Vaccines for Malaria

The present invention relates to a novel hybrid/fusion protein, methods for preparing and purifying the same, its use in medicine, particularly in the prevention of malarial infections, for example those caused by Plasmodium vivax (P. vivax), compositions/vaccines containing the protein or antibodies against the protein such as monoclonal or polyclonal antibodies and use of the same, particularly in therapy. The invention also extends to lipoprotein particles of said hybrid/fusion protein and formulations/vaccines comprising the same and use thereof.

Malaria is one of the world's major health problems with more than 2 to 4 million people dying from the disease each year. One of the most prevalent forms of the disease is caused by the protozoan parasite P. vivax, which is found in tropical and sub-tropical regions. Interestingly the parasite can complete its mosquito cycle at temperatures as low as 15 degrees Celsius, which has allowed the disease to spread in temperate climates.

The life cycle of P. vivax is complex, requiring two hosts, man and mosquito for completion. The infection of man is initiated by the introduction of sporozoites in the saliva of an infected mosquito. The sporozoites migrate to the liver and there infect hepatocytes where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells (RBC) to initiate cyclical replication in the asexual blood stage. The cycle is completed by the differentiation of a number of merozoites in the RBC 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.

Due to the fact that the disease caused by P. vivax is rarely lethal, efforts to prevent and treat malaria have been focused on the more deadly form of the disease caused by Plasmodium falciparum (P. falciparum) .

Although the disease caused by P. vivax does not usually result in death of the patient, due to the volume of cases, which seems to be increasing, the significant impact on the quality of life of the patient, the increasing reports of the severe incidences of the disease resulting in anemia and death, and the economic impact, an effective vaccination for the disease is still required.

A feature of the P. vivax is that some strains are capable of causing delayed infection by remaining latent in the liver before emerging into the peripheral circulation to manifest clinical symptoms. Thus individuals, for example when traveling through an infected area, may be infected and yet may not exhibit symptoms for several months. This has the potential to cause the spread of the disease and for this reason persons traveling to infected areas are not allowed to donate blood for transfusion for a defined period of time after traveling to the infected region.

P. vivax malaria infection remains latent within the liver while the parasite is undergoing pre-erthrocytic shizogony. If the parasite is controlled at this stage before it escapes the liver no clinical symptoms of the disease, are observed in the patient.

The sporozoite stage of P. vivax has been identified as a potential target of a malaria vaccine. Vaccination with deactivated (irradiated) sporozoite has been shown to induced protection against experimental human malaria (Am. J, Trop. Med. Hyg 24: 297-402, 1975). However, it is has not been possible practically and logistically to manufacture a vaccine for malaria for the general population based on this methodology, employing irradiated sporozoites.

The major surface protein of the sporozoite is known as circumsporozoite protein (CS protein). It is thought to be involved in the motility and invasion of the sporozoite during its passage from the initial site of inoculation by the mosquito into the circulation, where it migrates to the liver.

The CS protein of Plasmodia species is characterized by a central repetitive domain (repeat region) flanked by non-repetitive amino (N-terminus) and carboxy (C- terminus) fragments. The central domain of P.vivax is composed of several blocks of a repeat unit, generally of nine tandem amino acids.

In certain Asian strains, after the central repeat region, an additional sequence of approximately 12 amino acids is present (see SEQ ID No 11). The function of the latter is not known. However, it is hypothesized by some that said amino acids may be linked to the delayed onset of clinical symptoms of the disease, although this has not been investigated. It is thought that the N-terminus is characterised by a sequence of 5 amino acids known as region I (see SEQ ID No 1). It is also thought that the C- terminus is charcterised by comprising a sequence of 18 amino acids known as region II. The latter contains a cell-adhesive motif, which is highly conserved among all malaria CS protein (see SEQ ID No. 2).

Several groups have proposed subunit vaccines based on the circumsporozoite protein. Two of these vaccines have undergone clinical testing; one is a synthetic peptide, the other is a recombinant protein (Ballou et al Lancet: i 1277 (1987) and Herrington et al Nature 328:257 (1987)). These vaccines were successful in stimulating an anti-sporozoite response. Nonetheless, the magnitude of the response was disappointing, with some vaccinees not making a response at all. Furthermore, the absence of "boosting" of antibody levels on subsequent injections and results of in vitro lymphocyte proliferation assays suggested that T-cells of most of these volunteers did not recognise the immuno-dominant repeat. Nonetheless, one volunteer vaccinated in each study did not develop parasitemia.

WO 93/10152 and WO 98/05355 describe a vaccine derived from the CS protein of P. falciparum and it seems that there has been some progress made towards the vaccination against P. falciparum using the approach described therein, see also Heppner et al. 2005, Vaccine 23, 2243-50.

The CS protein in P. falciparum has a central repeat region that is conserved. In contrast at least two forms (designated VK210 or type I and VK247 or type II) of the CS protein for P. vivax are known. This renders it more difficult to identify a construct of the CS protein with all the desired properties such as immogenicity, which provides general protection against P. vivax regardless of the specific type of CS protein because antibodies directed the central repeating region of type I do not necessarily recognize epitopes on the corresponding region of type II and vice versa.

A recombinant P. vivax CS protein was expressed and tested as a vaccine in the 1980- 1990's with limited success (Collins et al, 1989. Am. J. Trop. Med. Hyg. 40, 455-64). Some work has been done to develop a vaccine based on Multiple Antigen Peptides (MAP) employing one or more epitopes that are cross-linked (Nardelli and Tarn, 1995, Pharm. Biotechnol. 6, 803-19).

The present invention provides a new, improved antigen for use in malaria vaccines, which is believed to produce a humoral response and also a cellular immune response. The antigen/particle is believed to induce the production of antibodies against the CS protein of type I and type II. The antigen/particle may also induce T helper cells for example ThI and/or Th2 cells.

Accordingly, the present invention provides an immunogenic hybrid fusion protein comprising: a. at least one repeat unit derived from the central repeat section of a type I circumsporozoite protein of P. vivax, b. at least one repeat unit derived from the central repeating section of a type II circumsporozoite protein of P. vivax, and c. surface antigen S derived from Hepatitis B virus.

Sequence Listing

SEQ. ID. No. 1 Region I in the N-terminus (described above)

SEQ. ID. No. 2 Motif from Region II in the C-terminus (described above)

SEQ. ID. No. 3-9 Various monomers of type I CS protein

SEQ. ID. No. 10 Major monomer from type II CS protein SEQ. ID. No. 11 Additional amino acids found in Asian strains

SEQ. ID. No. 12 Nucleotide sequence for the hybrid protein (optimized for expression in E CoIi)

SEQ. ID. No. 13 Amino acid sequence for the hybrid protein CSV

SEQ. ID. No. 14 Minor monomer from type II CS protein SEQ. ID. No. 15 Nucleotide sequence for the hybrid protein CSV

(optimized for expression in yeast)

SEQ. ID. No. 16 Nucleotide sequence for the hybrid fusion protein CSV-S

SEQ. ID. No. 17 Amino acid sequence for the hybrid fusion protein CSV-S

SEQ. ID No. 18 Nucleotide sequence for an RTS expression cassette and predicted RTS ₅ S protein.

SEQ. ID No. 19 Nucleotide sequence for the hybrid fusion gene CSV-S

(cloned into pHIL-D2 integrative Pichia pastoris expression vector)

SEQ. ID No. 20 Amino acid sequence for the hybrid fusion protein CSV-S expressed in Pichia pastoris SEQ ID No. 21 Shown in Fig 12 is a comparison of the sequence of a full length recombinant CSV-S protein and a truncated protein

(CSVtr-S)

Figures

Figure 1 Shows an electron micrograph of mul timer lipoprotein particles of the hybrid protein of the invention produced in yeast strain

S.cerevisiae (with a constitutive promoter)

Figure 2 Is a Western Blot of a hybrid protein of the invention and comparator proteins (part of gel) Figure 2a Is a Western Blot of a hybrid protein of the invention and comparator proteins (full gel of Figure 2, wherein lanes 1, 2 and 3 in Figure 2 are lanes 1, 2 and 8 in Figure 2a)

Figure 3 Plasmid map for pRIT15546 recombinant vector employed for introducing the sequence encoding for the CSV-S fusion protein into the yeast host cell (episomal vector). Figure 4 Plasmid map of pGFl-S2 a plasmid prepared by GSK employed in forming the fusion protein of a desired antigen with the S antigen from Hepatitis B.

Cloning heterologous DNA sequences between Smal sites (after excision of the 12bp Smal DNA fragment) creates in- frame fusion with the S gene.

Figure 5 Plasmid map of Prit 15607 employed in forming the fusion protein of a desired antigen with the S antigen from Hepatitis B

Digestion with Notl liberates a 6.8 kb linear DNA fragment carrying the CSV-S expression cassette plus the HIS4 selective marker, being used for insertion into the yeast chromosome.

Figure 6 Shows a Western Blot of recombinant protein expressed in

Yl 840 Figure 7 Shows an electron micrograph of multimer lipoprotein particles of the hybrid protein of the invention produced in Pichia pastoris

(a "non-conventional" yeast, a methylotrophic yeast, in which the recombinant expression is driven by a methanol inducible promoter).

CSV-S particles were purified from soluble extracts (based on

RTS ₅ S purification process) and submitted to electron microscopy analysis. Particles were visualized after negative staining with phosphotungstic acid. The scale is equivalent to 100 nm. Fig 8 Plasmid map of pRIT15582

Digestion with Xhol liberates a 8.5 kb linear DNA fragment carrying the CSV-S expression cassette plus the LEU2 selective marker, being used for insertion into the yeast chromosome. Fig 9 Restriction map of the linear Xhol fragment used to integrate CSV-S cassette

Fig 10 Western blot of recombinant proteins expressed in strain Y 1835.

Panel A : WB revealed with anti-S antibody

Samples loaded (lOOμg total prortein/well): 1: Y1631 (RTS,S producing strain, as comparison)

2 : Y1835 3 : Y1835 4 : Y1834

Panel B : WB revealed with anti-CSV antibody Samples loaded ( 100 μg total protein/well) :

1 : Yl 631 (RTS,S producing strain, as comparison) 2 : Y1295 3 : Y1835 4 : Y1834

5: nr (another construct CSVS) 6 : nr (another construct -S antigen only) Fig 11 Electron micrograph of CSV-S, S mixed particles produced in strain Yl 835 CSV-S,S particles were purified from soluble cell extracts ( based on RTS, S purification process) and submitted to electron microscopy analysis. Particles were visualized after negative staining with phosphotungstic acid. The scale is equivalent to lOOnm.

Fig 12 Shows a comparison of recombinant full length CS protein and a truncated version of the CS protein (CSVtr-S).

Hybrid protein herein refers to protein derived from P. vivax type I and type II.

Hybrid fusion protein herein refers to protein derived from P. vivax type I and type II fused to another protein or fragment thereof.

In one aspect the hybrid fusion protein of the invention comprises a hybrid protein derived from the CS proteins of P. vivax (CSV) and a surface antigen from Hepatitis B, generally the major surface protein known as the S antigen, such as the S antigen derived from an adw serotype.

The CSV derived antigen component (ie the hybrid protein) of the invention is generally fused to the amino terminal end of the S protein. More specifically the C- terminus end of the CSV fragment is fused the N-terminus of said S antigen.

It is believed that the presence of the surface antigen from Hepatitis B boosts the immunogenicity of the CS protein portion of the hybrid protein, aids stability, and/or assists reproducible manufacturing of the protein.

Generally the hybrid protein will also contain an N-terminus fragment from a CS protein of Plasmodium such as P. vivax (type I or II), for example a fragment comprising region I such as the amino acids shown in SEQ ID No. 1.

Alternatively the hybrid protein may contain an N-terminus fragment from the CS protein of P. falciparum.

In one aspect the hybrid protein may comprise one or more repeat units from the central region of P. falciparum. For example 1, 2, 3, 4, 5, 6, 7, 8, 9 or more repeat units.

Usually the hybrid protein will contain a C-terminus fragment from a CS protein of Plasmodium such as P. vivax (type I or II), for example a fragment comprising region II such as shown in SEQ ID No 2.

Alternatively the hybrid protein may contain a C-terminus fragment from the CS protein of P. falciparum.

Whilst not wishing to be bound by theory it is thought that the N and C terminal fragments include several T and B cell epitopes.

In recombinant proteins often unnatural amino acids are introduced in the cloning process and are observed in the final expressed protein. For example several such as 1 , 2, 3, 4 or 5 amino acids may be inserted at the beginning (N-terminal) of the protein. If 4 amino acids are inserted at the beginning of the protein they may for example be VIMAP. In addition to or alternatively 1, 2, or 3 such as 1 amino acids may be inserted into the body/middle of the protein at for example about amino acid, 259, 260, 261 or 262. The amino acid insert may be the amino acid with the symbol P. In one aspect the protein employed does not include any amino acids inserted by the cloning process in the body/middle of the protein.

The invention also extends to a so-called "truncated" version of the hybrid protein, for example as shown in Fig 12 and labeled CSVtr-S.

Thus the invention provides truncated hybrid protein wherein at least the arginine rich region found in the N-terminal of the CS protein (about amino acid 60) is removed/deleted.

In one aspect the invention provides a truncated hybrid protein, wherein at least amino acids 1 to 55 (or 1 to 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70) is

deleted. This truncated hybrid protein may include up to 4 amino acids inserted at the beginning of the protein such as MMAP.

In one aspect the invention relates to a hybrid proteins, which excludes amino 5 to 64 (inclusive) of the full length recombinant protein.

This/these truncated hybrid protein may be employed in other aspects of the invention described below.

Any suitable strain of P. vivax may be employed in the invention including: Latin America/South America (ie Sal 1, Belem), Korea, China, Thailand, Indonesia, India, and Vietnam. The construct in SEQ ID No 13 is based on a Korean strain (more specifically a South Korean strain).

P. vivax with type I CS proteins is more prevalent than P. vivax with type II CS proteins. Therefore in one aspect of the invention more repeat units from type I are included in the hybrid than repeat units of type II.

More specifically the hybrid protein of the invention may include 1 to 15 repeat units such as 9 repeat units from type I.

Examples of suitable monomers of type I CS proteins are given in SEQ ID No.s 3 to 9.

In one embodiment the invention provides a hybrid protein comprising a mixture of different repeat units of type I, such as one of each of those listed in SEQ ID No.s 3 to 9.

One or more repeat units may be duplicated in the hybrid, for example two monomers of SEQ ID No 3 and/or 4 may be incorporated into the construct.

a) In one aspect the CS protein comprises a unit of SEQ ID No 3.

b) In one aspect the CS protein comprises a unit of SEQ ID No 4, optionally in combination with a unit as described in paragraph a) directly above.

c) In one aspect the CS protein comprises a unit of SEQ ID No 5, optionally in combination with a unit as described in paragraph a) or b) directly above.

d) In one aspect the CS protein comprises a unit of SEQ ID No 6, optionally in combination with one or more units as described in paragraphs a) to c) directly above.

f) In one aspect the CS protein comprises a unit of SEQ ID No 7, optionally in combination with one or more units as described in paragraph a) to d) directly above.

g) In one aspect the CS protein comprises a unit of SEQ ID No 8, optionally in combination with one or more units as described in paragraph a) to f) directly above.

h) In one aspect the CS protein comprises a unit of SEQ ID No 9, optionally in combination with one or more units as described in paragraph a) to g) directly above.

Examples of suitable component repeat units of type II CS proteins are given in SEQ ID No.s 10 and 14, such as 10.

In one aspect of the invention there is provided a hybrid protein with 5 or less repeat units derived from type II such as one monomer, for example as shown in SEQ ID

No. 10.

The hybrid protein may also include the 12 amino acid insertion found at the end of the repeat region found in certain Asian strains of P. vivax, for example as shown in SEQ ID No. 11.

In one embodiment the hybrid protein comprises 257 amino-acids derived from P. vivax CS protein.

In one embodiment the hybrid fusion protein comprises 494 amino acids, for example 257 of which are derived from P. vivax CS protein.

In one aspect the CS protein comprises full length protein.

In one aspect the CS protein employed is a truncated version, for example as shown in Figure 12 or a corresponding truncated wherein the amino acids about 1 to about 67 are deleted.

In one embodiment between 1 and 5 additional amino acids are inserted at the beginning of the sequence as a result of the cloning process, for example MMAP or MMAPG inserted.

The hybrid protein and/or fusion protein may also include further antigens derived from P. falciparium and/or P. vivax, for example wherein the antigen is selected from DBP, PvTRAP, PvMSP2, PvMSP4, PvMSP5, PvMSPό, PvMSP7, PvMSP8, PvMSP9, PvAMAl and RBP or a fragment thereof.

In an embodiment the hybrid fusion protein (CSV-S) has substantially the amino acid sequence shown in SEQ ID No. 17. In the sequence, amino acids 6 to 262 are derived from CSV and 269 to 494 are derived from S. The remaining amino acids are introduced by genetic construction (these may, in particular, be varied if desired).

The properties of the CSV-S fusion protein of SEQ ID No. 17 are provided in the Tables below:

Whole Protein Composition Analysis

The properties of the CSV-S fusion protein of SEQ ID No. 20 are provided in the Tables below:

Nishikawa & Ooi 1987

Whole Protein Composition Analysis

The hybrid fusion protein CSV-S may, for example be prepared employing the plasmid pGFl-S2 (see Fig. 4 and the examples for further details), which when the appropriate sequence corresponding to CSV is inserted at the Smal cloning site processes the insertion to proved the fusion protein CSV-S.

. .. _ „ „ „ jj

The nucleotide sequence for the hybrid fusion protein of SEQ ID No 17 is given in SEQ ID No 16. The CSV portion of this sequence was codon optimized to optimize expression in yeast.

Alternatively the hybrid fusion protein CSV-S may, for example be prepared as described in Example 2.

An alternative nucleotide sequence for the hybrid fusion protein of the invention is given in SEQ ID No 19 and the amino acid sequence for the same is given in SEQ ID No 20.

The present invention also provides nucleotide sequences, for example DNA, encoding the proteins of the present invention.

The nucleotide sequences encoding the proteins of the present invention are, in one embodiment flanked by transcriptional control elements, preferably derived from yeast genes and incorporated into an expression vector.

An expression cassette for hybrid proteins of the invention may, for example, be constructed comprising the following features:

• A promoter sequence, derived from, for example, the S.cerevisiae TDH3 gene.

• A sequence encoding for the appropriate CSV-S hybrid fusion protein.

• A transcription termination sequence contained within the sequence, derived from, for example, the S. cerevisiae ARG3 gene.

An example of a specific promoter is the promoter from the S. cerevisiae TDH3 gene Musti et al.

The invention also extends to vectors employed in the preparation of the hybrid fusion protein.

A suitable plasmid can then be employed to introduce the sequence encoding for the hybrid fusion protein into a suitable host for synthesis. An example of a suitable plasmid is pRIT15546 a 2 micron-based vector for carrying the CSV-S expression cassette, see plasmid map of same in Figure 3.

Alternatively plasmids are known to person skilled in the art and/or described in the examples.

The plasmid will generally contain an in-built marker to assist selection, for example a gene encoding for antibiotic resistance or LEU and/or HIS auxotrophy.

In one aspect the plasmid is episomal ie the gene for the protein is not integrated into the DNA of the host.

The invention also relates to a host cell transformed with a vector according to the invention. Host cells can be prokaryotic or eukaryotic but preferably, are yeast, for example Saccharomyces (for example Saccharomyces cerevisiae such as DC5 in ATCC data base (accession number 20820), under the name RIT DC5 cir(o)). Depositor: Smith Kline-RIT,) and non- Saccharomyces yeasts. These include Schizosaccharomyces (eg Schizosaccharomyces pombe) Kluyveromyces (eg Kluyveromyces lactis), Pichia (eg Pichia pastoris), Hansenula (eg Hansenula polymorpha), Yarrowia (eg Yarrowia lipolytica) and Schwanniomyces (eg Schwanniomyces occidentalis).

In one aspect the invention relates to a recombinant yeast strain Yl 834 (and use thereof), which expresses a CSV-S hybrid fusion protein and/or immunogenic particles thereof, see Examples for preparation of the same.

In one aspect the invention relates to a recombinant yeast strain Yl 835 (and use thereof), which expresses a CSV-S hybrid fusion protein and/or immunogenic particles thereof, see Examples for preparation of the same.

In an alternative aspect the invention relates to a recombinant yeast strain Yl 840 (and use thereof), which expresses a CSV-S hybrid fusion protein and/or immunogenic particles thereof.

More specifically the invention relates to said yeast (or another yeast) comprising a nucleotide sequence encoding for the fusion protein of the invention, use of the yeasts for the preparation of said fusion protein, and processes involving the preparation of said fusion protein.

The nucleotide sequence (or a portion thereof, such as the portion encoding the hybrid protein but optionally not the portion encoding protein S) may be codon-optimized for expression in the relevant host such as yeast.

Codon-optimized in this context is intended to mean that the codons are modified to render them appropriate for expression in the relevant host. This may or may not be the most optimal codon selection, for example a sub-optimal optimized version may be selected if the expression of the same is greater than that of an optimal version.

In yeast cells once expressed, the hybrid fusion protein (comprising S antigen), is able to spontaneously assemble into a lipoprotein structure/particle composed of numerous monomers of said proteins.

These particles may also be referred to a Virus Like Particles (VLP). The particles may also be described as multimeric lipoprotein particles.

These particles can be prepared in a number of ways, for example by fusing each the Plasmodium derived antigens to another fusion partner, (for example the antigens of

Hepatitis B virus or a viral structural protein) and expressing the same in a suitable host such as yeast or bacteria.

When the chosen recipient yeast strain also carries in its genome one or more integrated copies of a hepatitis B S expression cassette, the resulting strain synthesizes hybrid protein as a fusion proteins, and also non- fused S antigen. These may spontaneously be assembled into lipoprotein particles comprising monomers of the hybrid fusion protein and monomers of the S antigen.

Whilst not wishing to be bound by theory, the particles, for example when expressed in Yl 834, are thought to consist essentially of units/monomers of the hybrid fusion protein, although it may be that the particle composition varies according the specific yeast employed.

Thus in some cases the lipoprotein particles may also contain the monomers of the unfused S antigen, for example as in the particles which are the result of preparation in the yeast Yl 835.

Thus in one aspect the invention provides an immunogenic particle comprising hybrid fusion proteins of the invention.

Thus in one aspect the particle is a 'so-called' simple particle in that it consists essentially of units of the hybrid fusion protein. In an alternative aspect of the invention the immunogenic lipoprotein particle comprises one or more hybrid fusion proteins and one or more unfused protein S units, in a so-called double or mixed particle.

Figures 1 and 7 are electron micrographs of a lipoprotein particle according to this aspect of the invention, albeit prepared in different yeast.

The particles shown in the micrograph were purified by classical techniques using a cesium chloride and sucrose gradient, in particular using successive cesium chloride and sucrose gradients, more specifically: 1 sucrose gradient followed by 3 successive

CsCl gradients.

The invention includes of methods of purifying said particles, for example employing a cesium chloride and sucrose gradient.

Additionally, it is hypothesized that, the surfactants used to liberate the protein from the cells may also assist in the stabilization of the lipoprotein particles. In a further aspect the lipoprotein particle comprises a surfactant. The surfactant may, for example be selected from Tween (such as Tween 20), briji, polyethylene glycol. The particle may, for example, comprise 1% or less, such as 0.5% or 0.1% by weight of surfactant.

It is hypothesized that the lipoprotein particles of the invention may contribute to further stimulating the immune response to the hybrid fusion protein in vivo.

The present invention also relates to vaccines comprising an immunoprotective amount of a protein or particle according to the invention in admixture with a suitable diluent or carrier.

The invention also extends to a composition comprising a hybrid/fusion protein or particle according to the invention and a viral vector comprising a malaria antigen, particularly a malaria antigen common with said particle, and optionally an adjuvant.

In the context of this specification, excipient refers to a component in a pharmaceutical formulation with no therapeutic effect in its own right. A diluent or carrier falls within the definition of an excipient.

In aspect the composition of the invention comprises a hybrid fusion protein and/or particle as described herein and an adjuvant.

In one embodiment the viral vector construct is as described in WO 2004/055187.

In one embodiment the said fusion proteins are in admixture and thus available as a single formulation. In an alternative embodiment the fusion proteins are prepared as separate formulations and are administered separately to the patient.

In a further aspect the invention relation to a combined treatment for P. falciparum and P. vivax, comprising: an immunogenic fusion protein comprising an antigen derived from a CS protein of P. vivax, and an immunogenic fusion protein comprising an antigen derived from CS protein of P. falciparum.

In this aspect of the invention, antigen derived from the CS protein of P. vivax component of the fusion maybe a naturally occurring (native) CS protein such as type I and or type II or a fragment thereof. Alternatively the antigen may be hybrid (chimeric) protein, as described above, such as the protein identified in SEQ ID No 13 and/or 15.

A fusion protein antigen component derived from P. falciparum may be prepared according to WO 93/10152 for example as RTS, S (wherein "S" represents an unfused monomer) or as RTS, both of which are suitable for use in this aspect of the invention.

In one embodiment the antigen derived from of P. falciparum comprises at least 4 repeat units of the central repeat region. More specifically this antigen comprises a sequence which contains at least 160 amino acids which is substantially homologous to the C-terminal portion of the CS protein. The CS protein may be devoid of the last 12 to 14 amino-acids from the C terminal.

More specifically the relevant fusion protein which comprises a portion of the CS protein of P. falciparum may substantial correspond to amino acids 207-395 of P. falciparum 7G8 fused in frame via a linear linker to the N-terminal of the S antigen. The linker may comprise a portion of preS2 from the S antigen.

More specifically the fusion protein derived from P. falciparium employed is that encoded for by the nucleotide sequence for the RTS expression cassette as provide in SEQ ID No 18.

Suitable S antigens, may comprise a preS2. An example of a suitable serotype is adw (Nature 280:815-819, 1979).

In one aspect the hybrid fusion proteins of the invention comprise a portion derived from a mutant S protein, for example as described in published US application No. 2006/194196 (also published as WO 2004/113369). This document describes a mutant labeled HDB05. In particular it describes comparisons of the mutant and the wild type protein in Figures 1 and 6 and genes for the mutant in Figures 4 and 5. Sequences 12 to 22 therein describe particular polypeptides of the mutant S protein. Each of the above is incorporated herein by reference.

The invention also relates to use of said combination for treatment, more particularly use as a vaccine for the prevention of malaria and method of treating a patient susceptible to Plasmodium infection comprising administering an effective amount of each of the components either simultaneously or sequentially, thereby to treat or prevent Malaria.

In a further aspect the invention provides a combination for treatment comprising: an immunogenic hybrid antigen comprising a. at least one repeat unit derived from the central repeat section of a type

I circumsporozoite protein of P. vivax, b. at least one repeat unit derived from the central repeating section of a type II circumsporozoite protein of P. vivax, and an immunogenic fusion protein comprising an antigen derived from the CS protein of P. falciparum.

For example, wherein said hybrid antigen and said fusion protein are administered concomitantly for example they may be in admixture for administration simultaneously or alternatively may be formulated for administration sequentially.

In a further aspect there is provided a combination for treatment comprising: an immunogenic hybrid antigen comprising a. at least one repeat unit derived from the central repeat section of a type

I circumsporozoite protein of P. vivax, b. at least one repeat unit derived from the central repeating section of a tvne TT circπmsnoπwoite nrotein of P vivnx anri

a viral vector encoding for a malaria antigen, such as said hybrid antigen.

For example, wherein said hybrid antigen and said viral vector are administered concomitantly, for example they may be admixed for administration simultaneously or alternatively may be formulated for administration sequentially.

As used herein the term "concomitantly" means wherein said combination of components are administered within a period of no more than 12 hours eg within a period of no more than 1 hour, typically on one occasion e.g. in the course of the same visit to the health professional, for example where they are administered sequentially or simultaneously.

The invention also includes a vaccine composition comprising said elements of each of these embodiments.

The invention also includes a vaccine kit comprising said elements for treating malaria.

The invention also extends to prime boost regimes comprising said elements, for example priming with antigen and/or particles from P. falciparium and boosting with antigen and/or particles from P. vivax and vice versa.

The prime boost regime may, for example comprise components derived solely or only from P. vivax.

In another embodiment the priming may be effected with plasmid/naked DNA and boosting is with antigen according to the invention and or vice versa.

Alternatively, priming may be effected with an appropriate viral vector and boosting with antigen according to the invention or vice versa.

General prime boost regimes are described below.

For example: • priming may be with an antigen (such as an adjuvanted antigen) comprising at least one type I repeat unit from the CS protein of P. vivax and at least one type II repeat unit from the CS protein of P. vivax and boosting may be with a viral vector encoding the same/corresponding antigen,

• priming may be with an antigen (such as an adjuvanted antigen) comprising at least one repeat unit or epitope from P. vivax and at least one repeat unit or epitope from the CS protein of P. falciparum and boosting may be with a viral protein encoding the same/corresponding antigen,

• priming may be with a viral vector encoding an antigen comprising at least one type I repeat unit from the CS protein of P.vivax and at least one type II repeat unit from the CS protein of P.vivax or comprising at least one repeat unit or epitope from P.vivax and at least one repeat unit or epitope from the CS

protein of P. falciparum and boosting with the same/corresponding adjuvanted antigen in the form of a protein and/or immunogenic particle.

Antigen in these contexts includes hybrid fusion protein and/or immunogenic particles of the invention. In these prime boost regimes antigen will usually be adjuvanted.

In this/these aspect(s) of the invention the preferences described above for the hybrid protein derived from P. vivax also apply. Thus in one aspect the hybrid protein has the sequence in SEQ ID No 13.

RTS ₅ S and antigen (in the form of an immunogenic particle) from P. falciparum can be prepared as described in WO 93/10152.

In one aspect the invention provides a replication deficient viral vector encoding a hybrid protein (or alternatively a hybrid fusion protein) according the invention.

Suitable viral vectors may be derived from adeno viral vectors, adeno-associated viral vectors (AAVs), measles, lentiviruses, alphaviruses, bacloviruses, herpes simplex virus, and poxviruses such as cowpox, fowlpox, pigeonpox, canarypox, suipox and sheeppox/goatpox. Methodology for preparing adeno viral vectors encoding a malaria antigen is, for example, described in WO 2004/055187.

The protein encoded by the vector may, for example, be modified to prevent glycosylation of the protein during expression, for example certain serines may be replaced by alanine residues to reduce glycosylation.

Adenovirus

Adenoviral vectors of the present invention comprise one or more heterologous polynucleotides (DNA) which encode one or more immunogenic polypeptides.

Adenoviral vectors of use in the present invention may be derived from a range of mammalian hosts.

Adenoviruses (herein referred to as "Ad" or "Adv") have a characteristic morphology with an icosahedral capsid consisting of three major proteins, hexon (II), penton base (III) and a knobbed fibre (IV), along with a number of other minor proteins, VI, VIII, IX, Ilia and IVa2 (Russell W.C. 2000, Gen Viriol, 81 :2573-2604). The virus genome is a linear, double-stranded DNA with a terminal protein attached covalently to the 5' termini, which have inverted terminal repeats (ITRs). The virus DNA is intimately associated with the highly basic protein VII and a small peptide termed mu. Another protein, V, is packaged with this DNA-protein complex and provides a structural link to the capsid via protein VI. The virus also contains a virus-encoded protease, which is necessary for processing of some of the structural proteins to produce mature infectious virus.

Over 100 distinct serotypes of adenovirus have been isolated which infect various mammalian sυecies. 51 of which are of human origin. Thus one or more of the

adenoviral vectors may be derived from a human adenovirus. Examples of such human-derived adenoviruses are AdI, Ad2, Ad4, Ad5, Ad6, AdI l, Ad 24, Ad34, Ad35, Ad50/51 particularly Ad5, AdI l and Ad35. The human serotypes have been categorised into six subgenera (A-F) based on a number of biological, chemical, immunological and structural criteria.

Although Ad5-based vectors have been used extensively in a number of gene therapy trials, there may be limitations on the use of Ad5 and other group C adenoviral vectors due to preexisting immunity in the general population due to natural infection. Ad5 and other group C members tend to be among the most seroprevalent serotypes. Immunity to existing vectors may develop as a result of exposure to the vector during treatment. These types of preexisting or developed immunity to seroprevalent vectors may limit the effectiveness of gene therapy or vaccination efforts. Alternative adenovirus serotypes, thus constitute very important targets in the pursuit of gene delivery systems capable of evading the host immune response.

One such area of alternative serotypes are those derived from non human primates, especially chimpanzee adenoviruses. See US Patent 6,083,716 which describes the genome of two chimpanzee adenoviruses.

It has been shown that chimpanzee ("Pan" or "C") adenoviral vectors induce strong immune responses to transgene products as efficiently as human adenoviral vectors (Fitzgerald et al. J. Immunol. 170:1416).

Non human primate adenoviruses can be isolated from the mesenteric lymph nodes of chimpanzees. Chimpanzee adenoviruses are sufficiently similar to human adenovirus subtype C to allow replication of El deleted virus in HEK 293 cells. Yet chimpanzee adenoviruses are phylogenetically distinct from the more common human serotypes (Ad2 and Ad5). Pan 6 is less closely related to and is serologically distinct from Pans 5, 7 and 9.

Thus one or more of the adenoviral vectors may be derived from a non-human primate adenovirus eg a chimpanzee adenovirus such as one selected from serotypes Pan5, Pan6, Pan7 and Pan9.

Adenoviral vectors may also be derived from more than one adenovirus serotype, and each serotype may be from the same or different source. For example they may be derived from more than one human serotype and/or more than one non-human primate serotype. Methods for constructing chimeric adenoviral vectors are disclosed in WO2005/001103.

There are certain size restrictions associated with inserting heterologous DNA into adenoviruses. Human adenoviruses have the ability to package up to 105% of the wild type genome length (Bett et al 1993, J Virol 67 (10), 5911-21). The lower packaging limit for human adenoviruses has been shown to be 75% of the wild type genome length (Parks et al 1995, J Virol 71(4), 3293-8).

One example of adenoviruses useful in the present invention are adenoviruses which are distinct from prevalent naturally occurring serotypes in the human population such as Ad2 and Ad5. This avoids the induction of potent immune responses against the vector which limits the efficacy of subsequent administrations of the same serotype by blocking vector uptake through neutralizing antibody and influencing toxicity.

Thus, the adenovirus may be an adenovirus which is not a prevalent naturally occurring human virus serotype. Adenoviruses isolated from animals have immunologically distinct capsid, hexon, penton and fibre components but are phylogenetically closely related. Specifically, the virus may be a non-human adenovirus, such as a simian adenovirus and in particular a chimpanzee adenovirus such as Pan 5, 6, 7 or 9. Examples of such strains are described in WO 03/000283 and are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and other sources. Desirable chimpanzee adenovirus strains are Pan 5 [ATCC VR-591 ], Pan 6 [ATCC VR-592], and Pan 7 [ATCC VR-593].

Use of chimpanzee adenoviruses is thought to be advantageous over use of human adenovirus serotypes because of the lack of pre-existing immunity, in particular the lack of cross-neutralising antibodies, to adenoviruses in the target population. Cross- reaction of the chimpanzee adenoviruses with pre-existing neutralizing antibody responses is only present in 2% of the target population compared with 35% in the case of certain candidate human adenovirus vectors. The chimpanzee adenoviruses are distinct from the more common human subtypes Ad2 and Ad5, but are more closely related to human Ad4 of subgroup E, which is not a prevalent subtype. Pan 6 is less closely related to Pan 5, 7 and 9.

The adenovirus of the invention may be replication defective. This means that it has a reduced ability to replicate in non-complementing cells, compared to the wild type virus. This may be brought about by mutating the virus e.g. by deleting a gene involved in replication, for example deletion of the EIa, EIb, E3 or E4 gene.

The adenoviral vectors in accordance with the present invention may be derived from replication defective adenovirus comprising a functional El deletion. Thus the adenoviral vectors according to the invention may be replication defective due to the absence of the ability to express adenoviral EIa and EIb, i.e., are functionally deleted in EIa and EIb. The recombinant adenoviruses may also bear functional deletions in other genes [see WO 03/000283] for example, deletions in E3 or E4 genes. The adenovirus delayed early gene E3 may be eliminated from the adenovirus sequence which forms part of the recombinant virus. The function of E3 is not necessary to the production of the recombinant adenovirus particle. Thus, it is unnecessary to replace the function of this gene product in order to package a recombinant adenovirus useful in the invention. In one particular embodiment the recombinant adenoviruses have functionally deleted El and E3 genes. The construction of such vectors is described in

Roy et al., Human Gene Therapy 15:519-530, 2004.

Recombinant adenoviruses may also be constructed having a functional deletion of the E4 gene, although it may be desirable to retain the E4 ORF6 function. Adenovirus vectors according to the invention may also contain a deletion in the delayed early gene E2a. Deletions may also be made in any of the late genes Ll through to L5 of the adenovirus genome. Similarly deletions in the intermediate genes IX and IVa may be useful.

Other deletions may be made in the other structural or non-structural adenovirus genes. The above deletions may be used individually, i.e. an adenovirus sequence for use in the present invention may contain deletions of El only. Alternatively, deletions of entire genes or portions thereof effective to destroy their biological activity may be used in any combination. For example in one exemplary vector, the adenovirus sequences may have deletions of the El genes and the E4 gene, or of the El, E2a and E3 genes, or of the El and E3 genes (such as functional deletions in EIa and EIb, and a deletion of at least part of E3), or of the El, E2a and E4 genes, with or without deletion of E3 and so on. Such deletions may be partial or full deletions of these genes and may be used in combination with other mutations, such as temperature sensitive mutations to achieve a desired result.

The adenoviral vectors can be produced on any suitable cell line in which the virus is capable of replication. In particular, complementing cell lines which provide the factors missing from the viral vector that result in its impaired replication characteristics (such as El and/or E4) can be used. Without limitation, such a cell line may be HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], HEK 293, KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells, among others. These cell lines are all available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209. Other suitable parent cell lines may be obtained from other sources, such as PER.C6© cells, as represented by the cells deposited under ECACC no. 96022940 at the European Collection of Animal Cell Cultures (ECACC) at the Centre for Applied Microbiology and Research (CAMR, UK) or Her 96 cells (Crucell).

The invention extends to use of known cell lines for the preparation of a viral vector encoding a protein of the present invention.

The polynucleotide sequences which encode immunogenic polypeptides may be codon optimised for mammalian cells. Such codon-optimisation is described in detail in WO 05/025614 for example, starting at page 37.

In one embodiment of the present invention wherein the viral vector comprising the polynucleotide constructs wherein said constructs comprise an N-terminal leader sequence. The signal sequence, transmembrane domain and cytoplasmic domain are individually all optionally present or deleted. In one embodiment of the present invention all these regions are present but modified.

A promoter for use in the adenoviral vector according to the invention may be the promoter from HCMV IE gene, for example wherein the 5' untranslated region of the HCMV IE gene comprising exon 1 is included and intron A is completely or partially excluded as described in WO 02/36792.

When several antigens are fused into a fusion protein, such protein would be encoded by a polynucleotide under the control of a single promoter.

In an alternative embodiment of the invention, several antigens may be expressed separately through individual promoters, each of said promoters may be the same or different. In yet another embodiment of the invention some of the antigens may form a fusion, linked to a first promoter and other antigen(s) may be linked to a second promoter, which may be the same or different from the first promoter.

Thus, the adenoviral vector may comprise one or more expression cassettes each of which encode one antigen under the control of one promoter. Alternatively or additionally it may comprise one or more expression cassettes each of which encode more than one antigen under the control of one promoter, which antigens are thereby expressed as a fusion. Each expression cassette may be present in more than one locus in the adenoviral vector.

The polynucleotide or polynucleotides encoding immunogenic polypeptides to be expressed maybe inserted into any of the adenovirus deleted regions, for example into the El deleted region.

Although two or more polynucleotides encoding immunogenic polypeptides may be linked as a fusion, the resulting protein may be expressed as a fusion protein, or it may be expressed as separate protein products, or it may be expressed as a fusion protein and then subsequently broken down into smaller subunits.

Immunogenic in the context of this specification is intended to refer to the ability to illicit an immune response. This response may be when the protein in administered in an appropriate formulation, which may include/require a suitable adjuvant. A booster comprising a dose similar or less than the original dose may be required to obtain the required immunogenic response (including boosting with a different entity ie heterologous boosting).

The composition/pharmaceutical formulations according to the invention may also include in admixture one or more further antigens, such as those derived from P. falciparium and/or P. vivax, for example wherein the antigen is selected from DBP, PvTRAP, PvMSP2, PvMSP4, PvMSP5, PvMSPό, PvMSP7, PvMSPδ, PvMSP9, PvAMAl and RBP or fragment thereof.

Other example, antigens derived from P falciparum include ,PfEMP-I, Pfs 16 antigen, MSP-I , MSP-3 , LSA- 1 , LS A-3 , AMA- 1 and TRAP . Other Plasmodium antigens include P. falciparum EBA, GLURP, RAPl, RAP2, Sequestrin, Pf332, STARP,

SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfs48/45, Pfs230 and their analogues in other Plasmodium spp.

In the vaccine of the invention, an aqueous solution of the hybrid antigen maybe used directly. Alternatively, the protein with or without prior lyophilisation can be mixed or absorbed with adjuvants, which include but are not limited to alum, muramyl dipeptide, saponins such as Quil A.

Particular adjuvants are those selected from the group of metal salts, oil in water emulsions, Toll like receptors agonist, (in particular Toll like receptor 2 agonist, Toll like receptor 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof with the proviso that metal salts are only used in combination with another adjuvant and not alone unless they are formulated in such a way that not more than about 60% of the antigen is adsorbed onto the metal salt. More specifically, not more than about 50%, for example 40% of the antigen is adsorbed onto the metal salt, and in one embodiment not more than about 30% of the antigen is adsorbed onto the metal salt. The level of antibody adsorbed onto the metal salt may be determined by techniques well known in the art. The level of free antigen may be increased by, for example, formulating the composition in the presence of phosphate ions, such as phosphate buffered saline, or by increasing the ratio of antigen to metal salt. In one embodiment the adjuvant does not include a metal salt as sole adjuvant. In one embodiment the adjuvant does not include a metal salt.

In an embodiment the adjuvant is a Toll like receptor (TLR) 4 ligand, for example an agonist such as a lipid A derivative, in particular monophosphoryl lipid A or more specifically 3 Deacylated monophoshoryl lipid A (3D - MPL).

3 Deacylated monophosphoryl lipid A is known from US patent No. 4,912,094 and UK patent application No. 2,220,211 (Ribi) and is available from Ribi Immunochem, Montana, USA.

3 D-MPL is sold under the trademark MPL® by Corixa corporation and primarily promotes CD4+ T cell responses with an IFN-g (ThI) phenotype. It can be produced according to the methods disclosed in GB 2 220 211 A. Chemically it is a mixture of 3 -deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Generally in the compositions of the present invention small particle 3D-MPL is used. Small particle 3D-MPL has a particle size such that it may be sterile- filtered through a 0.22μm filter. Such preparations are described in International Patent Application No. WO 94/21292.

Synthetic derivatives of lipid A are known and thought to be TLR 4 agonists including, but not limited to:

OM174 (2-deoxy-6-O-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylam ino]-4-o- phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoyl amino]-α-D- glucopyranosyldihydrogenphosphate), (WO 95/14026).

OM 294 DP (3S, 9 R) -3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)- [(R)-3 -hydroxytetradecanoylaminojdecan- 1 , 10-diol, 1 , 10-bis(dihydrogenophosphate) (WO 99 /64301 and WO 00/0462).

OM 197 MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylamino]-4-oxo-5- aza-9- [(R)-3 -hydroxytetradecanoylamino] decan- 1 , 10-diol, 1 -dihydrogenophosphate 10-(6-aminohexanoate) (WO 01/46127).

Typically when 3D-MPL is used the antigen and 3D-MPL are delivered with alum or presented in an oil in water emulsion or multiple oil in water emulsions. The incorporation of 3D-MPL is advantageous since it is a stimulator of effector T-cells responses. Alternatively the 3D-MPL may be formulated as liposomes.

Other TLR4 ligands which may be used are alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO 9850399 or US 6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in US 6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants.

Another immunostimulant for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quilaja Saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254). Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21). QS21 is a natural saponin derived from the bark of Quillaja saponaria Molina which induces CD8+ cytotoxic T cells (CTLs), ThI cells and a predominant IgG2a antibody response.

Particular formulations of QS21 have been described which further comprise a sterol (WO 96/33739). The ratio of QS21 : sterol will typically be in the order of 1 : 100 to 1 :1 weight to weight.

Generally an excess of sterol is present, the ratio of QS21 : sterol being at least 1 :2 w/w. Typically for human administration QS21 and sterol will be present in a vaccine in the range of about 1 μg to about 100 μg, such as about 10 μg to about 50 μg per dose.

The liposomes generally contain a neutral lipid, for example phosphatidylcholine, which is usually non-crystalline at room temperature, for example eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine or dilauryl phosphatidylcholine.

The liposomes may also contain a charged lipid which increases the stability of the lipsome-QS21 structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is often 1-20% w/w, such as 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), such as 20-25%.

These compositions may contain MPL (3-deacylated mono-phosphoryl lipid A, also known as 3D-MPL). 3D-MPL is known from GB 2 220 211 (Ribi) as a mixture of 3 types of de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana.

Generally in compositions of the invention small particle 3D-MPL, which has a particle size such that it may be sterile-filtered through a 0.22 μm filter, is employed. Such preparations are described in WO 94/21292.

The saponins may be separate in the form of micelles, mixed micelles (generally, but not exclusively with bile salts) or may be in the form of ISCOM matrices (EP 0 109 942 Bl), liposomes or related colloidal structures such as worm-like or ring- like multimeric complexes or lipidic/layered structures and lamellae when formulated with cholesterol and lipid, or in the form of an oil in water emulsion (for example as in WO 95/17210). The saponins may often be associated with a metallic salt, such as aluminium hydroxide or aluminium phosphate (WO 98/15287).

Usually, the saponin is presented in the form of a liposome, ISCOM or an oil in water emulsion.

Immunostimulatory oligonucleotides may also be used. Examples oligonucleotides for use in adjuvants or vaccines of the present invention include CpG containing oligonucleotides, generally containing two or more dinucleotide CpG motifs separated by at least three, more often at least six or more nucleotides. A CpG motif is a cytosine nucleotide followed by a guanine nucleotide. The CpG oligonucleotides are typically deoxynucleotides. In one embodiment the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention. Also included within the scope of the invention are oligonucleotides with mixed internucleotide linkages. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US5,666,153, US5,278,302 and WO 95/26204.

Examples of oligonucleotides are as follows: TCC ATG ACG TTC CTG ACG TT (CpG 1826)

TCT CCC AGC GTG CGC CAT (CpG 1758)

ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG

TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006)

TCC ATG ACG TTC CTG ATG CT (CpG 1668) TCG ACG TTT TCG GCG CGC GCC G (CpG 5456), the sequences may contain phosphorothioate modified internucleotide linkages.

Alternative CpG oligonucleotides may comprise one or more sequences above in that they have inconsequential deletions or additions thereto.

The CpG oligonucleotides may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such oligonucleotides maybe synthesized utilising an automated synthesizer.

Examples of a TLR 2 agonist include peptidoglycan or lipoprotein. Imidazoquinolines, such as Imiquimod and Resiquimod are known TLR7 agonists. Single stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), whereas double stranded RNA and poly IC (polyinosinic-polycytidylic acid - a commercial synthetic mimetic of viral RNA) are exemplary of TLR 3 agonists. 3D- MPL is an example of a TLR4 agonist whilst CpG is an example of a TLR9 agonist.

An immunostimulant may alternatively or in addition be included. In a one embodiment this immunostimulant will be 3 deacylated monophosphoryl lipid A (3D- MPL).

Adjuvants combinations include 3D-MPL and QS21 (EP 0 671 948 Bl), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D- MPL formulated with other carriers (EP 0 689 454 Bl) including liposomes. Other preferred adjuvant systems comprise a combination of 3D-MPL, QS21 and a CpG oligonucleotide as described in US 6558670 and US 6544518.

In one aspect the adjuvant comprises 3D-MPL.

In one aspect the adjuvant comprises QS21.

In one aspect the adjuvant comprises CpG.

In one aspect the adjuvant comprises QS21 and 3D-MPL.

In one aspect the adjuvant comprises QS21 , 3D-MPL and CpG

In one aspect the adjuvant is formulated as an oil in water emulsion.

In one aspect the adjuvant is formulated as liposomes.

In one embodiment of the present invention provides a vaccine comprising a hybrid protein as herein described, such as CSV-S in combination with 3D-MPL and a carrier. Typically the carrier will be an oil in water emulsion or alum. More specifically the hybrid protein is presented as a particle or mixed particle as herein described.

The protein of the present invention may also be encapsulated into microparticles such as liposomes.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.

The amount of the protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each does will comprise l-1000μg of protein, for example 1-200 μg such as 10-100μg more particularly 10-40 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists. The immune response to the protein of this invention is enhanced by the use of adjuvant and or an immunostimulant.

The amount of 3D-MPL used is generally small, but depending on the vaccine formulation may be in the region of l-1000μg per dose, generally l-500μg per dose, and more such as between 1 to lOOμg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90μg per dose).

The amount of CpG or immunostimulatory oligonucleotides in the adjuvants or vaccines of the present invention is generally small, but depending on the vaccine formulation maybe in the region of l-1000μg per dose, generally l-500μg per dose, and more such as between 1 to lOOμg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90μg per dose).

The amount of saponin for use in the adjuvants of the present invention may be in the region of l-1000μg per dose, generally l-500μg per dose, more such as l-250μg per dose, and more specifically between 1 to lOOμg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90μg per dose).

In a further aspect the invention provides a vaccine combination providing immunoprotection against malaria caused by P. faciparum and/or P.vivax comprising a hybrid protein derived from CS protein of P. vivax such as described herein.

In one aspect there is provided a vaccine comprising immunogenic particles as described herein, and optionally further comprising immunogenic particles of TRS, S in admixture (as described in WO 93/10152) and an appropriate immunostimulatory adjuvant.

The formulations of the present invention may be used in treatment for both prophylactic and therapeutic purposes.

Accordingly the invention provides use of any aspect of the invention described herein for treatment, in particular a vaccine composition as described herein for use in medicine.

A further aspect of the present invention is to provide a process for the preparation of hybrid protein of the invention, which process comprises expressing a nucleotide sequence encoding the protein, in a suitable host, preferably a yeast, and recovering the product.

The invention also extends to methods of preparing pharmaceutical composition such as vaccines employing one or more aspects described herein.

A further aspect of the invention lies in a method of treating a patient susceptible to Plasmodium infection by administering an effective amount of a vaccine as hereinbefore described.

Vaccine includes a parenteral vaccine.

Thus the invention extends to use of the antigens such as hybrid fusion proteins and/or lipoprotein particles of the invention and compositions comprising same for treatment and particular in use in the manufacture of a medicament for the treatment/prevention of malaria, such as malaria caused by P. vivax and/or P. falciparum.

Proteins and/or viral vectors encoding same may be administered in prime boost regimes, for example as specifically described in WO 2004/037189.

In the context of this specification "comprising" is to be interpreted as "including".

Aspects of the invention comprising certain elements are also intended to extend to said aspects "consisting" or "consisting essentially" of the relevant elements.

Examples

EXAMPLE 1

DESCRIPTION OF STRAIN Yl 834

The yeast recombinant strain Yl 834 expresses the CSV-S fusion protein. It consists of the Saccharomyces cerevisiae host strain DC5 transformed with the recombinant expression vector pRITl 5546.

DC5 is a laboratory yeast strain (ATCC No: 20820) with the following genotype: leu2-3, Ieu2-112, his3, canl-11. The double leu-2 mutation permits selection for the

uptake and maintenance of the pRIT 15546 vector which carries a functional LEU-2 gene copy. Only those cells carrying a vector with a LEU-2 gene can grow when leucine is absent from the growth medium.

The vector pRITl 5546 is a yeast episomal expression vector (2μ-based vector) carrying the CSV-S expression cassette. The recombinant expression is driven by a promoter derived from the yeast TDH3 gene (constitutive expression). The construction of pRIT15546 vector is detailed below.

Construction of ρRIT15546 vector.

A CSV synthetic gene, with an appropriate codon usage for yeast expression was constructed and sub-cloned into pUC57 vector (GenBank/EMBL accession number Y14837). The resulting plasmid pUC57/CSV and the yeast expression vector pGfl -S2 were both restricted with the appropriate enzyme.

The vector pGfl-S2 was constructed (at GSK) by a multistep cloning procedure. This vector, which already carries an S expression cassette, allows the construction of fusion genes, as N-terminal in-frame fusion with the S gene of Hepatitis B virus. The final expression vector, after sequence verification, was named pRITl 5546 (Fig 3)

Transformation of strain DC5.

The leu- and his-auxotrophic DC5 strain was transformed with the recombinant plasmid pRITl 5546, by using yeast standard protocol.

Transformed cells were plated on agar selective plates. One transformant was selected and received the official designation Yl 834.

EXPRESSION OF THE RECOMBINANT PROTEIN:

Yl 834 is grown, at 30°C, in YNB (Yeast Nitrogen Base available from Kracker Scientific Inc) minimal medium supplemented to a final concentration 8μg/ml histidine to an O.D (620nm) of about 0.5 (here 0.770). Then cells are harvested and cellular extracts are prepared.

EXTRACT PREPARATION:

Cells are resuspended in Breaking Buffer and mechanically disrupted (glass beads). Extract is centrifuged for 5-10 minutes at 5000 rpm. Supernatant fraction is run on SDS - PAGE 4-20%.

Breaking Buffer: 5OmM phosphate Na buffer (PH:7.5)

4mMEDTA Tween-20 0.5%

+ proteases inhibitor cocktail (Complete/ ROCHE)

Cell concentration: 100 ml culture (OD: 0.5) in 5 ml breaking buffer

= concentration of 10 OD unit /ml.

Crude extract clarification: extract centrifuged 5-10 minutes/5000 rpm

DETECTION OF RECOMBINANT PROTEIN

Clarified extracts are run on SDS-PAGE 4-20%, proteins transferred to nitrocellulose membrane and subjected to immunostaining. See Fig. 2

Western blot analysis :

Reagent = Mouse monoclonal antibody anti-S (prepared by GSK

Biologicals)- (dilution: 1/500)

Anti-S antibodies which are commercially available may be substituted for those employed in this method. Alternatively anti-CSV antibodies may be employed, for example those known as MR4 available from NIH.

CSV-S fusion protein:

MW theoretical: 51794 Daltons MW apparent: 55 kDa

EXAMPLE 2:

DESCRIPTION OF STRAIN Yl 840

The Pichia pastoris strain Yl 840 expresses the CSV-S fusion protein. Strain Yl 840 contains four copies of the CSV-S fusion gene, integrated in the genome. To obtain a strain expressing the CSV-S fusion protein, the Pichia pastoris strain GS 115 was transformed with an integrative linear DNA fragment which carries the CSV-S cassette and the functional HIS4 gene.

GSl 15 is a laboratory yeast strain (ATCC No: 20864) with the following genotype: his4. The his4 mutation permits selection for the uptake of the pRITl 5607-derived linear DNA fragment which carries the CSV-S cassette and the functional HIS4 gene.

The vector pRITl 5607 is a Pichia pastoris integrative expression vector carrying the CSV-S expression cassette. The recombinant expression is driven by the strong, tightly regulated methanol inducible AOXl promoter. The construction of pRIT15607 vector is detailed below.

Construction of pRIT15607 vector.

The CSV-S fusion gene, present on pRITl 5546, was amplified by PCR and cloned into the pGEM-T Easy intermediate vector (Promega, cat N°: # Al 360). After sequence verification the recombinant plasmid was digested

with the appropriate restriction enzymes and cloned into the pHIL-D2 Pichia pastoris integrative vector. The final expression vector, after sequence verification, was named pRIT 15607 (Fig 5). The CSV-S fusion protein encoded by pRIT 15607 plasmid is almost identical to the sequence detailed in SEQ ID No 17, except that methionine 2 is replaced by a valine ( see SEQ. ID.

Nol9 and SEQ.IDNo 20). Digestion of pRIT15607 with Notl endonuclease liberates a 6816 bp linear fragment which can be integrated into the yeast genome by homologous recombination at the resident AOXl locus.

Transformation of strain GS 115.

The GSl 15 host strain was transformed with the recombinant plasmid pRIT15607. Prior to transformation the integrative vector was restricted with Notl in order to release a linear DNA fragment carrying the expression cassette and the HIS4 selective marker. Notl restriction will favor integration at the AOXl locus. Transformed cells were plated on agar selective plates. Multicopy integrant clones were selected by quantitative dot blot analysis. Among the clones selected has having a high copy number of integrated CSV- S expression cassettes, one of them showing the highest expression level for CSV-S recombinant protein was selected and given the official designation

Yl 840. This clone carries four copies of the CSV-S fusion gene.

EXPRESSION OF THE RECOMBINANT PROTEIN:

Yl 840 is grown, at 30°C, in YNB (Yeast Nitrogen Base available from Kracker Scientific Inc) minimal medium supplemented with 1% glycerol as a carbon source to an O.D. (620nm) of about 0.5 (0.709 in this case). Then cells are harvested and resuspended in the same volume of YNB medium supplemented with 1% methanol as a carbon source (as inducer) and incubated at 30°C for about 16 hours.

EXTRACT PREPARATION: Methanol induced cells are centrifuged, cell pellets resuspended in Breaking

Buffer and mechanically disrupted (glass beads or French press). Extract is centrifuged for 5-10 minutes at 5000 rpm. Supernatant fraction is run on SDS - PAGE 12.5%.

Breaking Buffer: 6OmM Na ₂ HPO ₄

4OmM NaH ₂ PO ₄

ImM MgSO ₄

10Mm KCl

Tween-20 0.5% 2Mm PMSF

Cell concentration: 100 ml culture (OD:0.5) in 2.5 ml breaking buffer = concentration of 20 OD unit /ml.

Crude extract clarification: extract centrifuged 5-10 minutes/5000 rpm DETECTION OF RECOMBINANT PROTEIN

Clarified extracts are run on SDS-PAGE 12.5 %, proteins transferred to nitrocellulose membrane and subjected to immuno staining. See Figure 6.

Western blot analysis: Reagent = Mouse monoclonal antibody anti-S (prepared by GSK

Biologicals)- (dilution: 1/250)

Anti-S antibodies which are commercially available may be substituted for those employed in this method. Alternatively anti-CSV antibodies may be employed, for example those known as MR4 available from NIH.

CSV-S fusion protein: MW theoretical : 51762 Daltons

MW apparent: 55 kDa

Example 3: DESCRIPTION OF STRAIN Y1835

The yeast recombinant strain Yl 835 simultaneously expresses the CSV-S fusion protein and the S antigen. To obtain a strain co-expressing CSV-S and S proteins, the Saccharomyces cerevisiae strain Yl 295, which already carries five integrated copies of S expression cassettes, was transformed with the recombinant integrative expression vector pRIT15582.

The strain Y1295 was constructed at GSK by a multistep transformation procedure. The construction of Y1295 strain is described in WO 93/10152. Strain Y1295 has the following genotype: leu2-3, Ieu2-112, gall. The leu-2 mutation permits selection for the uptake of pRITl 5582-derived linear DNA fragment which carries the CSV-S cassette and the functional LEU2 gene.

The vector pRITl 5582 is a yeast integrative expression vector (Ty-based vector) carrying the CSV-S expression cassette. The recombinant expression is driven by a promoter derived from the yeast TDH3 gene (constitutive expression). The construction of pRIT15582 vector is detailed below.

Construction of pRIT15582 integrative vector.

The starting material used to construct pRIT15582 vector was the expression plasmid pRIT15546 (Fig 3). The construction of this plasmid is described in example 1. Digestion of pRIT 15546 with HindIII endonuclease liberates a 3706 bp long DNA fragment corresponding to the complete CSV-S expression cassette (pTDH3 + CSV-S + tARG3).This HindIII DNA fragment (after filling

with T4 DNA polymerase) was inserted on the Ty-based integrative vector pRIT13144 at the unique Sail cloning site (Sail restricted/T4 treated).The resulting plasmid pRIT 15582 contains, in addition to the expression cassette, the yeast LEU2 gene as selective marker (Fig 8). Digestion of pRIT 15582 with Xhol endonuclease liberates a 8500 bp linear fragment shown in figure 4 which can be integrated into the yeast genome by homologous recombination of the free ends with resident Ty elements.

Transformation of strain Y 1295.

To obtain a strain expressing both S and CSV-S proteins, strain Y 1295 was transformed with the 8500bp linear Xhol fragment (Fig 9) with selection for Leu+ colonies. Several integrants containing sets of both expression cassettes present in the genome at various ratio were obtained. One transformant carrying four copies of CSV-S cassettes was selected and given the official designation Yl 835.

EXPRESSION OF THE RECOMBINANT PROTEIN:

Yl 835 is grown, at 30°C, in YNB (Yeast Nitrogen Base available from Kracker Scientific Inc) minimal medium to an O.D (620nm) of about 0.5 (0.8). Then cells are harvested and cellular extracts are prepared.

ANALYSIS OF EXPRESSION PRODUCTS BY IMMUNOBLOTTING:

EXTRACT PREPARATION:

Cells are re-suspended in Breaking Buffer and mechanically disrupted (glass beads). Extract is centrifuged for 5-10 minutes at 5000 rpm. Supernatant fraction is run on SDS - PAGE 12.5%.

Breaking Buffer: 5OmM phosphate Na buffer (PH:7.5)

4mMEDTA Tween-20 0.5%

+ proteases inhibitor cocktail (Complete/ ROCHE)

Cell concentration: 100 ml culture (OD:0.5) in 2.5 ml breaking buffer = concentration of 20 OD unit/ml.

Crude extract clarification: extract centrifuged 5-10 minutes/5000 rpm

DETECTION OF RECOMBINANT PROTEIN Clarified extracts are run on SDS-PAGE 12.5%, proteins transferred to nitrocellulose membrane and subjected to immunostaining

Western blot analysis (Fig 10):

Reagents : 1 /Mouse monoclonal antibody anti-S (prepared by GSK Biologicals)- (dilution: 1/250) 2/ Rabbit polyclonal antibody anti-CSV (kindly provided by WRAIR)-dilution 1/20,000.

Anti-S antibodies as well as anti-P.vzvαx/CSP antibodies which are commercially available may be substituted for those employed in this method.

Reference

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