SEQ ID NO: l in WO2005/071093) or ChAdOxl (Antrobus et al Molecular Therapy 22: 668 - 674, 2014; Dicks et al PLoS One 2012 7| e40385; WO2012/172277). WO2008/122769 describes recombinant replication-defective simian adenovirus vectors encoding TRAP or variants thereof, including ChAd63 encoding the ME-TRAP antigen. The TRAP-plasmodial-antigen-encoding nucleotide vector is administered in an amount sufficient to generate an immune response in the human subject. In one embodiment, between 1 x 10 ¹⁰ and 1 x 10 ¹¹ , such as 5 xlO ¹⁰ viral particles are administered per dose.

In some embodiments, the method or use of the invention comprises two administrations of a nucleotide vector encoding a TRAP plasmodial antigen. In such embodiments, the second administration (booster) may boost the immune response induced by the first administration (primer). In a preferred embodiment, the method or use comprises:

i) administering an adenoviral vector which encodes a TRAP plasmodial antigen as described herein above, and

ii) administering a non-adenoviral vector which encodes a TRAP plasmodial antigen, which is a

TRAP protein or an immunogenic fragment or variant thereof.

In one embodiment, said non-adenoviral vector is a recombinant pox virus vector, such as modified vaccinia Ankara (MVA). MVA is a highly attenuated strain of vaccinia virus that underwent multiple, fully characterised, spontaneous deletions during more than 570 passages in chicken embryo fibroblast (CEF) cells. These included host range genes and genes encoding cytokine receptors. The virus is unable to replicate efficiently in human and most other mammalian cells but the replication defect occurs at a late stage of virion assembly such that viral and recombinant gene expression is unimpaired making MVA an efficient single-round expression vector incapable of causing disseminated infection in mammals. The entire DNA sequence of MVA has been published (Antoine et al. (1998) Virology 244:365).

In a further embodiment, the adenoviral vector and the non-adenoviral vector both encode ME-

TRAP. WO2008122769 describes an MVA vector encoding ME-TRAP. In one embodiment, the non- adenoviral vector is MVA and between 1 x 10 ⁸ and 1 x 10 ⁹ , such as 2 x 10 ⁸ pfu are administered per dose.

In some embodiments, the non-adenoviral vector encoding a TRAP plasmodial antigen, such as MVA encoding ME-TRAP is administered more than once, such as 2, 3 or more times.

Immunization regimens

In some embodiments, the method of the invention or use of the invention comprises two or more, such as three, administrations of CS plasmodial antigen.

In a preferred embodiment, wherein the method or use comprises three administrations of CS plasmodial antigen, wherein the CS plasmodial antigen is the same in all three administrations and the CS plasmodial antigen is adjuvanted in all three administrations with an adjuvant comprising 3D-MPL and QS21 in a liposomal formulation. Most preferably, the CS plasmodial antigen is RTS,S.

In one embodiment, the time interval between each of the administrations of CS plasmodial antigen is between 1 weeks and 1 year, e.g. between 2 weeks and 6 months, such as between 2 weeks and 6 weeks, e.g. 4 weeks. An accelerated three-dose regimen could be administration of CS plasmodial antigen with 5-9 day intervals, such as administration on Day 0, Day 7 and Day 14. In some embodiments, the dose of CS plasmodial antigen and the dose of adjuvant are kept constant across all administrations.

In other embodiments, however:

a. the amount of CS plasmodial antigen is lower in the second administration, and/or one of the subsequent administrations, as compared to the amount of CS plasmodial antigen in the first administration

and/or

b. the amount of adjuvant is lower in the second administration, and/or one of the subsequent administrations, as compared to the amount of adjuvant in the first administration. For example, in one such embodiment, two or more administrations of CS plasmodial antigen, such as RTS,S, are combined with an adjuvant comprising 3D-MPL and QS21 in a liposomal formulation, but the amount of adjuvant is lower in the second administration, or one of the subsequent administrations, as compared to the amount of adjuvant in the first administration. For example, the lower amount is an amount at least 10% lower, such as at least 25% lower, e.g. at least two fold lower, such as at least three fold lower, e.g. at least four fold lower, such as at least five fold lower, e.g. at least six fold lower, such as at least seven fold lower, e.g. at least eight fold lower, such as at least nine fold lower, e.g. at least ten fold lower, such as at least 15 fold lower, e.g. at least 20 fold lower.

In another embodiment, the lower amount of adjuvant is an amount between 2 and 50 fold lower, such as between 2 and 20 fold lower, e.g. between 2 and 15 fold lower, such as between 2 and 10 fold lower, e.g. between 3 and 7 fold lower, such as between 4 and 6 fold lower.

In one embodiment, the first administration comprises between 25 and 75, such as 50 micrograms, of 3D-MPL and between 25 and 75, such as 50 micrograms of QS21 in a liposomal formulation and one or more of the subsequent administrations comprises between 5 and 15, such as 10 micrograms of 3D-MPL and between 5 and 15, such as 10 micrograms of QS21 in a liposomal formulation.

In another embodiment, the first administration comprises between 12.5 and 37.5, such as 25 micrograms, of 3D-MPL and between 12.5 and 37.5, such as 25 micrograms of QS21 in a liposomal formulation and one or more of the subsequent administrations comprises between 2.5 and 7.5, such as 5 micrograms of 3D-MPL and between 2.5 and 7.5, such as 5 micrograms of QS21 in a liposomal formulation.

In further embodiments, the amount of CS plasmodial antigen is lower in the second administration, or one of the subsequent administrations, as compared to the amount of CS plasmodial antigen in the first administration. For example, the lower amount is an amount at least 10% lower, such as at least 25% lower, e.g. at least two fold lower, such as at least three fold lower, e.g. at least four fold lower, such as at least five fold lower, e.g. at least six fold lower, such as at least seven fold lower, e.g. at least eight fold lower, such as at least nine fold lower, e.g. at least ten fold lower, such as at least 15 fold lower, e.g. at least 20 fold lower. In another embodiment, the lower amount is an amount between 2 and 50 fold lower, such as between 2 and 20 fold lower, e.g. between 2 and 15 fold lower, such as between 2 and 10 fold lower, e.g. between 3 and 7 fold lower, such as between 4 and 6 fold lower. In embodiments comprising administration of an adenoviral vector which encodes a TRAP plasmodial antigen as described herein above, followed by administration of a non-adenoviral vector which encodes a TRAP plasmodial antigen, wherein the TRAP plasmodial antigen is a TRAP protein or an immunogenic fragment or variant thereof, the non-adenoviral vector may e.g. be administered at least two weeks, e.g. between 2 and 12 weeks, after the adenoviral vector, such as 8 weeks after the adenoviral vector.

In one embodiment, the method or use of the invention comprises the following administrations, optionally in the specified order:

a. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, b. Administration of ChAd63 encoding ME-TRAP

c. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation d. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation e. Administration of MVA encoding ME-TRAP.

In a further embodiment hereof, the time interval between step a. and b. is between 1 and 3 weeks, such as 2 weeks, and wherein the time interval between step b. and c. is between 1 and 3 weeks, such as 2 weeks, and wherein the time interval between step c. and d. is between 1 and 8 weeks, such as 4 weeks, and wherein the time interval between step d. and e. is between 1 and 3 weeks, such as 2 weeks.

In further embodiments, the administration of one or all TRAP-encoding vectors is performed simultaneously, or essentially simultaneously, with the administration of a CS plasmodial antigen. In this context, "essentially simultaneously" means administration during the same visit to the clinic, i.e. on the same day, such as within 1 hour of one another, e.g. within 20 minutes, such as within 5 minutes of one another .

In particular embodiments, the TRAP-encoding vectors are co-administered both essentially simultaneously and at the same site on the body as the CS plasmodial antigen (e.g., in one arm).

In alternative embodiments, the invention specifically excludes the administration of one or all TRAP-encoding vectors simultaneously, or essentially simultaneously, with the administration of the CS plasmodial antigen. Again, in this exclusionary context, "essentially simultaneously" means administration during the same visit to the clinic, i.e. on the same day, such as within 1 hour of one another.

In particular embodiments, the invention specifically excludes the co-administration of the TRAP- encoding vector(s) and the CS plasmodial antigen at the same site on the body (e.g, in one arm) and at the essentially the same time. Thus, in such embodiments, the administrations are at distinct body sites. Administration at a "distinct body site" when used herein in the context of administration of two or more compositions to a subject means that the compositions are given a different sites, typically contra- laterally or at different limbs or in any event at sufficient distance from each other so that any local effects of the administrations do not interfere with each other. For example, the distance may be at least 0.5 cm, such as at least 1 cm, e.g. at least 5 cm.

Thus, in some embodiments of the method or use of the invention, the administration of one or all TRAP-encoding vectors is not performed essentially simultaneously with the administration of a CS plasmodial antigen.

In other embodiments, the administration of the one or all TRAP-encoding vectors is to a distinct body site from that those used for the administration of the CS plasmodial antigen.

In further embodiments, any nucleotide vector encoding TRAP plasmodial antigen is administered at a distinct body site, or given 1 or more days apart, from any administration of a CS plasmodial antigen, such as more than 2 days apart, e.g. more than 3, 4, 5, 6, or 7 days apart, such as 14 or more days apart.

In even further embodiments, if the method comprises two or more administrations of nucleotide vectors encoding a TRAP plasmodial antigen, the second and further administrations are given at a distinct body site, or given 1 or more days apart, from any administration of a CS plasmodial antigen, such as more than 2 days apart, e.g. more than 3, 4, 5, 6, or 7 days apart, such as 14 or more days apart.

In even further embodiments, if the method comprises administration with a non-adenoviral vector encoding a TRAP plasmodial antigen, said administration is given at a distinct body site, or given 1 or more days apart, from any administration of a CS plasmodial antigen, such as more than 2 days apart, e.g. more than 3, 4, 5, 6, or 7 days apart, such as 14 or more days apart.

In even further embodiments, if the method comprises administration with a MVA vector encoding a TRAP plasmodial antigen, said administration is given at a distinct body site, or given 1 or more days apart, from any administration of a CS plasmodial antigen, such as more than 2 days apart, e.g. more than 3, 4, 5, 6, or 7 days apart, such as 14 or more days apart.

In one embodiment, the method or use of the invention comprises the following administrations, optionally in the specified order:

a. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or essentially simultaneously, with administration of ChAd63 encoding ME- TRAP

b. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation simultaneously, or essentially simultaneously, with administration of MVA encoding ME-TRAP, and

c. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation simultaneously, or essentially simultaneously, with administration of MVA encoding ME-TRAP.

In another embodiment, the method or use of the invention comprises the following administrations, optionally in the specified order: a. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or essentially simultaneously, with administration of ChAd63 encoding ME- TRAP

b. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation simultaneously, or essentially simultaneously, with administration of MVA encoding ME-TRAP, and

c. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation.

In another embodiment, the method or use of the invention comprises the following administrations, optionally in the specified order:

a. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or essentially simultaneously, with administration of ChAd63 encoding ME- TRAP

b. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, and c. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation simultaneously, or essentially simultaneously, with administration of MVA encoding ME-TRAP.

In another embodiment, the method or use of the invention comprises the following administrations, optionally in the specified order:

a. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or essentially simultaneously, with administration of ChAd63 encoding ME- TRAP

b. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, and c. Administration of RTS,S adjuvanted with 3D-MPL and QS21 in a liposomal formulation.

In some embodiments, one or more TRAP-encoding vectors are mixed with the CS plasmodial antigen prior to administration.

For example, RTS,S and the ME-TRAP-encoding vector (such as ChAd63 encoding ME-TRAP) may be provided together in a vial in lyophilized form and be re-constituted with the adjuvant solution prior to administration. Alternatively, the components of the composition may be provided in a liquid formulation and be mixed prior to administration.

Thus, in a further aspect, the invention relates to a composition, such as a lyophilised or liquid composition, comprising:

i) a circumsporozoite (CS) plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof; and

ii) a nucleotide vector encoding a thrombospondin-related adhesive protein (TRAP) plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof.

In one embodiment, the CS is antigen is RTS,S and the nucleotide vector encoding a TRAP plasmodial antigen is ChAd63 encoding ME-TRAP. The human subject to be treated using the method of the invention may be of any age. The method of the invention could be used as part of an elimination program for malaria in which case immunisation of essentially the whole population, i.e. any age groups, might be useful. In one embodiment, however, the human subject is more than 18 years of age when the first composition is administered. In another embodiment, the human subject is less than five years of age when the first composition is administered. In a further embodiment, the subject is aged 6-12 weeks or less or 5-17 months. A further particularly suitable target population includes travellers to regions where malaria is endemic.

The antigens may be administered via various suitable routes, including parenteral, such as intramuscular, intradermal or subcutaneous administration.

In a further aspect, the invention relates to a kit comprising

i) a first container, such as a vial, comprising a circumsporozoite (CS) plasmodial antigen, which is a CS protein or an immunogenic fragment or variant thereof and

ii) a second container, such as a vial, comprising a nucleotide vector encoding a thrombospondin- related adhesive protein (TRAP) plasmodial antigen, which is a TRAP protein or an immunogenic fragment or variant thereof, and

iii) optionally, a separate third container with adjuvant for CS antigen administration and/or instructions for use of the kit in the immunisation of human subjects against malaria according to any of the methods described herein.

Immunogenic compositions used in the invention may be made by admixing the antigen(s) and the adjuvant. The antigen(s) may be provided in a lyophilized form or in a liquid formulation. For each composition, a kit may be provided comprising a first container comprising the antigen(s) and a second container comprising the adjuvant. In another embodiment, both components are provided in a single formulation.

Suitably, the immunogenic compositions according to the present invention have a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.5 ml, in particular a dose volume of about 0.5 ml, or 0.7 ml. The volume of the second immunogenic composition may be reduced, and e.g. be between 0.05 ml and 0.5 ml, such as between 0.1 and 0.2 ml. The volumes of the compositions used may depend on the delivery route with smaller doses being given by the intradermal route.

The teachings of all references in the present application, including patent applications and granted patents, are herein incorporated by reference in their entireties. 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, example:

EXAMPLE 1 A phase I/IIa sporozoite challenge study to assess the safety and protective efficacy of the combination malaria vaccine candidate regimen of RTS,S/ ASOIB + ChAd63 and MVA encoding ME-TRAP and also RTS,S/AS01B alone.

A phase I/IIa clinical trial was performed in which the safety, immunogenicity and protective efficacy of two candidate immunization regimens was tested.

The first immunization regimen (Group 1) consisted of 3 administrations of RTS,S/AS01B, one administration of ChAd63 encoding ME-TRAP and one administration of MVA encoding ME-TRAP. The second immunization regimen (Group 2) consisted of 3 administrations of RTS,S/AS01B alone. Furthermore, unvaccinated controls (Group 3) were included.

The study was an open label, partially randomised, multi-centre phase I/IIa controlled human malaria infection (CHMI) study. The study population was healthy adults aged 18-45 years. Group sizes: Groups 1, 2 and 3 contained 17, 16 and 6 volunteers at the time of challenge, respectively

Study vaccines

RTS,S/AS01B: RTS,S was produced in yeast (S. cerevisiae) essentially as described in WO 93/10152. 3D- MPL/QS21 liposomal adjuvant (AS01) was produced essentially as described in Example II in WO2007/068907. 50 micrograms (meg) RTS,S with sucrose as cryoprotectant, presented as a lyophilized pellet in a single dose vial was reconstituted with AS01B adjuvant in liquid form. AS01B adjuvant contained 50 meg of MPL and 50 meg of QS21 with liposomes. The injectable volume after reconstitution was 0.5 ml. Each RTS,S/ AS01B dose was given intramuscularly.

ChAd63 ME-TRAP was made as describe previously (O'Hara et al. 2012 J Infect Dis 205:772; Ewer et al 2013 Nat Commun 4:2836. doi: 10.1038/ncomms3836). ChAd63 ME-TRAP was given intramuscularly at a dose of 5 x 10 ¹⁰ vp. MVA ME-TRAP was made as described previously (McConkey et al. 2003 Nat Med 9:729; O'Hara et al. 2012 J Infect Dis 205:772; Ewer et al 2013 Nat Commun 4:2836. doi: 10.1038/ncomms3836). MVA ME-TRAP was given intramuscularly at a dose of 2 x 10 ⁸ pfu.

Vaccination regimens

The vaccination regimens that were used are indicated in the following table:

CHMI

CHMI by sporozoite challenge (mosquito bite) was performed at week 12. CHMI was performed as described in Ewer et al 2013 Nat Commun 4:2836. doi: 10.1038/ncomms3836. Post-CHMI, twice daily and daily visits were scheduled to enable measurement of the time to the efficacy endpoints of 20 P. falciparum parasites/ml in peripheral blood by qPCR (performed as described in Andrews et al. 2005 Am J Trap Med Hyg 73:191), 500 parasites/ml in peripheral blood by qPCR, and blood stage infection defined by a composite of symptoms, blood film result and parasitaemia.

Immunogenicity

T cell responses to ME-TRAP were measured by ELISpot assays and by flow cytometry as described in O'Hara et al. 2012 J Infect Dis 205:772 and Ewer et al 2013 Nat Commun 4:2836. doi: 10.1038/ ncomms3836. CS antibody responses were measured by ELISA as described in Kester et al J Infect Dis 2009. Results

Figure la shows the percentage of subjects remaining aparasitaemic by blood film and by PCR over time subsequent to sporozoite challenge.

Protection - sterile efficacy

Subjects who remained aparasitaemic by blood film and by PCR after 20 days were considered protected. The results for the different groups were as follows:

Group 1: 14 out of 17 volunteers were protected = 82.4%

Group 2: 12 out of 16 volunteers were protected = 75%

Group 3: 0 out of 6 volunteers were protected = 0%

Statistical significance:

Group 1 vs Group 2 vs Controls (log-rank) P = 1 x 10-8

Group 1 vs Controls: Hazard ratio (log-rank) = 0.065 [0.003-0.19], P < 10-7

Group 2 vs Controls: Hazard ratio (log-rank) = 0.122 [0.004- 0.13], P < 10-3

Group 1 vs Group 2: Hazard ratio (log-rank) = 0.65 [0.15 - 2.86], P = 0.57

Vaccine Efficacy (protection + significant delay in time to patency of malaria infection)

In the control group (Group 3), all 6 subjects became positive by microscopic examination of blood film slides at day 11 (1 subject), 11.5 (1 subject), 12 (1 subject), 12.5 (1 subject) and 13 (2 subjects), respectively. This gives a mean of 12.17 +/- 0.8 days (Standard Deviation, SD). Vaccine recipients that are delayed to more than the mean plus two standard deviations of the control group are considered "delayed in time to patency" and show partial vaccine efficacy. In this trial this would be a delay to later than 12.17 plus (2 x 0.8) days = 13.77 days.

In Group 1, the 3 subjects that did become positive before day 21 became positive at day 14 (1 subject), 14.5 (1 subject) and 16 (1 subject). Thus, in all 3 subjects, there was a significant delay in this mean (14.83 days) as compared to the mean of Group 3 (12.17 days) of 2.66 days. This delay of 2.66 days is more than 2 times the standard deviation of the control group (0.8 days) and therefore considered statistically significant. All three subjects were delayed to beyond day 13.77. In Group 2 the 4 subjects that did become positive before day 21 became positive at day 11.5 (1 subject), 12.5 (1 subject), 14 (1 subject) and 19 (1 subject). Thus, in 2 subjects, there was a significant delay as compared to the mean of Group 3 because 2 subjects were delayed to beyond day 13.77.

Thus, if subjects in which a significant delay of infection is observed are added to the sterilely protected subjects, Figure la, the vaccine efficacies become as follows:

Group 1: 17/17 show efficacy = 100%

Group 2: 14/16 show efficacy = 87.5%

Group 3: 0 / 6 show efficacy = 0% as expected

Statistical significance:

Group 1 vs Group 2 vs Controls (chi-square) P < Iff ⁶

Group 1 vs Group 2 (chi-square): P = 0.066 (i.e. 0/17 vs 2/16) A one-tailed P value is used as the prior hypothesis being tested was that Group 1 would be better protected than Group 2.

Immunogenicity

The most likely mechanism of relevance to the protective efficacy of the RTS,S vaccine is the induction of high titre antibodies. These were induced to a very similar level in Groups 1 and 2 (Figure lb) and these levels appeared similar to those in previous trials of RTS,S / AS01 in the USA (Kester et al. (2008) Vaccine 26, 2191 2202; Kester et al (2009) J. Infect. Dis. 200, 337 346). Antibodies to TRAP correlated with TRAP T cell immunogenicity in Group 1 (Figure 2a); but the much lower level of T cells induced to CS did not correlate with CS antibodies (Figure 2b). The CS antibody levels across individual vaccine recipients also did not correlate with levels of T cells to TRAP in Group 1 (Figure 2c). Antibodies to TRAP in Group 1 were found at a similar level to those in a previous vaccine trial (Vac045, Hodgson et al submitted) using ME-TRAP in the same vectors used alone (Figure 2d), suggesting no interference with TRAP antibody immunogenicity from the administration of RTS,S to the same individuals. Antibodies to CS correlated positively with all three measures of vaccine efficacy (Figure 2e, 2f, 2g), i.e. at time of diagnosis (Figure 2e), time to 20 parasites per ml measured by PCR (Figure 2 and time to 500 parasites per ml measured by PCR (Figure 2g). This supports a protective role of high titre antibodies to CS induced by these vaccination regimes, consistent with previous studies. T cells to TRAP measured by ELISPOT on the day before challenge showed a mean level in Group 1 vaccine recipients of 1372 spot forming cells per million but did not correlate with efficacy (not shown), although power to detect such a correlation was very low.

Main conclusions

The highest percentage of sterile protection (82.4%) was achieved in Group 1. To our knowledge, 82.4% protection is the highest protection rate ever detected in any vaccine trial group with a sample size > 10 in any malaria challenge study.

When delayed infections were included in the vaccine efficacy, the efficacy in Group 1 became 100%. To our knowledge, this is the first vaccine group to show 100% efficacy in any challenge study with a group size of 10 or more. Re-Challenge Data

The ability to provide sustained and durable efficacy is a key feature of immunisation. This can be a limiting feature in overall vaccine efficacy. For example in young infants in the phase III trial of RTS,S/AS01, the reduction in incidence of clinical malaria by 6-mo period was 47% (95% CI 39% to 54%) during months 1-6, 23% (95% CI 15% to 31%) during months 7-12, and 12% (95% CI 1% to 21%) during months 13-18 following dose 3. This may relate to the reduction in efficacy of the same vaccine when used in challenge studies in the USA in adults. Vaccine efficacy in the first month post vaccination (typically at 3 weeks after the last dose) was 50% in the Kester et al 2009 trial. But on re- challenge at 6 months only 4 of 9 individuals (44%) were again protected. This can be calculated to represent about 22% overall sterile efficacy at six months (50% x 44% = 22%). A previous re-challenge study with RTSS/AS02 (Stoute et al 1997 N Engl J Med 336(2):86, Stoute et al 1998 J Infect Dis 178(4): 1139) found an overall efficacy at 6 months of 17%. Similarly in a recent phase Ila study of an irradiated sporozoite vaccine by Sanaria vaccine efficacy in the first month appears high with 12/15 vaccine recipients showing no infection on challenge (Seder et al 2013, Science 341, 1359-1365). Allowing for the failure of infection in one challenge control this amounted to an overall estimated vaccine sterile efficacy rate of 76% (Seder et al 2013 Science 341, 1359-1365, see Figure 2 legend). However on re-challenge of six of these protected individuals at 5 months only 2 were again protected (Richie et al, presented at ICOPA meeting, Mexico City, August 2014) amounting to an overall vaccine sterile efficacy rate of (76% x 33% = 25%) 25%. Of the 14 protected volunteers in Group 1, 8 underwent re-challenge. Of the 12 protected vaccine recipients in Group 2, 6 underwent re-challenge. Re-challenge followed the standard five bite protocol (Ewer et al. Nat Communications 20134:2836. doi: 10.1038/ncomms3836) with P. falciparum and was undertaken six months after the last vaccination along with five new control volunteers. All control volunteers developed malaria with a mean time to patency of 12.4 days (Figure 3). The individual volunteers were diagnosed at days 11.5, 11.5, 12.5, 13, 13.5 respectively (standard deviation = 0.76 days). For Group 1, 7 out of 8 vaccine recipients were protected (87.5%) and the remaining volunteer was diagnosed at day 17.5. For Group 2 5/6 were protected (83.3%) and the remaining volunteer was diagnosed at day 14.5.

Overall Vaccine Sterile Efficacy at six months can thus be calculated for Group 1 as 82.4% x 87.5% = 72.1%. Overall Vaccine Sterile Efficacy at six months can be calculated for Group 2 as 75% x 83.3% = 62.5%

Comparison of Durable Protection Rates

To determine whether these improved levels of durable efficacy are significantly better than those reported previously, we compared the Group 1 and the Group 2 data to those reported by Kester et al. 2009 using RTS,S/AS01 in a previous trial.

Group 2 vs AS01 in Kester et a I 2009

Group 2:- 5 / (6 x 16/12) = 5 / 8 original vaccine recipients protected

AS01 Kester et al.:- 4 / (9 x 36/18) = 4 / 18 original vaccine recipients protected

Chi square test (http://www.openepi.com), mid-P exact value, 2 tailed, P = 0.07

Group 1 vs AS01 in Kester et a I 2009

Group 1:- 7 / (8 x 17/14) = 7 / 9.71 vaccine recipients protected

AS01 Kester et al:- 4 / (9 x 36/18) = 4 / 18 original vaccine recipients protected

Chi square test, mid-P exact value, 2 tailed, P < 0.02

This analysis indicated that the RTS,S/AS01 alone group was not significantly different to the use of the same vaccine regimen by Kester et al. However, Group 1 is significantly improved over RTS,S/AS01 used by Kester et a I (2009).

We conclude that the rate of sterile protection at 6 months for Group 1 is the highest ever reported at 72% and this appears to represent a very worthwhile improvement over RTS,S used alone. We also note that at both the first challenge and the re-challenge every volunteer in Group 1 showed significant vaccine efficacy: 17/17 in the first challenge and 8/8 in the re-challenge. Again, to our knowledge this level of durable efficacy is an unprecedented result in 30 years of malaria vaccine phase II trials.

EXAMPLE 2 Safety, immunogenicity and efficacy of the combination malaria vaccine regimen of RTS,S/AS01 concomitantly administered with ChAd-MVA viral vectors expressing ME-TRAP.

In a further study, concomitant administration of ChAd63 ME-TRAP and MVA ME-TRAP with RTS,S/AS01 was tested. The study was an open label, partially randomised, multi-centre phase I/IIa controlled human malaria infection (CHMI) study. The study population was healthy malaria-naive adults aged 18-45 years. Group sizes: Groups 1, 2, 3, 4 and 5 contained 8, 9, 10, 9 and 4 volunteers at the time of challenge, respectively.

Study vaccines

The study vaccines were the same as in Example 1, except that the 3 ^rd dose of RTS,S/AS01 given in two of the groups was only l/5 ^th of the standard dose. The vaccines were administered intramuscularly to the deltoid muscle of the left arm. When two vaccines were administered concomitantly these were given at essentially the same location and one administration immediately followed the other.

Vaccination regimens

The vaccination regimens that were used are indicated in the following table:

CHMI and immunogenicity assay

CHMI and immunogenicity assays were performed as described in Example 1.

Results

Participants

Ten subjects were enrolled to Group 1, Group 2 and Group 4. Eleven subjects were enrolled to Group 3. A total of 5 subjects withdrew from the study after enrolment, but before challenge (2 subjects from Group 1, and 1 subject from each of Groups 2, 3 and 4). Efficacy

Numbers of subjects exhibiting sterile protection in each group is summarized below.

Sterile protection by day 16/17:

Group 1: 6/8 subjects (75%)

Group 2: 8/9 subjects (88.9%)

Group 3: 6/10 subjects (60%)

Group 4: 5/9 subjects (55.6%)

Group 1&2 pooled: 14/17 (82.4%)

Group 3&4 pooled: 11/19 (57.9%)

Group 5 mean time to malaria diagnosis (+/-SD) = 11.63 days (+/- 0.22)

Group comparisons:

The data show that contrary to the increased efficacy observed in Example 1, concomitant administration of ChAd63 ME-TRAP and MVA ME-TRAP with RTS,S/AS01 did not result in increased efficacy over RTS,S/AS01 alone.

Vaccine Safety

No safety concerns were raised during the trial. No protocol defined stopping or holding rules were activated. A total of 2 serious adverse events (SAEs) occurred, but neither was related to vaccination. No suspected unexpected serious adverse reactions (SUSARs) occurred. A higher frequency of adverse events (AEs) was reported in groups 3 & 4, and the highest frequency of AEs in all groups was reported following the 2nd round of vaccinations. Frequency of adverse events in Groups 3 and 4 were still broadly comparable to previous trials in which RTS,S/AS01B and ChAd63/MVA ME-TRAP were given alone. Pain, feverishness, fatigue, myalgia, malaise were the most commonly reported AEs.

Immunoaenicity

CS Ab titres failed to boost after the 3rd dose of vaccine (Day 56) in Groups 3 and 4 and titres measured on the day before CHMI (C-l) was approximately 2.5 fold greater in Groups 1 and 2 than Group 3 and 4. (Figure 7 (VAC59 panel)). Significantly higher CS Ab titres were induced by RTS,S/AS01 alone (Example 2, Groups 1 and 2 (VAC59 panel in Figure 7) and Example 1, Group 2 (VAC55 panel in Figure 7)) or RTS,S/AS01 and viral vectors staggered (Example 1, Group 1 (VAC55 panel in Figure 7) than in Example 2, Groups 3 and 4 (VAC59 panel, Figure 7).

There was also only very limited boosting in TRAP Abs following the 3rd vaccination (Day 56) in Groups 3 and 4, compared with that observed in the Example 1 study (data not shown). T-Cell responses to TRAP as measured by Interferon-γ ELISPOT were not significantly different to those measured in the Group 1 subjects in the Example 1 study who received RTS,S/AS01B with ChAd63/MVA ME-TRAP, but with vaccinations staggered as opposed to given concomitantly (data not shown).