Vaccine

Abstract

The invention relates to a replication deficient simian adenoviral vector C7 encoding a protein comprising CS protein from P. falciparum or a fragment thereof, for example as shown in Seq ID No: 1 or Seq ID No: 3. The invention also relates to processes of preparing said viral vector and use of the viral vector in the treatment/prevention of malaria infection. Compositions, vaccines and kits comprising said viral vector are also described. In one aspect the invention employs a synthetic C7 viral vector. The C7 viral vector according to the invention may be co-administered or co-formulated with a malaria antigen such as RTS,S optionally in the presence of an adjuvant for example comprising 3D-MPL and/or a saponin such as QS21.

Description

[0001] The present invention relates to a simian derived adenoviral vector particularly encoding a new malaria antigen derived from the circumsporozoite protein of Plasmodium falciparum. The invention further relates to processes of preparing said viral vector and use of same in the treatment/prevention of malaria infection.

[0002] Malaria, is one of the world's major health problems with more than 2 to 4 million people dying from the disease each year.

[0003] One of the most acute forms of the disease is caused by the protozoan parasite, Plasmodium falciparum (P. falciparum) which is responsible for most of the mortality attributable to malaria.

[0004] The life cycle of P. falciparum is complex, requiring two hosts, man and mosquito for completion. The infection of man is initiated by the inoculation of sporozoites in the bloodstream through the bite 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.

[0005] The sporozoite stage of Plasmodium has been identified as a potential target of a malaria vaccine. Vaccination with deactivated (irradiated) sporozoite has been shown to induce 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.

[0006] 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.

[0007] 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.

[0008] To date the most advanced malaria vaccine in the clinic is based on a lipoprotein particle (also known as a virus like particle) referred to as RTS,S. This particle contains a portion of the CS protein of P. falciparum substantially as corresponding to amino acids 207-395 of the CS protein of P. falciparum (strain NF54[3D7]) fused to the N-terminal of the S antigen from Hepatitis B. The S antigen may comprise a portion of the prcS2.

[0009] The RTS,S particle is usually delivered along with a strong adjuvant.

[0010] Nevertheless malaria vaccines have been proposed employing recombinant adenoviral vectors, for example WO 2004/055187 describes certain viral vectors including specific adeno 5 (Ad5) and adeno 35 (Ad 35) vectors, both derived from human adeno viruses, encoding CS protein.

[0011] There are more than 40 different serotypes of human adeno viruses, which vary in their pathogenicity, for example Ad5 is associated with mild respiratory infections in children, Ad4 and Ad7 are thought to be associated with respiratory infections in adults, and Ad40 is thought to cause diarrhoea in infants.

[0012] Immunity to adenovirus infections is thought to be life-long following infection. It is thought that pre-existing immunity to particularly Ad5 and Ad35 may result in the neutralisation of therapeutic adenoviral vectors based on human adeno viruses. This may reduce the therapeutic effectiveness of the vector as the vector is prevented from entering cells and manufacturing the relevant antigen in vivo.

[0013] The present invention is thought to reduce the issues of pre-existing immunity by providing a vaccine for prevention and/or treatment of malaria comprising:

a replication deficient simian adenoviral vector C7 (also referred to as Pan 7 or CV-33) encoding a protein comprising CS protein from P. falciparum or a fragment thereof, for example as shown in Seq ID No: 1 or Seq ID No: 3.

BRIEF DESCRIPTION OF THE SEQUENCES

[0014] Seq ID No: 1 An amino acid sequence of a protein/antigen derived from CS protein of P. falciparum (referred to herein as Ade2 protein) [0015] Seq ID No: 2 A nucleic acid sequence encoding the protein of Seq ID No: 1 (referred herein as Ade2 gene) [0016] Seq ID No: 3 An alternative amino acid sequence of a protein/antigen derived from CS protein of P. falciparum (referred herein as Ade1 protein) [0017] Seq ID No: 4 A nucleic acid sequence encoding the protein of Seq ID No: 3 (referred herein as Ade1 gene) [0018] Seq ID No: 5 Capsid protein sequence from Chimp Adeno 7 (seq ID No 17 from WO 03/046124) [0019] Seq ID No: 6 An amino acid sequence from Chimp Adeno 7 (seq ID No 20 from WO 03/046124) [0020] Seq ID No: 7 An amino acid sequence from P. falciparum CS protein [0021] Seq ID No: 8 An amino acid sequence from P. falciparum CS protein [0022] Seq ID No: 9 An amino acid sequence from P. falciparum CS protein [0023] Seq ID No: 10 An amino acid sequence from P. falciparum CS protein [0024] Seq ID No: 11 Nucleotide sequence for CpG 1826 [0025] Seq ID No: 12 Nucleotide sequence for CpG 1758 [0026] Seq ID No: 13 Nucleotide sequence for a CpG [0027] Seq ID No: 14 Nucleotide sequence for CpG 2006 [0028] Seq ID No: 15 Nucleotide sequence for CpG 1668 [0029] Seq ID No: 16 Nucleotide sequence for CpG 5456 [0030] Seq ID No: 17 Shows the nucleotide sequence of an alternative expression cassette to Ade2 expression cassette and cloned into C7 adenoviral vector [0031] Seq ID No: 18 Shows the nucleotide sequence of the Ade2 expression cassette, cloned into C7 adenoviral vector [0032] Seq ID No: 19 Shows the complete nucleotide sequence of the synthetic recombinant vector C7-Ade2.

BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1 Shows a plasmid map for pCR2.1-Adc2

[0034] FIG. 2 Shows the plasmid map for pShuttle6-Ade2

[0035] FIG. 3 Shows plasmid maps for pC7000-CMV Ade2

[0036] FIGS. 4 to 7 Show comparison between the CS-specific T cell responses induced by C7 Ade1 & C7 Ade2 in C57B1/6 mice.

[0037] FIGS. 8 to 11 Show comparison between the CS-specific T cell responses induced by C7 Ade2 and Ad5 Ade2 in C57B1/6 mice.

[0038] FIGS. 12 to 15 Show comparison between the CS-specific T cell responses induced by C7 Ade1 and Ad5 Ade1 in C57B1/6 mice.

[0039] FIG. 16 Shows anti-CS antibody responses determined by ELISA in C57B1/6 mice.

[0040] FIGS. 17 and 18 Show kinetics of the CS-specific CD8 T cell responses induced by C7-Ade2 in CB6F1 mice.

[0041] FIGS. 19 and 20 Show kinetics of the CS-specific CD4 T cell responses induced by C7 Ade2 in CB6F1 mice.

[0042] FIGS. 21 and 22 Show cytokine profile of the CS-specific CD8 T cell responses induced by C7-Ade2 in CB6F1 mice.

[0043] FIGS. 23 and 24 Show cytokine profile of the CS-specific CD4 T cell responses induced by C7-Ade2 in CB6F1 mice.

[0044] FIGS. 25 and 26 Show kinetics of the CS-specific CD8 T cell responses induced by C7-Adc2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0045] FIGS. 27 and 28 Show kinetics of the CS-specific CD4 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0046] FIG. 29 Shows kinetics of the HBs-specific CD8 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0047] FIG. 30 Shows kinetics of the HBs-specific CD4 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0048] FIGS. 31 and 32 Show cytokine profile of the CS-specific CD8 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0049] FIG. 33 Cytokine profile of the HBs-specific CD8 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0050] FIGS. 34 and 35 Cytokine profile of the CS-specific CD4 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0051] FIG. 36 Cytokine profile of the HBs-specific CD4 T cell responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0052] FIGS. 37 and 38 Show antibody responses antibody responses induced by C7-Ade2 in prime/boost or co-formulation with RTS,S/AS01B in CB6F1 mice.

[0053] FIGS. 39 to 41 Show kinetics of the CS- & HBs-specific CD8 T cell responses induced by co-formulation C7-Ade2+ RTS,S/AS01B in CB6F1 mice.

[0054] FIGS. 42 to 44 Show kinetics of the CS- & HBs-specific CD4 T cell responses induced by co-formulation C7-Ade2+ RTS,S/AS01B in CB6F1 mice.

[0055] FIGS. 45 to 47 Show cytokine profile of the CS- & HBs-specific CD8 T cell responses induced by co-formulation C7-Ade2+ RTS,S/AS01B in CB6F1 mice.

[0056] FIGS. 48 to 50 Show cytokine profile of the CS- & HBs-specific CD4 T cell responses induced by co-formulation C7-Ade2+ RTS,S/AS01B in CB6F1 mice.

[0057] FIGS. 51 and 52 Show antibody responses induced by co-formulation C7-Ade2+ RTS,S/AS01B in CB6F1 mice.

[0058] FIGS. 53 to 55 Show kinetics of the CS- & HBs-specific CD8 T cell responses induced by C7-Ade2, RTS,S and AS01B responses in CB6F1 mice.

[0059] FIGS. 56 to 58 Show kinetics of the CS- & HBs-specific CD4 T cell responses induced by C7-Adc2, RTS,S and AS01B responses in CB6F1 mice.

[0060] FIGS. 59 to 61 Show cytokinc profile of the CS- & HBs-specific CD8 T cell responses induced by C7-Ade2, RTS,S and AS01B responses in CB6F1 mice.

[0061] FIGS. 62 to 64 Show cytokine profile of the CS- & HBs-specific CD4 T cell responses induced by C7-Ade2, RTS,S and AS01B responses in CB6F1 mice.

[0062] FIGS. 65 and 66 Show antibody responses induced by C7-Ade2, RTS,S and AS01B responses in CB6F1 mice.

[0063] FIGS. 67 and 68 Show kinetics of the CS-specific CD8 T cell responses induced by synthetic C7 Ade2 in CB6F1 mice.

[0064] FIGS. 69 and 70 Show kinetics of the CS-specific CD4 T cell responses induced by synthetic C7 Ade2 in CB6F1 mice.

[0065] FIGS. 71 and 72 Show cytokine profile of the CS-specific CD8 T cell responses induced by synthetic C7 Ade2 in CB6F1 mice.

[0066] FIGS. 73 and 74 Show cytokine profile of the CS-specific CD4 T cell responses induced by synthetic C7 Ade2 in CB6F1 mice.

[0067] The sequence and preparation of C7 is described in WO 2003/046124. Sequence ID Nos: 6 (penton sequence), 9 (nucleic acid sequence), 10 & 11 (hexon sequence), & 12 (fibre protein) of WO 2003/046124 are incorporated by reference. The deposit number for C7 is [ATCC VR-593].

[0068] The characteristics and properties of any given adenoviral vector are often individual, although there is a hypothesis that vectors may be grouped into families and that adenoviral vectors within a given family may have similar characteristics.

[0069] Employing C7 is thought to be particularly advantageous as it seems to be more stable once the protein encoding gene is inserted than certain other known vectors, for example C6 also described in WO 2003/046124. That is to say C7 is thought to be less prone to re-organisation. Of course it is very important that any adenoviral vector employed in a vaccine is stable because pharmaceutical products need to be well characterised and shown to be stable and safe before they can be marketed.

[0070] Pre-existing immunity to C7 is thought to be very low and thus the risk of neutralisation of the viral vector after the first administration to a patient is low.

[0071] Furthermore, there are thought to be one or more other properties of C7 that are likely to make it particularly suitable for administration to humans and/or for generating a favourable immune response in vivo.

[0072] In one aspect the invention employs a synthetic C7 viral vector, which may be particularly suitable for gaining regulatory approval for administration to humans.

[0073] In one aspect the malaria antigen component from the CS protein has the last 12 to 14 amino acids removed.

[0074] In one aspect the malaria antigen encoded by the adenoviral vector is modified to remove potential glycosylation sites, for example the amino acid alanine may replace a serine, such as shown in position about 379 of Seq ID No: 1.

[0075] In one aspect the invention the protein/antigen employed comprises the following amino acids;

TABLE-US-00001 NNGDNGREGKDEDKRDGNN [Seq ID No: 7]

optionally located at about amino acid 81 to 99.

[0076] In one aspect the protein/antigen encoded comprises the amino acids:

TABLE-US-00002 AIGL [Seq ID No: 8]

for example at the C terminus.

[0077] In one aspect the invention employs a protein comprising the following amino acids:

TABLE-US-00003 [Seq ID No: 9] PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPN ANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPN

[0078] In one aspect the invention employs a protein comprising the following amino acids:

TABLE-US-00004 [Seq ID No: 10] NANP NVDP NANP NVDP NANP NVDP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NVDP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP

[0079] In a further aspect the protein/antigen employed comprises the sequences of Seq ID No. 7 and/or Seq ID No. 8 and/or Seq ID No 9.

[0080] In a further aspect the protein/antigen employed comprises the sequences of Seq ID No. 7 and/or Seq ID No. 8 and/or Seq ID No 10.

[0081] In one aspect the protein/antigen encoded is Seq ID No: 1 or 3.

[0082] The protein sequence given in Seq ID No: 1 is new and forms an aspect of the invention.

[0083] Polynucleotide encoding the protein sequence of Seq ID No:1 also forms an aspect of the invention, in particular the polynucleotide sequence of Seq ID No: 2. This polynucleotide sequence (ID No: 2) is already codon-optimized for expression in, humans.

[0084] Optionally a polynucleotide sequence encoding the protein of Seq ID No: 1 may be codon-optimized.

[0085] The invention also extends to vectors/plasmids/hosts employed in the preparation of the novel hybrid fusion protein of Seq ID No: 1 or employed in the preparation of a viral vector according to the invention.

[0086] When preparation and isolation of the protein is required a suitable plasmid can be employed to insert the sequence encoding for the protein into a suitable host for synthesis. An example of a suitable plasmid is pRIT15546 a 2 micron-based vector for carrying a suitable expression cassette. The plasmid will generally contain an in-built marker to assist selection, for example a gene encoding for antibiotic resistance or LEU2 or HIS auxotrophy.

[0087] 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).

[0088] In one aspect the invention provides use of the vectors according to the invention or a protein of Seq ID No 1 for the treatment or prevention of malaria.

[0089] In one aspect the invention provides a pharmaceutical formulation comprising a viral vector according to the invention and an excipient such as an isotonic carrier suitable for injection. Suitable excipients are discussed in more detail below.

[0090] In one embodiment a formulation comprises: [0091] an adeno viral vector according to the invention, [0092] a malaria antigen such as a lipoprotein particle particularly RTS,S, and [0093] optionally an adjuvant for example comprising a saponin and/or 3D-MPL.

[0094] When the vector encodes the sequence of Seq ID No: 1 the vector is particularly suitable for use in a treatment regime with the protein known as RTS,S. This is because the protein encoded by the adenoviral vector corresponds as closely as possible to the "RT" component in RTS,S. Use of the vector in a regime with RTS,S is thought to have the ability to reinforce efficiently the efficacy of RTS,S.

[0095] The viral vectors described herein are suitable for use as component for a malaria vaccine. The viral vectors of the invention may need to be used in combination with other components including other antigens to provide adequate protection against infection. Nevertheless the vectors of the present invention are suitable for use at least as a component of vaccine or treatment regime.

RTS,S

[0096] RTS,S can be prepared as described in WO 93/10152 (eg from P. falciparum NF54/3D7 strain). The nucleotide sequence for the RTS expression cassette and predicted translation product is provided in FIG. 9 of WO 93/10152 (referred to therein as RTS*).

[0097] 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. Suitable carriers include PBS, saline and the like. Adjuvants are also within this definition of excipient because whilst adjuvants may have a physiological effect in vivo this effect is general and in the absence of a therapeutic component is not a specific therapeutic effect.

Adjuvants

[0098] 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.

[0099] In an embodiment the adjuvant is a Toll like receptor (TLR) 4 ligand, for example an agonist such as a lipid A derivative particularly monophosphoryl lipid A or more particularly 3-deacylated monophoshoryl lipid A (3D-MPL).

[0100] 3-Deacylated monophosphoryl lipid A is known from U.S. Pat. No. 4,912,094 and UK patent application No. 2,220,211 (Ribi) and is available from Ribi Immunochem, Montana, USA.

[0101] 3D-MPL is sold under the trademark MPL.RTM. by Corixa corporation and primarily promotes CD4+ T cell responses with an IFN-g (Th1) 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 0.22 .mu.m filter. Such preparations are described in WO 94/21292. Synthetic derivatives of lipid A are known and thought to be TLR agonists including, but not limited to: [0102] OM174 (2-deoxy-6-O-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-O-phos- phono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.alpha.- -D-glucopyranosyldihydrogenphosphate), (WO 95/14026) [0103] OM 294 DP (3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-h- ydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate) (WO99/64301 and WO 00/0462), [0104] OM 197 MP-Ac DP (3S-, 9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxyt- etradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate 10-(6-aminohexanoate) (WO 01/46127).

[0105] Typically when 3D-MPL is used the antigen and 3D-MPL are delivered 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.

[0106] Other TLR4 ligands which may be used are alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO 9850399 or U.S. Pat. No. 6,303,347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in U.S. Pat. No. 6,764,840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants.

[0107] 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 Quilaja saponaria Molina which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response.

[0108] 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 .mu.g to about 100 .mu.g, such as about 10 .mu.g to about 50 .mu.g per dose.

[0109] Liposomal formulations 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%.

[0110] 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.

[0111] The saponins may 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), 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).

[0112] Usually, the saponin is presented in the form of a liposomal formulation, ISCOM or an oil in water emulsion.

[0113] 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 preferably 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 U.S. Pat. No. 5,666,153, U.S. Pat. No. 5,278,302 and WO 95/26204.

Examples of oligonucleotides are as follows:

TABLE-US-00005 [Seq ID No: 11] TCC ATG ACG TTC CTG ACG TT (CpG 1826) [Seq ID No: 12] TCT CCC AGC GTG CGC CAT (CpG 1758) [Seq ID No: 13] ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG [Seq ID No: 14] TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) [Seq ID No: 15] TCC ATG ACG TTC CTG ATG CT (CpG 1668) [Seq ID No: 16] TCG ACG TTT TCG GCG CGC GCC G (CpG 5456),

the sequences may contain phosphorothioate modified internucleotide linkages.

[0114] Alternative CpG oligonucleotides may comprise one or more sequences above in that they have inconsequential deletions or additions thereto.

[0115] The CpG oligonucleotides may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer.

[0116] Examples of a TLR 2 agonist include peptidoglycan or lipoprotein.

[0117] 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 agonists. 3D-MPL is an example of a TLR4 agonist whilst CpG is an example of a TLR9 agonist.

[0118] An immunostimulant may alternatively or in addition be included. In a one embodiment this immunostimulant will be 3-deacylated monophosphoryl lipid A (3D-MPL).

[0119] In one aspect the adjuvant comprises 3D-MPL.

[0120] In one aspect the adjuvant comprises QS21.

[0121] In one aspect the adjuvant comprises CpG.

[0122] In one aspect the adjuvant is formulated as an oil in water emulsion.

[0123] In one aspect the adjuvant is formulated as liposomes.

[0124] Adjuvants combinations include 3D-MPL and QS21 (EP 0 671 948 B1) oil in water emulsions or liposomal formulations comprising 3D-MPL and QS21 or 3D-MPL formulated with other carriers (EP 0 689 454 B1). Other preferred adjuvant systems comprise a combination of 3D-MPL, QS21 and a CpG oligonucleotide as described in U.S. Pat. No. 6,558,670 and U.S. Pat. No. 6,544,518.

Formulations

[0125] Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877.

[0126] The formulations of the present invention may be used for both prophylactic and therapeutic purposes. Accordingly the invention provides a vaccine composition as described herein for use in medicine, for example, for the treatment and/or prophylaxis of malaria.

[0127] In one aspect the invention provides a composition comprising a C7 adenoviral vector according to the invention and a malaria antigen such as RTS,S or the novel antigen of Seq ID No: 1 or virus like particles of the same and an excipient, optionally in the presence of an adjuvant.

[0128] Immunogenic in the context of this specification is intended to refer to the ability to elicit an immune response, wherein said response is specific to a malaria component in the relevant formulation. This response may require the presence of a suitable adjuvant and/or boosting. A booster, for example, comprising a dose similar or less than the original dose, may be required to obtain an appropriate immunogenic response.

[0129] 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, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PvAMA1 and RBP or fragment thereof.

[0130] Other example, antigens derived from P. falciparum include, PfEMP-1, Pfs 16 antigen, MSP-1, MSP-3, LSA-1, LSA-3, AMA-1 and TRAP. Other Plasmodium antigens include P. falciparum EBA, GLURP, RAP1, RAP2, Sequestrin, Pf332, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs48/45, Pfs230 and their analogues in other Plasmodium spp.

[0131] The invention also relates to use of C7 for encoding a malaria antigen, for example particularly as described herein for the treatment and/or prevention of malaria, or for the manufacture of a medicament for same.

[0132] The invention also includes a method of treatment comprising administering a therapeutically effective amount of one or more aspects of the invention. Optionally the C7 viral vector according to the invention may be co-administered or co-formulated with a malaria antigen such as RTS,S or the antigen of Seq ID No. 1, optionally in the presence of an adjuvant for example comprising 3D-MPL and/or a saponin such as QS21.

[0133] The C7 vector may also be co-administered or co-formulated with another adenoviral vector of a different serotype and/or origin, encoding the same of different antigens.

[0134] The invention also extends to use of any aspect defined herein in a prime boost regime, for example wherein the priming dose or doses is/are given at a timepoint zero (and subsequent primes within for example 3 months) and a boost is given, for example at about 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the last priming dose, optionally with a further boosting shot or shots given up to one year after said first boosting shot.

[0135] Advantageously one or more aspects of the invention, including the combination vaccine described above, stimulate specific humoral (that is antibody responses) and/or cellular immune responses (such as CD8+ and/or CD4+) such as antibody responses and CD 8+ and/or CD4+ responses, particularly CD8+ and antibody responses. That is to say responses specific to the CS protein and/or S antigen (as appropriate).

[0136] This type of balanced immune response may be required to give so called sterile protection against malarial infection.

[0137] Furthermore antibody responses for combinations may be augmented in relation to antibody responses to adjuvanted protein only regime schemes.

[0138] In one embodiment the invention provides use of C7 as the prime or boost in a prime boost regime with: [0139] a C7 adenoviral vector encoding the same or different malaria antigen, [0140] another viral vector such as a human adenoviral vector such as Ad5 or Ad35 encoding a malaria antigen such as a CS protein from P. falciparum or simian adenoviral vector of a different serotype (ie not C7), and/or [0141] a malaria antigen such as RTS,S and an adjuvant, for example comprising a saponin and/or 3D-MPL, as the complementary component of the regime.

[0142] The invention also provides any of the aspects herein described for the manufacture of a medicament for the treatment and/or prevention of malarial infection.

Quantities

[0143] The amount of 3D-MPL used is generally small, but depending on the vaccine formulation may be in the region of 1-1000 .mu.g per dose, for example 1-500 .mu.g per dose, and such as in the range 1 to 100 .mu.g per dose, such as 50 or 25 .mu.g per dose.

[0144] 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 may be in the region of 1-1000 .mu.g per dose, for example 1-500 .mu.g per dose, and such as in the range 1 to 100 .mu.g per dose.

[0145] The amount of saponin for use in the adjuvants of the present invention may be in the region of 1-1000 .mu.g per dose, for example 1-500 .mu.g per dose, such as 1-250 .mu.g per dose, and particularly in the range 1 to 100 .mu.g per dose such as 50 or 25 .mu.g per dose.

[0146] When protein is administered the dose may, for example be 1 to 500 .mu.g such as 10 to 100 .mu.g, particularly 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 .mu.g per dose.

[0147] When adenoviral vectors are administered the dose may, for example be 10.sup.3 to 10.sup.16 vpu such as 10.sup.6 to 10.sup.10 vpu.

[0148] When a combination is employed then the amounts employed for each component of the combination may correspond to the dose given for that component alone.

[0149] The invention also extends to kits comprising the elements employed in combinations according to the invention.

[0150] The invention further relates to a process for preparing an adenoviral vector according to the invention and formulations comprising the same.

[0151] The invention also relates to a method of producing the protein of Seq ID No 1.

[0152] In the context of this specification comprising is to be interpreted as including.

[0153] The invention extends to embodiments which correspond to embodiments described herein as comprising certain element but consisting or consisting essentially of the relevant elements.

[0154] Discussion in the background section of this specification is provided for the purpose of putting the invention in context. It is not to be taken as admission about what is known in the art and in particular is not an admission of what constitutes common general knowledge.

[0155] The examples below are shown to illustrate the methodology, which may be employed in the invention.

EXAMPLES

Example 1

[0156] The synthetic gene was prepared by a company Medigenomix. The gene was cloned into pCR2.1-TOPO-TA cloning vector (Invitrogen see FIG. 1). This vector was digested with NotI and BamHI and a recombinant shuttle plasmid vector (-Ade2) was created. The map of the shuttle plasmid is shown in FIG. 2.

Description of the Expression Cassette and Methodology for Virus Rescue

[0157] The expression cassette contains the cytomegalovirus (CMV) early promoter and first exon, an intron derived from the plasmid pCI (purchased from Promega) the DNA encoding Ade2, and the rabbit globin polyadenylation signal. The complete cassette is flanked by recognition sites for the restriction enzymes I-CeuI and PI-SceI respectively. The expression cassettes were excised from the shuttle plasmid using I-CeuI and PI-SceI and introduced into a plasmid molecular clone of an E1 deleted genome of SAdV-24 (ie C7)-pC7 000 pkGFP as described (Roy et al. Hum Gene Ther. (2004) 5:519-530) to obtain the plasmid shown in FIG. 3 The plasmid molecular clone DNA was linearized by digesting with the restriction enzyme PacI and transfected into HEK 293 cells to rescue recombinant adenovirus. The adenoviruses were propagated, amplified and purified using standard techniques.

[0158] The sequences of the expression cassette for (Ade2) from the I-CeuI to the PI-Sce recognition sites is shown in Seq ID Nos: 18.

Example 2

Immunogenicity of the C7-Ade1 & C7-Ade2 in C57B1/6 Mice

[0159] C57B1/6 mice were immunized once intramuscularly with a dose range (10e10, 10e9, 10e8 viral particles) of the C7 chimpadenoviruses expressing either of the construct Ade1 or Ade2. As positive controls, some mice were immunized with the human adenovirus 5 (at the dose of 10e9 and 10e8) expressing either of the construct Ade1 or Ade2. As negative controls, some mice were immunized with empty C7 & empty Ad5 viral vectors.

[0160] Peripheral blood was collected and pooled on days 14, 28, 34 and 49 post-immunization and the Ag-specific CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest, i.e. the N-terminal region (N-term) or C-terminal region (C-term) of the CS protein. As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.

[0161] The results indicate that under the experimental conditions described above, both constructs induced CS-specific CD4 and CD8 T cell responses (FIGS. 4 to 15). Of note, C-term specific CD8 T cell responses were only detected in mice immunized with adenoviruses carrying the Ade2 insert (FIGS. 4 & 8). In particular, these C-term specific CD8 T cell responses were similar in mice immunized with either 10e10vp of C7 Ade2 or 10e9vp of Ad5 Ade2 (FIG. 8).

[0162] In addition, the anti-CS antibody responses were determined by ELISA on sera collected 48 days post-immunization. In particular, it is the total Ig response against the R32LR polypeptide (i.e. which covers the middle portion of P. falciparum CSP) that was measured (Mettens et al., Vaccine 2008). The results indicate that a single immunization with C7 Ade1 or C7 Ade2 induces low levels of R32LR-specific antibody response. The intensity of this response correlates with the number of viral particles used for immunization (dose range effect).

Example 3

Immunogenicity of the C7-Ade2 in CB6F1 Mice

[0163] CB6F1 mice were immunized once intramuscularly with a dose range (10e10, 10e9, 10e8 viral particles) of the C7 chimpadenovirus expressing the Ade2 construct (5 pools of mice/group). Peripheral blood was collected and pooled on days 21, 28 and 35 post-immunization and the CS C-term and CS N-term specific CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest, i.e. the N-terminal region (N-term) or C-terminal region (C-term) of the CS protein. As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.

[0164] The results indicate that in this mouse strain, a single immunization of either 10e9vp or 10c10vp of C7 Adc2 induce C-term and N-term specific CD4 and CD8 T cell responses (FIGS. 17 to 20). In particular, the intensity of the average observed response is equal if not higher when the 10e9 vp dose is used. The CS-specific CD8 T cell responses are mainly N term-specific while the CS-specific CD4 T cell responses are equally targeting the N-term and C-term region of the CS protein.

[0165] The cytokine profile of the CS-specific CD4 and CD8 T cell response was also determined and it was similar across all the tested timepoints. The profile displayed on d28 post-immunization is shown in FIGS. 21 to 24 and is representative of the other tested timepoints. Briefly, the CS-specific CD8 T cell responses are mostly composed of CD8 T cells producing IFNg only (FIGS. 21 & 22). The CS-specific CD4 T cell responses are also composed of IFNg producing CD4 T cells but also and to a lesser extent of CD4 T cells producing IL2 only or both IL2 & IFNg (FIGS. 23 & 24).

Example 4

Immunogenicity of the C7-Ade2 in Prime/Boost or Co-Formulation with RTS,S/AS01B in CB6F1 Mice

[0166] We have tested the immunogenicity of the C7 chimpadenovirus expressing the Ade2 construct in either prime-boost or co-formulation (combo) with RTS,S/AS01B in CB6F1 mice (4 pools of mice/group). AS01B is an adjuvant system containing 3D-MPL and QS21 formulated with liposomes. Mice were immunized intramuscularly on d0, 14 and 28 as follows:

TABLE-US-00006 d 0 d 14 d 28 Group 1 A P P Group 2 P P A Group 3 P P P Group 4 C C C Where, A = 10e9 vp of C7 Ade2 P = 5 .mu.g RTS, S/50 .mu.l AS01B C = 10e10 vp of C7 Ade2 + 5 .mu.g RTS, S/50 .mu.l AS01B

[0167] Peripheral blood was collected and pooled on days 21 (7d pII), 35 (7d pIII), 49(21d pIII), 63 (35d pIII),77 (49d pIII) post-immunization and the CS C-term, CS N-term and HBs specific CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest (CS N-term, CS C-term or HBs). As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.

The results indicate that: [0168] APP, PPA and CCC all induce N-term specific CD8 T cell responses. At the 7d pII timepoint, N-term specific CD8 T cell responses are observed in the CCC group, followed by the APPgroup. At the later timepoints tested, (21d pIII, 35d pIII & 49d pIII), these response are & remain of similar intensity in the APP, PPA and CCC groups (FIG. 25). [0169] APP, PPA and CCC induce C-term specific CD8 T cell responses that still persist 49 days post 3.sup.rd immunization (FIG. 26) [0170] N-term specific CD4 T cell responses are mainly detected in mice immunized with APP or CCC with higher intensities of such responses in the APP group (FIG. 27) [0171] All groups display C-term specific CD4 T cell responses. However, APP and CCC induce similar higher levels of C-term specific CD4 T cell responses and these about 2 to 3 times higher than the ones induced by PPA and PPP at all the timepoints tested (FIG. 28). [0172] The HBs-specific CD4 and CD8T cell responses are higher in the animals immunized with PPP than with APP, PPA or CCC (FIGS. 29 and 30). [0173] The CCC treatment regimen is the only one that is associated with the simultaneous induction of CS and HBs-specific CD4 and CD8 T cell responses.

[0174] The cytokine profiles of the CS- and HBs- specific CD4 and CD8 T cell responses were also determined and were similar across the timepoints tested. The ones from the 21d pIII timepoint are shown below as representative of all timepoints tested (FIGS. 31 to 36). Briefly, the Ag-specific CD8 T cell responses are mostly composed of CD8 T cells producing IFNg (FIGS. 31 to 33). In contrast, the Ag-specific CD4 T cell responses are composed of a mixture of CD4 T cells producing IFNg, IFNg and IL-2 and to a lesser extent CD4 T cells producing IL-2 (FIGS. 34 to 36).

[0175] In addition, the Ag-specific antibody responses were determined by ELISA on sera collected 14 and 42 days post-3.sup.rd immunization. In particular, the total Ig responses against the R32LR polypeptide (i.e. which covers the middle portion of P. falciparum CSP) and against HBs were measured (Mettens et al., Vaccine 2008). All immunization regimens did elicit R32LR and HBs-specific antibody responses that persisted up to the last timepoint tested, i.e. 42 days post 3.sup.rd immunization (FIGS. 37 & 38).

Example 5

Immunogenicity of Co-Formulation C7-Ade2+ RTS,S/AS01B in CB6F1 Mice

[0176] In this experiment, we compared the immunogenicity of the chimpadenovirus C7 Ade2 co-formulated (Combo) with RTS,S/AS01B. In particular, we compared the immune response elicited by 1, 2 or 3 injections of the combo in CB6F1 mice (4 pools of mice/group). In addition, different intervals between the 2 injections of the combo were evaluated (i.e. 14 & 21 days). Finally, a group of mice immunized with A-P-P served as control in the experiment. The experimental design can be summarized as follows:

TABLE-US-00007 d 0 d 7 d 14 d 28 Group 1 C C Group 2 C-C (14 d interval) C C Group 3 C-C (21 d interval) C C Group 4 C-C-C C C C Group 5 A-P-P A P P Where, A = 10e9 vp of C7 Ade2 P = 5 .mu.g RTS, S/50 .mu.l AS01B C = 10e9 vp of C7 Ade2 + 5 .mu.g RTS, S/50 .mu.l AS01B

[0177] Peripheral blood was collected and pooled on days 35, 42, 49, 63 and 98 and the CS C-term, CS N-term and HBs specific CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest, i.e. the N-terminal region (N-term), the C-terminal region (C-term) of the CS protein or HBs. As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulatcd). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulatcd cells from the average cytokine response produced by the peptide-stimulated cells.

[0178] The results indicate that under these experimental conditions, 2 immunizations of the combo were required to simultaneously induce CS C-term, CS N-term and HBs-specific CD4 and CD8 T cell responses. The interval between the 2 immunizations of the combo did not seem to significantly impact the levels of Ag-specific T cell responses detected. There was a trend for higher HBs-specific CD4 and CD8 T cell responses in mice immunized 3 times with the combo. The kinetics of the Ag-specific T cell responses are shown in FIGS. 39 to 44.

[0179] The cytokine profiles of the CS- and HBs- specific CD4 and CD8 T cell responses were also determined and were similar across the timepoints tested. The ones from the day 42 of the study are shown below as representative of all timepoints tested (FIGS. 42 to 47). Briefly, the Ag-specific CD8 T cell responses are mostly composed of CD8 T cells producing IFNg (FIGS. 45 to 47). In contrast, the Ag-specific CD4 T cell responses are composed of a mixture of CD4 T cells producing IFNg, IFNg and IL-2 and to a lesser extent CD4 T cells producing IL-2 (FIGS. 48 to 50).

[0180] In addition, the Ag-specific antibody responses were determined by ELISA on sera collected on day 56 and 99 of the study. In particular, the total Ig responses against the R32LR polypeptide (i.e. which covers the middle portion of P. falciparum CSP) and against HBs were measured (Mettens et al., Vaccine 2008). All immunization regimens did elicit R32LR and HBs-specific antibody responses: within each group, these responses were of similar intensity at both timepoints tested. In addition, when the groups immunized with the combo were compared, there was a trend for higher responses in groups immunized with 3 doses of the combo (FIGS. 51 & 52).

Example 6

Evaluation of the Need for Each Component of the Combo, i.e. C7-Ade2, RTS,S and AS01B to elicit both CS & HBs T Cell and Ab Responses Simultaneously in CB6F1 Mice

[0181] In this experiment, we evaluated the need for each component of the combo (i.e. C7 Ade2, RTS,S and AS01B) to elicit CS and HBs-specific CD4 and CD8 T cell responses simultaneously. In this experiment, CB6F1 mice (6 pools of mice/group) were immunized twice intramuscularly (on days 0 & 14) with the combo or components thereof as shown below:

TABLE-US-00008 d 0 d 14 Group 1 C C Group 2 P P Group 3 RTS, S/buffer RTS, S/buffer Group 4 RTS, S/A/buffer RTS, S/A/buffer Group 5 A A Group 6 A/AS01B A/AS01B Group 7 RTS, S/C7 empty/AS01B RTS, S/C7 empty/AS01B Group 8 RTS, S/C7 empty/buffer RTS, S/C7 empty/buffer Where: A = 10e9 vp of C7 Ade2 P = 5 .mu.g RTS, S/50 .mu.l AS01B C = 10e9 vp of C7 Ade2 + 5 .mu.g RTS, S/50 .mu.l AS01B C7 empty = 10e9 vp of C7 empty vector (no insert) RTS, S = 5 .mu.g of RTS, S AS01B = GSK Proprietary Adjuvant System 1B Buffer = AS01B buffer (no immunostimulant)

[0182] Peripheral blood was collected and pooled on days 14, 28, 70, 91 & 112 and the CS C-term, CS N-term and HBs specific CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest, i.e. the N-terminal region (N-term), the C-terminal region (C-term) of the CS protein or HBs. As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.

[0183] In addition, the Ag-specific antibody responses were determined by ELISA on sera collected on day 42 and 84 of the study. In particular, the total Ig responses against the R32LR polypeptide (i.e. which covers the middle portion of P. falciparum CSP) and against HBs were measured (Mettens et al., Vaccine 2008).

[0184] The results indicate that each component of the combo is required to simultaneously elicit CS(N-term & C-term) CD4 and CD8 T cell responses (FIGS. 53 to 58) as well as R32LR and HBs antibody responses (FIGS. 65 and 66).

[0185] The average cytokine profiles of the CS- and HBs- specific CD4 and CD8 T cell responses were also determined at each timepoint of the study and these are shown in FIGS. 59 to 64. Briefly, the Ag-specific CD8 T cell responses are mostly composed of CD8 T cells producing IFNg (FIGS. 59 to 61). In contrast, the Ag-specific CD4 T cell responses are composed of a mixture of CD4 T cells producing IFNg, IFNg and IL-2 and to a lesser extent CD4 T cells producing IL-2 (FIGS. 62 to 64).

Example 7

Immunogenicity of a Synthetic C7 Ade2 in CB6F1 Mice

[0186] A synthetic C7 chimpadenovirus expressing the Ade2 construct was made available and its immunogenicity in mice was compared to the one of the original C7 Ade2. In this experiment, CB6F1 mice (6 pools of mice/group) were immunized with 10e9 vp of the original C7 Ade2 or its synthetic counterpart.

[0187] Peripheral blood was collected and pooled on days 21, 28 & 35 post-immunization and the CS C-term and CS N-term CD4 & CD8 T cell responses producing IL-2 and/or IFN-gamma were measured by flow cytometry, after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest, i.e. the N-terminal region (N-term), the C-terminal region (C-term) of the CS protein. As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The Ag-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.

[0188] The results indicate that both C7 adenoviruses elicited similar levels of N-term and C-term specific CD4 and CD8 T cell responses. In particular, and regardless of the viral vector used (original or synthetic), the Ag-specific CD8 T cell responses were mainly N-term specific (FIGS. 67 and 68). The N-term and C-term specific CD4 T cell responses were of similar intensity regardless of the viral vector used (FIGS. 69 and 70).

[0189] The cytokine profiles of the CS-specific CD4 and CD8 T cell responses were also determined and are shown in FIGS. 71 to 74. Briefly, the Ag-specific CD8 T cell responses were mostly composed of CD8 T cells producing IFNg (FIGS. 71 & 72). In contrast, the Ag-specific CD4 T cell responses were composed of a mixture of CD4 T cells producing IFNg, IFNg and IL-2 and to a lesser extent CD4 T cells producing IL-2 (FIGS. 73 to 74).

TABLE-US-00009 Amino acid sequence SEQ ID NO 1 MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNE 50 LEMNYYGKQENWYSLKKNSRSLGENDDGNNNNGDNGREGKDEDKRDGNNE 100 DNEKLRKPKHKKLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNAN 150 PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNANPN 200 ANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN 250 PNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKN 300 NNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPK 350 DELDYANDIEKKICKMEKCSSVFNVVNSAIGL 382 Nucleotide sequence SEQ ID NO 2 ATGATGAGAAAACTTGCCATCCTCAGCGTCAGCTCTTTCCTGTTCGTGGA 50 GGCCCTCTTCCAGGAGTATCAGTGCTACGGAAGCAGCAGCAATACAAGGG 100 TCCTGAACGAGCTCAACTATGACAACGCTGGAACGAACCTGTATAACGAG 150 CTGGAGATGAACTACTATGGCAAGCAGGAGAACTGGTATAGCCTGAAGAA 200 GAACAGCCGGTCCCTGGGCGAGAACGACGACGGCAACAACAACAACGGCG 250 ACAACGGCAGGGAGGGCAAAGATGAGGACAAGAGGGACGGGAACAACGAG 300 GATAACGAGAAGCTGCGGAAGCCCAAGCACAAGAAACTCAAGCAGCCCGC 350 CGACGGGAACCCGGACCCCAATGCAAATCCCAACGTCGACCCAAACGCAA 400 ACCCTAACGTGGACCCCAACGCCAATCCCAACGTCGATCCTAATGCCAAT 450 CCAAATGCCAACCCTAACGCAAATCCTAATGCAAACCCCAACGCCAATCC 500 TAACGCCAACCCAAATGCCAACCCAAACGCTAACCCCAACGCTAACCCAA 550 ATGCAAATCCCAATGCTAACCCAAACGTGGACCCTAACGCTAACCCCAAC 600 GCAAACCCTAACGCCAATCCTAACGCAAACCCCAATGCAAACCCAAACGC 650 AAATCCCAACGCTAACCCTAACGCAAACCCCAACGCCAACCCTAATGCCA 700 ACCCCAATGCTAACCCCAACGCCAATCCAAACGCAAATCCAAACGCCAAC 750 CCAAATGCAAACCCCAACGCTAATCCCAACGCCAACCCAAACGCCAATCC 800 TAACAAGAACAATCAGGGCAACGGGCAGGGCCATAACATGCCGAACGACC 850 CTAATCGGAATGTGGACGAGAACGCCAACGCCAACAGCGCCGTGAAGAAC 900 AACAACAACGAGGAGCCCTCCGACAAGCACATCAAGGAATACCTGAACAA 950 GATCCAGAACAGTCTGAGCACCGAGTGGTCCCCCTGCTCCGTGACCTGCG 1000 GCAACGGCATCCAGGTGAGGATCAAGCCCGGCTCCGCCAACAAGCCCAAG 1050 GACGAGCTGGACTACGCCAACGACATCGAGAAGAAGATCTGCAAGATGGA 1100 GAAATGCAGCTCTGTGTTCAACGTCGTGAACTCCGCCATCGGCCTGTGA 1149 Seq ID NO 3 MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNE 50 LEMNYYGKQENWYSLKKNSRSLGENDDGNNNNGDNGREGKDEDKRDGNNE 100 DNEKLRKPKHKKLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNAN 150 PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNANPN 200 ANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN 250 PNANPNANPNANKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEP 300 SDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYA 350 NDIEKKICKMEKCSSVFNVVNS 372 Seq ID NO 4 Atgatgaggaaactggccatcctgagcgtgagcagcttcctgttcgtgga 50 Ggccctgtttcaggagtaccagtgctacggcagcagcagcaacacccggg 100 Tgctgaacgagctgaactacgacaacgccggcaccaacctgtacaacgag 150 Ctggagatgaactactacggcaagcaggagaactggtacagcctgaagaa 200 Gaacagccggtctctgggcgagaacgacgacggcaacaacaacaacggcg 250 Acaacggccgggagggcaaggacgaggacaagcgggacggcaacaacgag 300 Gacaacgagaagctgcggaagcccaagcacaagaaacttaagcagcccgc 350 Cgacggcaaccccgaccccaacgccaaccccaacgtggaccccaacgcca 400 Atcctaatgtcgaccccaatgccaatccgaacgttgatcccaatgcgaat 450 Cctaacgctaaccccaatgccaacccaaatgccaatccaaatgcaaatcc 500 Caacgccaatccaaacgcaaaccctaatgctaatccaaacgctaatccta 550 Atgccaatcccaatgctaacccaaacgtcgatcctaacgcaaatccgaac 600 Gctaaccccaacgcaaatcccaacgctaacccgaacgcaaaccctaacgc 650 Caatccgaatgccaacccaaacgccaacccgaacgctaatccgaatgcta 700 Acccgaatgctaatcctaacgcaaacccaaatgcaaaccccaatgcaaac 750 Ccgaacgccaatcccaacgccaatcctaatgccaacaagaacaatcaggg 800 Caacggccagggccacaacatgcccaacgaccccaaccggaacgtggacg 850 Agaacgccaacgccaacagcgccgtgaagaacaacaacaacgaggagccc 900 Agcgacaagcacatcaaggagtacctgaacaagatccagaacagcctgag 950 Caccgagtggagcccctgcagcgtgacctgcggcaacggcattcaggtgc 1000 Ggatcaagcccggcagcgccaacaagcccaaggacgagctggactacgcc 1050 Aatgacatcgagaagaagatctgcaagatggagaagtgcagcagcgtgtt 1100 Caacgtggtgaactcctga 1119 Chimp Adeno 7(seq ID No 17 from WO 03/046124) Seq ID No. 5 Ala Pro Lys Gly Ala Pro Asn Thr Cys Gln Trp Thr Tyr Lys Ala Gly 1 5 10 15 Asp Thr Asp Thr Glu Lys Thr Tyr Thr Tyr Gly Asn Ala Pro Val Gln 20 25 30 Gly Ile Ser Ile Thr Lys Asp Gly Ile Gln Leu Gly Thr Asp Ser Asp 35 40 45 Gly Gln Ala Ile Tyr Ala Asp Glu Thr Tyr Gln Pro Glu Pro Gln Val 50 55 60 Gly Asp Ala Glu Trp His Asp Ile Thr Gly Thr Asp Glu Lys Tyr Gly 65 70 75 80 Gly Arg Ala Leu Lys Pro Asp Thr Lys Met Lys Pro Cys Tyr Gly Ser 85 90 95 Phe Ala Lys Pro Thr Asn Lys Glu Gly Gly Gln Ala Asn Val Lys Thr 100 105 110 Glu Thr Gly Gly Thr Lys Glu Tyr Asp Ile Asp Met Ala Phe Phe Asp 115 120 125 Asn Arg Ser Ala Ala Ala Ala Gly Leu Ala Pro Glu Ile Val Leu Tyr 130 135 140 Thr Glu Asn Val Asp Leu Glu Thr Pro Asp Thr His Ile Val Tyr Lys 145 150 155 160 Ala Gly Thr Asp Asp Ser Ser Ser Ser Ile Asn Leu Gly Gln Gln Ser 165 170 175 Met Pro Asn Arg Pro Asn Tyr Ile Gly Phe Arg Asp Asn Phe Ile Gly 180 185 190 Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln 195 200 205 Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu 210 215 220 Leu Ser Tyr Gln Leu Leu Leu Asp Ser Leu Gly Asp Arg Thr Arg Tyr 225 230 235 240 Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg 245 250 255 Ile Ile Glu Asn His Gly Val Glu Asp Glu Leu Pro Asn Tyr Cys Phe 260 265 270 Pro Leu Asp Ala Val Gly Arg Thr Asp Thr Tyr Gln Gly Ile Lys Ala 275 280 285 Asn Gly Asp Asn Gln Thr Thr Trp Thr Lys Asp Asp Thr Val Asn Asp 290 295 300 Ala Asn Glu Leu Gly Lys Gly Asn Pro Phe 305 310 Chimp Adeno 7(seq ID No 20 from WO 03/046124) Seq ID No. 6 Thr Leu Trp Thr Thr Ala Asp Pro Ser Pro Asn Cys Lys Ile Tyr Ser 1 5 10 15 Glu Lys Asp Ala Lys Leu Thr Leu Cys Leu Thr Lys Cys Gly Ser Gln 20 25 30 Ile Leu Gly Thr Val Thr Val Leu Ala Val Asn Asn Gly Ser Leu Asn 35 40 45 Pro Ile Thr Asn Thr Val Ser Thr Ala Leu Val Ser Leu Lys Phe Asp 50 55 60 Ala Ser Gly Val Leu Leu Ser Ser Ser Thr Leu Asp Lys Glu Tyr Trp 65 70 75 80 Asn Phe Arg Lys Gly Asp Val Thr Pro Ala Glu Pro Tyr Thr Asn Ala 85 90 95 Ile Gly Phe Met Pro Asn Ile Lys Ala Tyr Pro Lys Asn Thr Ser Ala 100 105 110 Ala Ser Lys Ser His Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Glu 115 120 125 Ala Lys Pro Leu Met Leu Ile Ile Thr Phe Asn Glu Thr Glu Asp Ala 130 135 140 Thr Cys Thr Tyr Ser Ile Thr Phe Gln Trp Lys Trp Asp Ser Thr Lys 145 150 155 160 Tyr Thr Gly Glu Thr Leu Ala Thr Ser Ser Phe Thr Phe Ser Tyr Ile 165 170 175 Ala Gln Glu [Seq ID No: 7] NNGDNGREGKDEDKRDGNN [Seq ID No: 8] AIGL [Seq ID No: 9] PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPN ANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPN [Seq ID No: 10] NANP NVDP NANP NVDP NANP NVDP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NVDP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP NANP

[Seq ID No: 11] TCC ATG ACG TTC CTG ACG TT (CpG 1826) [Seq ID No: 12] TCT CCC AGC GTG CGC CAT (CpG 1758) [Seq ID No: 13] ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG [Seq ID No: 14] TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) [Seq ID No: 15] TCC ATG ACG TTC CTG ATG CT (CpG 1668) [Seq ID No: 16] TCG ACG TTT TCG GCG CGC GCC G (CpG 5456), Seq ID No: 17 Sequence of a expression cassette for a protein of Seq ID No. 3. The protein encoding region is underlined. TAACTATAACGGTCCTAAGGTAGCGAAAGCTCAGATCGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTT CCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGC CCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCG GATTCCCCGTGCCAAGAGTGCGGCCAGCTTTATTGCGGTAGTTTATCACAGTTAAAT TGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTG GTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTAT TGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAA TTACAGCTCTTAAGGCTAGAGTGGCCGCACCATGATGAGGAAACTGGCCATCCTGAG CGTGAGCAGCTTCCTGTTCGTGGAGGCCCTGTTTCAGGAGTACCAGTGCTACGGCAG CAGCAGCAACACCCGGGTGCTGAACGAGCTGAACTACGACAACGCCGGCACCAACCT GTACAACGAGCTGGAGATGAACTACTACGGCAAGCAGGAGAACTGGTACAGCCTGAA GAAGAACAGCCGGTCTCTGGGCGAGAACGACGACGGCAACAACAACAACGGCGACAA CGGCCGGGAGGGCAAGGACGAGGACAAGCGGGACGGCAACAACGAGGACAACGAGAA GCTGCGGAAGCCCAAGCACAAGAAACTTAAGCAGCCCGCCGACGGCAACCCCGACCC CAACGCCAACCCCAACGTGGACCCCAACGCCAATCCTAATGTCGACCCCAATGCCAA TCCGAACGTTGATCCCAATGCGAATCCTAACGCTAACCCCAATGCCAACCCAAATGC CAATCCAAATGCAAATCCCAACGCCAATCCAAACGCAAACCCTAATGCTAATCCAAA CGCTAATCCTAATGCCAATCCCAATGCTAACCCAAACGTCGATCCTAACGCAAATCC GAACGCTAACCCCAACGCAAATCCCAACGCTAACCCGAACGCAAACCCTAACGCCAA TCCGAATGCCAACCCAAACGCCAACCCGAACGCTAATCCGAATGCTAACCCGAATGC TAATCCTAACGCAAACCCAAATGCAAACCCCAATGCAAACCCGAACGCCAATCCCAA CGCCAATCCTAATGCCAACAAGAACAATCAGGGCAACGGCCAGGGCCACAACATGCC CAACGACCCCAACCGGAACGTGGACGAGAACGCCAACGCCAACAGCGCCGTGAAGAA CAACAACAACGAGGAGCCCAGCGACAAGCACATCAAGGAGTACCTGAACAAGATCCA GAACAGCCTGAGCACCGAGTGGAGCCCCTGCAGCGTGACCTGCGGCAACGGCATTCA GGTGCGGATCAAGCCCGGCAGCGCCAACAAGCCCAAGGACGAGCTGGACTACGCCAA TGACATCGAGAAGAAGATCTGCAAGATGGAGAAGTGCAGCAGCGTGTTCAACGTGGT GAACTCCTGAGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAA GCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGT GTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGCGATCTGAATTCATCTATGTCGGG TGCGGAGAAAGAGGTAATGAAATGGCA Seq ID No: 18 Sequence of Ade2 expression cassette is below. The protein encoding region is underlined. TAACTATAACGGTCCTAAGGTAGCGAAAGCTCAGATCGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTT CCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGC CCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCG GATTCCCCGTGCCAAGAGTGCGGCCAGCTTTATTGCGGTAGTTTATCACAGTTAAAT TGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTG GTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTAT TGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAA TTACAGCTCTTAAGGCTAGAGTGGCCGCACCATGATGAGAAAACTTGCCATCCTCAG CGTCAGCTCTTTCCTGTTCGTGGAGGCCCTCTTCCAGGAGTATCAGTGCTACGGAAG CAGCAGCAATACAAGGGTCCTGAACGAGCTCAACTATGACAACGCTGGAACGAACCT GTATAACGAGCTGGAGATGAACTACTATGGCAAGCAGGAGAACTGGTATAGCCTGAA GAAGAACAGCCGGTCCCTGGGCGAGAACGACGACGGCAACAACAACAACGGCGACAA CGGCAGGGAGGGCAAAGATGAGGACAAGAGGGACGGGAACAACGAGGATAACGAGAA GCTGCGGAAGCCCAAGCACAAGAAACTCAAGCAGCCCGCCGACGGGAACCCGGACCC CAATGCAAATCCCAACGTCGACCCAAACGCAAACCCTAACGTGGACCCCAACGCCAA TCCCAACGTCGATCCTAATGCCAATCCAAATGCCAACCCTAACGCAAATCCTAATGC AAACCCCAACGCCAATCCTAACGCCAACCCAAATGCCAACCCAAACGCTAACCCCAA CGCTAACCCAAATGCAAATCCCAATGCTAACCCAAACGTGGACCCTAACGCTAACCC CAACGCAAACCCTAACGCCAATCCTAACGCAAACCCCAATGCAAACCCAAACGCAAA TCCCAACGCTAACCCTAACGCAAACCCCAACGCCAACCCTAATGCCAACCCCAATGC TAACCCCAACGCCAATCCAAACGCAAATCCAAACGCCAACCCAAATGCAAACCCCAA CGCTAATCCCAACGCCAACCCAAACGCCAATCCTAACAAGAACAATCAGGGCAACGG GCAGGGCCATAACATGCCGAACGACCCTAACCGGAATGTGGACGAGAACGCCAACGC CAACAGCGCCGTGAAGAACAACAACAACGAGGAGCCCTCCGACAAGCACATCAAGGA ATACCTGAACAAGATCCAGAACAGTCTGAGCACCGAGTGGTCCCCCTGCTCCGTGAC CTGCGGCAACGGCATCCAGGTGAGGATCAAGCCCGGCTCCGCCAACAAGCCCAAGGA CGAGCTGGACTACGCCAACGACATCGAGAAGAAGATCTGCAAGATGGAGAAATGCAG CTCTGTGTTCAACGTCGTGAACTCCGCCATCGGCCTGTGAGGATCCGATCTTTTTCC CTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTA ATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACT CGGAAGCGATCTGAATTCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA T

Sequence CWU 1

1

191382PRTArtificial SequenceAmino acid sequence of Ade2 protein 1Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val1 5 10 15Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asn Asn65 70 75 80Asn Asn Gly Asp Asn Gly Arg Glu Gly Lys Asp Glu Asp Lys Arg Asp 85 90 95Gly Asn Asn Glu Asp Asn Glu Lys Leu Arg Lys Pro Lys His Lys Lys 100 105 110Leu Lys Gln Pro Ala Asp Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn 115 120 125Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 130 135 140Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn145 150 155 160Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 165 170 175Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 180 185 190Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 195 200 205Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 210 215 220Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn225 230 235 240Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 245 250 255Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Lys Asn Asn Gln 260 265 270Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp Pro Asn Arg Asn Val 275 280 285Asp Glu Asn Ala Asn Ala Asn Ser Ala Val Lys Asn Asn Asn Asn Glu 290 295 300Glu Pro Ser Asp Lys His Ile Lys Glu Tyr Leu Asn Lys Ile Gln Asn305 310 315 320Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly 325 330 335Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn Lys Pro Lys Asp Glu 340 345 350Leu Asp Tyr Ala Asn Asp Ile Glu Lys Lys Ile Cys Lys Met Glu Lys 355 360 365Cys Ser Ser Val Phe Asn Val Val Asn Ser Ala Ile Gly Leu 370 375 38021149DNAArtificial SequenceAde2 nucleic acid sequence 2atgatgagaa aacttgccat cctcagcgtc agctctttcc tgttcgtgga ggccctcttc 60caggagtatc agtgctacgg aagcagcagc aatacaaggg tcctgaacga gctcaactat 120gacaacgctg gaacgaacct gtataacgag ctggagatga actactatgg caagcaggag 180aactggtata gcctgaagaa gaacagccgg tccctgggcg agaacgacga cggcaacaac 240aacaacggcg acaacggcag ggagggcaaa gatgaggaca agagggacgg gaacaacgag 300gataacgaga agctgcggaa gcccaagcac aagaaactca agcagcccgc cgacgggaac 360ccggacccca atgcaaatcc caacgtcgac ccaaacgcaa accctaacgt ggaccccaac 420gccaatccca acgtcgatcc taatgccaat ccaaatgcca accctaacgc aaatcctaat 480gcaaacccca acgccaatcc taacgccaac ccaaatgcca acccaaacgc taaccccaac 540gctaacccaa atgcaaatcc caatgctaac ccaaacgtgg accctaacgc taaccccaac 600gcaaacccta acgccaatcc taacgcaaac cccaatgcaa acccaaacgc aaatcccaac 660gctaacccta acgcaaaccc caacgccaac cctaatgcca accccaatgc taaccccaac 720gccaatccaa acgcaaatcc aaacgccaac ccaaatgcaa accccaacgc taatcccaac 780gccaacccaa acgccaatcc taacaagaac aatcagggca acgggcaggg ccataacatg 840ccgaacgacc ctaatcggaa tgtggacgag aacgccaacg ccaacagcgc cgtgaagaac 900aacaacaacg aggagccctc cgacaagcac atcaaggaat acctgaacaa gatccagaac 960agtctgagca ccgagtggtc cccctgctcc gtgacctgcg gcaacggcat ccaggtgagg 1020atcaagcccg gctccgccaa caagcccaag gacgagctgg actacgccaa cgacatcgag 1080aagaagatct gcaagatgga gaaatgcagc tctgtgttca acgtcgtgaa ctccgccatc 1140ggcctgtga 11493372PRTArtificial SequenceAmino acid sequence of Ade1 protein 3Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val1 5 10 15Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asn Asn65 70 75 80Asn Asn Gly Asp Asn Gly Arg Glu Gly Lys Asp Glu Asp Lys Arg Asp 85 90 95Gly Asn Asn Glu Asp Asn Glu Lys Leu Arg Lys Pro Lys His Lys Lys 100 105 110Leu Lys Gln Pro Ala Asp Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn 115 120 125Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 130 135 140Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn145 150 155 160Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 165 170 175Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 180 185 190Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 195 200 205Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 210 215 220Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn225 230 235 240Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 245 250 255Ala Asn Pro Asn Ala Asn Lys Asn Asn Gln Gly Asn Gly Gln Gly His 260 265 270Asn Met Pro Asn Asp Pro Asn Arg Asn Val Asp Glu Asn Ala Asn Ala 275 280 285Asn Ser Ala Val Lys Asn Asn Asn Asn Glu Glu Pro Ser Asp Lys His 290 295 300Ile Lys Glu Tyr Leu Asn Lys Ile Gln Asn Ser Leu Ser Thr Glu Trp305 310 315 320Ser Pro Cys Ser Val Thr Cys Gly Asn Gly Ile Gln Val Arg Ile Lys 325 330 335Pro Gly Ser Ala Asn Lys Pro Lys Asp Glu Leu Asp Tyr Ala Asn Asp 340 345 350Ile Glu Lys Lys Ile Cys Lys Met Glu Lys Cys Ser Ser Val Phe Asn 355 360 365Val Val Asn Ser 37041119DNAArtificial SequenceAde1 nucleic acid sequence 4atgatgagga aactggccat cctgagcgtg agcagcttcc tgttcgtgga ggccctgttt 60caggagtacc agtgctacgg cagcagcagc aacacccggg tgctgaacga gctgaactac 120gacaacgccg gcaccaacct gtacaacgag ctggagatga actactacgg caagcaggag 180aactggtaca gcctgaagaa gaacagccgg tctctgggcg agaacgacga cggcaacaac 240aacaacggcg acaacggccg ggagggcaag gacgaggaca agcgggacgg caacaacgag 300gacaacgaga agctgcggaa gcccaagcac aagaaactta agcagcccgc cgacggcaac 360cccgacccca acgccaaccc caacgtggac cccaacgcca atcctaatgt cgaccccaat 420gccaatccga acgttgatcc caatgcgaat cctaacgcta accccaatgc caacccaaat 480gccaatccaa atgcaaatcc caacgccaat ccaaacgcaa accctaatgc taatccaaac 540gctaatccta atgccaatcc caatgctaac ccaaacgtcg atcctaacgc aaatccgaac 600gctaacccca acgcaaatcc caacgctaac ccgaacgcaa accctaacgc caatccgaat 660gccaacccaa acgccaaccc gaacgctaat ccgaatgcta acccgaatgc taatcctaac 720gcaaacccaa atgcaaaccc caatgcaaac ccgaacgcca atcccaacgc caatcctaat 780gccaacaaga acaatcaggg caacggccag ggccacaaca tgcccaacga ccccaaccgg 840aacgtggacg agaacgccaa cgccaacagc gccgtgaaga acaacaacaa cgaggagccc 900agcgacaagc acatcaagga gtacctgaac aagatccaga acagcctgag caccgagtgg 960agcccctgca gcgtgacctg cggcaacggc attcaggtgc ggatcaagcc cggcagcgcc 1020aacaagccca aggacgagct ggactacgcc aatgacatcg agaagaagat ctgcaagatg 1080gagaagtgca gcagcgtgtt caacgtggtg aactcctga 11195314PRTchimpanzee adenovirus Pan7 5Ala Pro Lys Gly Ala Pro Asn Thr Cys Gln Trp Thr Tyr Lys Ala Gly1 5 10 15Asp Thr Asp Thr Glu Lys Thr Tyr Thr Tyr Gly Asn Ala Pro Val Gln 20 25 30Gly Ile Ser Ile Thr Lys Asp Gly Ile Gln Leu Gly Thr Asp Ser Asp 35 40 45Gly Gln Ala Ile Tyr Ala Asp Glu Thr Tyr Gln Pro Glu Pro Gln Val 50 55 60Gly Asp Ala Glu Trp His Asp Ile Thr Gly Thr Asp Glu Lys Tyr Gly65 70 75 80Gly Arg Ala Leu Lys Pro Asp Thr Lys Met Lys Pro Cys Tyr Gly Ser 85 90 95Phe Ala Lys Pro Thr Asn Lys Glu Gly Gly Gln Ala Asn Val Lys Thr 100 105 110Glu Thr Gly Gly Thr Lys Glu Tyr Asp Ile Asp Met Ala Phe Phe Asp 115 120 125Asn Arg Ser Ala Ala Ala Ala Gly Leu Ala Pro Glu Ile Val Leu Tyr 130 135 140Thr Glu Asn Val Asp Leu Glu Thr Pro Asp Thr His Ile Val Tyr Lys145 150 155 160Ala Gly Thr Asp Asp Ser Ser Ser Ser Ile Asn Leu Gly Gln Gln Ser 165 170 175Met Pro Asn Arg Pro Asn Tyr Ile Gly Phe Arg Asp Asn Phe Ile Gly 180 185 190Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln 195 200 205Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu 210 215 220Leu Ser Tyr Gln Leu Leu Leu Asp Ser Leu Gly Asp Arg Thr Arg Tyr225 230 235 240Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg 245 250 255Ile Ile Glu Asn His Gly Val Glu Asp Glu Leu Pro Asn Tyr Cys Phe 260 265 270Pro Leu Asp Ala Val Gly Arg Thr Asp Thr Tyr Gln Gly Ile Lys Ala 275 280 285Asn Gly Asp Asn Gln Thr Thr Trp Thr Lys Asp Asp Thr Val Asn Asp 290 295 300Ala Asn Glu Leu Gly Lys Gly Asn Pro Phe305 3106179PRTchimpanzee adenovirus Pan7 6Thr Leu Trp Thr Thr Ala Asp Pro Ser Pro Asn Cys Lys Ile Tyr Ser1 5 10 15Glu Lys Asp Ala Lys Leu Thr Leu Cys Leu Thr Lys Cys Gly Ser Gln 20 25 30Ile Leu Gly Thr Val Thr Val Leu Ala Val Asn Asn Gly Ser Leu Asn 35 40 45Pro Ile Thr Asn Thr Val Ser Thr Ala Leu Val Ser Leu Lys Phe Asp 50 55 60Ala Ser Gly Val Leu Leu Ser Ser Ser Thr Leu Asp Lys Glu Tyr Trp65 70 75 80Asn Phe Arg Lys Gly Asp Val Thr Pro Ala Glu Pro Tyr Thr Asn Ala 85 90 95Ile Gly Phe Met Pro Asn Ile Lys Ala Tyr Pro Lys Asn Thr Ser Ala 100 105 110Ala Ser Lys Ser His Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Glu 115 120 125Ala Lys Pro Leu Met Leu Ile Ile Thr Phe Asn Glu Thr Glu Asp Ala 130 135 140Thr Cys Thr Tyr Ser Ile Thr Phe Gln Trp Lys Trp Asp Ser Thr Lys145 150 155 160Tyr Thr Gly Glu Thr Leu Ala Thr Ser Ser Phe Thr Phe Ser Tyr Ile 165 170 175Ala Gln Glu719PRTPlasmodium falciparum 7Asn Asn Gly Asp Asn Gly Arg Glu Gly Lys Asp Glu Asp Lys Arg Asp1 5 10 15Gly Asn Asn84PRTArtificial SequenceAmino acid sequence derived from P. falciparum CS protein 8Ala Ile Gly Leu19122PRTArtificial SequenceAmino acid sequence derived from P. falciparum CS protein 9Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn1 5 10 15Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 20 25 30Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Val Asp 35 40 45Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 50 55 60Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn65 70 75 80Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 85 90 95Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 100 105 110Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 115 12010144PRTArtificial SequenceAmino acid sequence derived from P. falciparum CS protein 10Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro1 5 10 15Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro 20 25 30Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 35 40 45Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 50 55 60Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro65 70 75 80Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 85 90 95Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 100 105 110Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 115 120 125Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 130 135 1401120DNAArtificial SequenceCpG oligonucleotide 11tccatgacgt tcctgacgtt 201218DNAArtificial SequenceCpG oligonucleotide 12tctcccagcg tgcgccat 181330DNAArtificial SequenceCpG oligonucleotide 13accgatgacg tcgccggtga cggcaccacg 301424DNAArtificial SequenceCpG oligonucleotide 14tcgtcgtttt gtcgttttgt cgtt 241520DNAArtificial SequenceCpG oligonucleotide 15tccatgacgt tcctgatgct 201622DNAArtificial SequenceCpG oligonucleotide 16tcgacgtttt cggcgcgcgc cg 22172250DNAArtificial SequenceAde 1 expression cassette 17taactataac ggtcctaagg tagcgaaagc tcagatcggc tgaccgccca acgacccccg 60cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 120acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 180tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 240ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc 300tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc 360acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 420tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag 480gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg 540gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg 600cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgcgg ccagctttat 660tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc 720gaacttaagc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag 780acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt 840ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca 900ctcccagttc aattacagct cttaaggcta gagtggccgc accatgatga ggaaactggc 960catcctgagc gtgagcagct tcctgttcgt ggaggccctg tttcaggagt accagtgcta 1020cggcagcagc agcaacaccc gggtgctgaa cgagctgaac tacgacaacg ccggcaccaa 1080cctgtacaac gagctggaga tgaactacta cggcaagcag gagaactggt acagcctgaa 1140gaagaacagc cggtctctgg gcgagaacga cgacggcaac aacaacaacg gcgacaacgg 1200ccgggagggc aaggacgagg acaagcggga cggcaacaac gaggacaacg agaagctgcg 1260gaagcccaag cacaagaaac ttaagcagcc cgccgacggc aaccccgacc ccaacgccaa 1320ccccaacgtg gaccccaacg ccaatcctaa tgtcgacccc aatgccaatc cgaacgttga 1380tcccaatgcg aatcctaacg ctaaccccaa tgccaaccca aatgccaatc caaatgcaaa 1440tcccaacgcc aatccaaacg caaaccctaa tgctaatcca aacgctaatc ctaatgccaa 1500tcccaatgct aacccaaacg tcgatcctaa cgcaaatccg aacgctaacc ccaacgcaaa 1560tcccaacgct aacccgaacg caaaccctaa cgccaatccg aatgccaacc caaacgccaa 1620cccgaacgct aatccgaatg ctaacccgaa tgctaatcct aacgcaaacc caaatgcaaa 1680ccccaatgca aacccgaacg ccaatcccaa cgccaatcct aatgccaaca agaacaatca 1740gggcaacggc cagggccaca acatgcccaa cgaccccaac cggaacgtgg acgagaacgc 1800caacgccaac agcgccgtga agaacaacaa caacgaggag cccagcgaca agcacatcaa 1860ggagtacctg aacaagatcc agaacagcct gagcaccgag tggagcccct gcagcgtgac 1920ctgcggcaac ggcattcagg tgcggatcaa gcccggcagc gccaacaagc ccaaggacga 1980gctggactac gccaatgaca tcgagaagaa gatctgcaag atggagaagt gcagcagcgt 2040gttcaacgtg gtgaactcct gaggatccga tctttttccc tctgccaaaa attatgggga 2100catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc 2160aatagtgtgt tggaattttt tgtgtctctc actcggaagc gatctgaatt catctatgtc 2220gggtgcggag aaagaggtaa tgaaatggca

2250182281DNAArtificial SequenceAde2 expression cassette 18taactataac ggtcctaagg tagcgaaagc tcagatcggc tgaccgccca acgacccccg 60cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 120acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 180tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 240ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc 300tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc 360acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 420tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag 480gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg 540gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg 600cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgcgg ccagctttat 660tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc 720gaacttaagc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag 780acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt 840ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca 900ctcccagttc aattacagct cttaaggcta gagtggccgc accatgatga gaaaacttgc 960catcctcagc gtcagctctt tcctgttcgt ggaggccctc ttccaggagt atcagtgcta 1020cggaagcagc agcaatacaa gggtcctgaa cgagctcaac tatgacaacg ctggaacgaa 1080cctgtataac gagctggaga tgaactacta tggcaagcag gagaactggt atagcctgaa 1140gaagaacagc cggtccctgg gcgagaacga cgacggcaac aacaacaacg gcgacaacgg 1200cagggagggc aaagatgagg acaagaggga cgggaacaac gaggataacg agaagctgcg 1260gaagcccaag cacaagaaac tcaagcagcc cgccgacggg aacccggacc ccaatgcaaa 1320tcccaacgtc gacccaaacg caaaccctaa cgtggacccc aacgccaatc ccaacgtcga 1380tcctaatgcc aatccaaatg ccaaccctaa cgcaaatcct aatgcaaacc ccaacgccaa 1440tcctaacgcc aacccaaatg ccaacccaaa cgctaacccc aacgctaacc caaatgcaaa 1500tcccaatgct aacccaaacg tggaccctaa cgctaacccc aacgcaaacc ctaacgccaa 1560tcctaacgca aaccccaatg caaacccaaa cgcaaatccc aacgctaacc ctaacgcaaa 1620ccccaacgcc aaccctaatg ccaaccccaa tgctaacccc aacgccaatc caaacgcaaa 1680tccaaacgcc aacccaaatg caaaccccaa cgctaatccc aacgccaacc caaacgccaa 1740tcctaacaag aacaatcagg gcaacgggca gggccataac atgccgaacg accctaaccg 1800gaatgtggac gagaacgcca acgccaacag cgccgtgaag aacaacaaca acgaggagcc 1860ctccgacaag cacatcaagg aatacctgaa caagatccag aacagtctga gcaccgagtg 1920gtccccctgc tccgtgacct gcggcaacgg catccaggtg aggatcaagc ccggctccgc 1980caacaagccc aaggacgagc tggactacgc caacgacatc gagaagaaga tctgcaagat 2040ggagaaatgc agctctgtgt tcaacgtcgt gaactccgcc atcggcctgt gaggatccga 2100tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc 2160tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc 2220actcggaagc gatctgaatt catctatgtc gggtgcggag aaagaggtaa tgaaatggca 2280t 22811938398DNAArtificial SequenceSynthetic recombinant vector C7-Ade2. 19catcatcaat aatatacctc aaacttttgg tgcgcgttaa tatgcaaatg agctgtttga 60atttggggag ggaggaaggt gattggccga gagacgggcg accgttaggg gcggggcggg 120tgacgttttg atgacgtggc cgtgaggcgg agccggtttg caagttctcg tgggaaaagt 180gacgtcaaac gaggtgtggt ttgaacacgg aaatactcaa ttttcccgcg ctctctgaca 240ggaaatgagg tgtttctggg cggatgcaag tgaaaacggg ccattttcgc gcgaaaactg 300aatgaggaag tgaaaatctg agtaatttcg cgtttatggc agggaggagt atttgccgag 360ggccgagtag actttgaccg attacgtggg ggtttcgatt accgtatttt tcacctaaat 420ttccgcgtac ggtgtcaaag tccggtgttt ttacatcatt tccccgaaaa gtgccacctg 480acgtaactat aacggtccta aggtagcgaa agctcagatc ggctgaccgc ccaacgaccc 540ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 600ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 660tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 720tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat 780cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga 840ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 900aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg 960taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 1020ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct 1080ccgcggccgg gaacggtgca ttggaacgcg gattccccgt gccaagagtg cggccagctt 1140tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt 1200ctcgaactta agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac 1260aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg 1320tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt 1380ccactcccag ttcaattaca gctcttaagg ctagagtggc cgcaccatga tgagaaaact 1440tgccatcctc agcgtcagct ctttcctgtt cgtggaggcc ctcttccagg agtatcagtg 1500ctacggaagc agcagcaata caagggtcct gaacgagctc aactatgaca acgctggaac 1560gaacctgtat aacgagctgg agatgaacta ctatggcaag caggagaact ggtatagcct 1620gaagaagaac agccggtccc tgggcgagaa cgacgacggc aacaacaaca acggcgacaa 1680cggcagggag ggcaaagatg aggacaagag ggacgggaac aacgaggata acgagaagct 1740gcggaagccc aagcacaaga aactcaagca gcccgccgac gggaacccgg accccaatgc 1800aaatcccaac gtcgacccaa acgcaaaccc taacgtggac cccaacgcca atcccaacgt 1860cgatcctaat gccaatccaa atgccaaccc taacgcaaat cctaatgcaa accccaacgc 1920caatcctaac gccaacccaa atgccaaccc aaacgctaac cccaacgcta acccaaatgc 1980aaatcccaat gctaacccaa acgtggaccc taacgctaac cccaacgcaa accctaacgc 2040caatcctaac gcaaacccca atgcaaaccc aaacgcaaat cccaacgcta accctaacgc 2100aaaccccaac gccaacccta atgccaaccc caatgctaac cccaacgcca atccaaacgc 2160aaatccaaac gccaacccaa atgcaaaccc caacgctaat cccaacgcca acccaaacgc 2220caatcctaac aagaacaatc agggcaacgg gcagggccat aacatgccga acgaccctaa 2280tcggaatgtg gacgagaacg ccaacgccaa cagcgccgtg aagaacaaca acaacgagga 2340gccctccgac aagcacatca aggaatacct gaacaagatc cagaacagtc tgagcaccga 2400gtggtccccc tgctccgtga cctgcggcaa cggcatccag gtgaggatca agcccggctc 2460cgccaacaag cccaaggacg agctggacta cgccaacgac atcgagaaga agatctgcaa 2520gatggagaaa tgcagctctg tgttcaacgt cgtgaactcc gccatcggcc tgtgaggatc 2580cgatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac 2640ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct 2700ctcactcgga agcgatctga attcatctat gtcgggtgcg gagaaagagg taatgaaatg 2760gcattatggg tattatgggt ctgcattaat gaatcggcca gatatgctgg ccaccgtgca 2820tgtgacctcg cacccccgca agacatggcc cgagttcgag cacaacgtca tgacccgatg 2880caatgtgcac ctggggtccc gccgaggcat gttcatgccc taccagtgca acatgcaatt 2940tgtgaaggtg ctgctggagc ccgatgccat gtccagagtg agcctgacgg gggtgtttga 3000catgaatgtg gagctgtgga aaattctgag atatgatgaa tccaagacca ggtgccgggc 3060ctgcgaatgc ggaggcaagc acgccaggct tcagcccgtg tgtgtggagg tgacggagga 3120cctgcgaccc gatcatttgg tgttgtcctg caacgggacg gagttcggct ccagcgggga 3180agaatctgac tagagtgagt agtgtttggg ggaggtggag ggcttgtatg aggggcagaa 3240tgactaaaat ctgtgttttt ctgtgtgttg cagcagcatg agcggaagcg cctcctttga 3300gggaggggta ttcagccctt atctgacggg gcgtctcccc tcctgggcgg gagtgcgtca 3360gaatgtgatg ggatccacgg tggacggccg gcccgtgcag cccgcgaact cttcaaccct 3420gacctacgcg accctgagct cctcgtccgt ggacgcagct gccgccgcag ctgctgcttc 3480cgccgccagc gccgtgcgcg gaatggccct gggcgccggc tactacagct ctctggtggc 3540caactcgact tccaccaata atcccgccag cctgaacgag gagaagctgc tgctgctgat 3600ggcccagctc gaggccctga cccagcgcct gggcgagctg acccagcagg tggctcagct 3660gcaggcggag acgcgggccg cggttgccac ggtgaaaacc aaataaaaaa tgaatcaata 3720aataaacgga gacggttgtt gattttaaca cagagtcttg aatctttatt tgatttttcg 3780cgcgcggtag gccctggacc accggtctcg atcattgagc acccggtgga ttttttccag 3840gacccggtag aggtgggctt ggatgttgag gtacatgggc atgagcccgt cccgggggtg 3900gaggtagctc cattgcaggg cctcgtgctc gggggtggtg ttgtaaatca cccagtcata 3960gcaggggcgc agggcgtggt gctgcacgat gtccttgagg aggagactga tggccacggg 4020cagccccttg gtgtaggtgt tgacgaacct gttgagctgg gagggatgca tgcgggggga 4080gatgagatgc atcttggcct ggatcttgag attggcgatg ttcccgccca gatcccgccg 4140ggggttcatg ttgtgcagga ccaccagcac ggtgtatccg gtgcacttgg ggaatttgtc 4200atgcaacttg gaagggaagg cgtgaaagaa tttggagacg cccttgtgac cgcccaggtt 4260ttccatgcac tcatccatga tgatggcgat gggcccgtgg gcggcggcct gggcaaagac 4320gtttcggggg tcggacacat cgtagttgtg gtcctgggtg agctcgtcat aggccatttt 4380aatgaatttg gggcggaggg tgcccgactg ggggacgaag gtgccctcga tcccgggggc 4440gtagttgccc tcgcagatct gcatctccca ggccttgagc tcggaggggg ggatcatgtc 4500cacctgcggg gcgatgaaaa aaacggtttc cggggcgggg gagatgagct gggccgaaag 4560caggttccgg agcagctggg acttgccgca gccggtgggg ccgtagatga ccccgatgac 4620cggctgcagg tggtagttga gggagagaca gctgccgtcc tcgcggagga ggggggccac 4680ctcgttcatc atctcgcgca catgcatgtt ctcgcgcacg agttccgcca ggaggcgctc 4740gccccccagc gagaggagct cttgcagcga ggcgaagttt ttcagcggct tgagcccgtc 4800ggccatgggc attttggaga gggtctgttg caagagttcc agacggtccc agagctcggt 4860gatgtgctct agggcatctc gatccagcag acctcctcgt ttcgcgggtt ggggcgactg 4920cgggagtagg gcaccaggcg atgggcgtcc agcgaggcca gggtccggtc cttccagggt 4980cgcagggtcc gcgtcagcgt ggtctccgtc acggtgaagg ggtgcgcgcc gggctgggcg 5040cttgcgaggg tgcgcttcag gctcatccgg ctggtcgaga accgctcccg gtcggcgccc 5100tgcgcgtcgg ccaggtagca attgagcatg agttcgtagt tgagcgcctc ggccgcgtgg 5160cccttggcgc ggagcttacc tttggaagtg tgtccgcaga cgggacagag gagggacttg 5220agggcgtaga gcttgggggc gaggaagacg gactcggggg cgtaggcgtc cgcgccgcag 5280ctggcgcaga cggtctcgca ctccacgagc caggtgaggt cgggccggtt ggggtcaaaa 5340acgaggtttc ctccgtgctt tttgatgcgt ttcttacctc tggtctccat gagctcgtgt 5400ccccgctggg tgacaaagag gctgtccgtg tccccgtaga ccgactttat gggccggtcc 5460tcgagcgggg tgccgcggtc ctcgtcgtag aggaaccccg cccactccga gacgaaggcc 5520cgggtccagg ccagcacgaa ggaggccacg tgggaggggt agcggtcgtt gtccaccagc 5580gggtccacct tctccagggt atgcaagcac atgtccccct cgtccacatc caggaaggtg 5640attggcttgt aagtgtaggc cacgtgaccg ggggtcccgg ccgggggggt ataaaagggg 5700gcgggcccct gctcgtcctc actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg 5760ggtaggtatt ccctctcgaa ggctggcata acctcggcac tcaggttgtc agtttctaga 5820aacgaggagg atttgatatt gacggtgccg ttggagacgc ctttcatgag cccctcgtcc 5880atctggtcag aaaagacgat ctttttgttg tcgagcttgg tggcgaagga gccgtagagg 5940gcgttggaga ggagcttggc gatggagcgc atggtctggt tcttttcctt gtcggcgcgc 6000tccttggcgg cgatgttgag ctgcacgtac tcgcgcgcca cgcacttcca ttcggggaag 6060acggtggtga gctcgtcggg cacgattctg acccgccagc cgcggttgtg cagggtgatg 6120aggtccacgc tggtggccac ctcgccgcgc aggggctcgt tggtccagca gaggcgcccg 6180cccttgcgcg agcagaaggg gggcagcggg tccagcatga gctcgtcggg ggggtcggcg 6240tccacggtga agatgccggg cagaagctcg gggtcgaagt agctgatgca ggtgtccaga 6300tcgtccagcg ccgcttgcca gtcgcgcacg gccagcgcgc gctcgtaggg gctgaggggc 6360gtgccccagg gcatggggtg cgtgagcgcg gaggcgtaca tgccgcagat gtcgtagacg 6420tagaggggct cctcgaggac gccgatgtag gtggggtagc agcgcccccc gcggatgctg 6480gcgcgcacgt agtcgtacag ctcgtgcgag ggcgcgagga gccccgtgcc gaggttggag 6540cgttgcggct tttcggcgcg gtagacgatc tggcggaaga tggcgtggga gttggaggag 6600atggtgggcc tctggaagat gttgaagtgg gcgtggggca ggccgaccga gtccctgatg 6660aagtgggcgt aggagtcctg cagcttggcg acgagctcgg cggtgacgag gacgtccagg 6720gcgcagtagt cgagggtctc ttggatgatg tcgtacttga gctggccctt ctgcttccac 6780agctcgcggt tgagaaggaa ctcttcgcgg tccttccagt actcttcgag ggggaacccg 6840tcctgatcgg cacggtaaga gcccaccatg tagaactggt tgacggcctt gtaggcgcag 6900cagcccttct ccacggggag ggcgtaagct tgtgcggcct tgcgcaggga ggtgtgggtg 6960agggcgaagg tgtcgcgcac catgaccttg aggaactggt gcttgaagtc gaggtcgtcg 7020cagccgccct gctcccagag ctggaagtcc gtgcgcttct tgtaggcggg gttgggcaaa 7080gcgaaagtaa catcgttgaa gaggatcttg cccgcgcggg gcatgaagtt gcgagtgatg 7140cggaaaggct ggggcacctc ggcccggttg ttgatgacct gggcggcgag gacgatctcg 7200tcgaagccgt tgatgttgtg cccgacgatg tagagttcca cgaatcgcgg gcggccctta 7260acgtggggca gcttcttgag ctcgtcgtag gtgagctcgg cggggtcgct gagcccgtgc 7320tgctcgaggg cccagtcggc gacgtggggg ttggcgctga ggaaggaagt ccagagatcc 7380acggccaggg cggtctgcaa gcggtcccgg tactgacgga actgctggcc cacggccatt 7440ttttcggggg tgacgcagta gaaggtgcgg gggtcgccgt gccagcggtc ccacttgagc 7500tggagggcga ggtcgtgggc gagctcgacg agcggcgggt ccccggagag tttcatgacc 7560agcatgaagg ggacgagctg cttgccgaag gaccccatcc aggtgtaggt ttccacatcg 7620taggtgagga agagcctttc ggtgcgagga tgcgagccga tggggaagaa ctggatctcc 7680tgccaccagt tggaggaatg gctgttgatg tgatggaagt agaaatgccg acggcgcgcc 7740gagcactcgt gcttgtgttt atacaagcgt ccgcagtgct cgcaacgctg cacgggatgc 7800acgtgctgca cgagctgtac ctgggttcct ttgacgagga atttcagtgg gcagtggagc 7860gctggcggct gcatctggtg ctgtactacg tcctggccat cggcgtggcc atcgtctgcc 7920tcgatggtgg tcatgctgac gagcccgcgc gggaggcagg tccagacttc ggctcggacg 7980ggtcggagag cgaggacgag ggcgcgcagg ccggagctgt ccagggtcct gagacgctgc 8040ggagtcaggt cagtgggcag cggcggcgcg cggttgactt gcaggagctt ttccagggcg 8100cgcgggaggt ccagatggta cttgatctcc acggcgccgt tggtggcgac gtccacggct 8160tgcagggtcc cgtgcccctg gggcgccacc accgtgcccc gtttcttctt gggcgctgct 8220tccatgccgg tcagaagcgg cggcgaggac gcgcgccggg cggcaggggc ggctcgggac 8280ccggaggcag gggcggcagg ggcacgtcgg cgccgcgcgc gggcaggttc tggtactgcg 8340cccggagaag actggcgtga gcgacgacgc gacggttgac gtcctggatc tgacgcctct 8400gggtgaaggc cacgggaccc gtgagtttga acctgaaaga gagttcgaca gaatcaatct 8460cggtatcgtt gacggcggcc tgccgcagga tctcttgcac gtcgcccgag ttgtcctggt 8520aggcgatctc ggtcatgaac tgctcgatct cctcctcctg aaggtctccg cggccggcgc 8580gctcgacggt ggccgcgagg tcgttggaga tgcggcccat gagctgcgag aaggcgttca 8640tgccggcctc gttccagacg cggctgtaga ccacggctcc gtcggggtcg cgcgcgcgca 8700tgaccacctg ggcgaggttg agctcgacgt ggcgcgtgaa gaccgcgtag ttgcagaggc 8760gctggtagag gtagttgagc gtggtggcga tgtgctcggt gacgaagaag tacatgatcc 8820agcggcggag cggcatctcg ctgacgtcgc ccagggcttc caagcgctcc atggcctcgt 8880agaagtccac ggcgaagttg aaaaactggg agttgcgcgc cgagacggtc aactcctcct 8940ccagaagacg gatgagctca gcgatggtgg cgcgcacctc gcgctcgaag gccccggggg 9000gctcctcttc ttccatctct tcctcctcca ctaacatctc ttctacttcc tcctcaggag 9060gcggcggcgg gggaggggcc ctgcgtcgcc ggcggcgcac gggcagacgg tcgatgaagc 9120gctcgatggt ctccccgcgc cggcgacgca tggtctcggt gacggcgcgc ccgtcctcgc 9180ggggccgcag cgtgaagacg ccgccgcgca tctccaggtg gccgccgggg gggtctccgt 9240tgggcaggga gagggcgctg acgatgcatc ttatcaattg gcccgtaggg actccgcgca 9300aggacctgag cgtctcgaga tccacgggat ccgaaaaccg ctgaacgaag gcttcgagcc 9360agtcgcagtc gcaaggtagg ctgagcccgg tttcttgttc ttcggggatt tcgggaggcg 9420ggcgggcgat gctgctggtg atgaagttga agtaggcggt cctgagacgg cggatggtgg 9480cgaggagcac caggtccttg ggcccggctt gctggatgcg cagacggtcg gccatgcccc 9540aggcgtggtc ctgacacctg gcgaggtcct tgtagtagtc ctgcatgagc cgctccacgg 9600gcacctcctc ctcgcccgcg cggccgtgca tgcgcgtgag cccgaacccg cgctggggct 9660ggacgagcgc caggtcggcg acgacgcgct cggcgaggat ggcctgctgt atctgggtga 9720gggtggtctg gaagtcgtcg aagtcgacga agcggtggta ggctccggtg ttgatggtat 9780aggagcagtt ggccatgacg gaccagttga cggtctggtg gccgggtcgc acgagctcgt 9840ggtacttgag gcgcgagtag gcgcgcgtgt cgaagatgta gtcgttgcag gtgcgcacga 9900ggtactggta tccgacgagg aagtgcggcg gcggctggcg gtagagcggc catcgctcgg 9960tggcgggggc gccgggcgcg aggtcctcga gcatgaggcg gtggtagccg tagatgtacc 10020tggacatcca ggtgatgccg gcggcggtgg tggaggcgcg cgggaactcg cggacgcggt 10080tccagatgtt gcgcagcggc aggaagtagt tcatggtggc cgcggtctgg cccgtgaggc 10140gcgcgcagtc gtggatgctc tagacatacg ggcaaaaacg aaagcggtca gcggctcgac 10200tccgtggcct ggaggctaag cgaacgggtt gggctgcgcg tgtaccccgg ttcgaatctc 10260gaatcaggct ggagccgcag ctaacgtggt actggcactc ccgtctcgac ccaagcctgc 10320taacgaaacc tccaggatac ggaggcgggt cgttttttgg ccttggtcgc tggtcatgaa 10380aaactagtaa gcgcggaaag cgaccgcccg cgatggctcg ctgccgtagt ctggagaaag 10440aatcgccagg gttgcgttgc ggtgtgcccc ggttcgagcc tcagcgctcg gcgccggccg 10500gattccgcgg ctaacgtggg cgtggctgcc ccgtcgtttc caagacccct tagccagccg 10560acttctccag ttacggagcg agcccctctt tttcttgtgt ttttgccaga tgcatcccgt 10620actgcggcag atgcgccccc accctccacc tcaaccgccc ctaccgccgc agcagcagca 10680acagccggcg cttctgcccc cgccccagca gcagccagcc actaccgcgg cggccgccgt 10740gagcggagcc ggcgttcagt atgacctggc cttggaagag ggcgaggggc tggcgcggct 10800gggggcgtcg tcgccggagc ggcacccgcg cgtgcagatg aaaagggacg ctcgcgaggc 10860ctacgtgccc aagcagaacc tgttcagaga caggagcggc gaggagcccg aggagatgcg 10920cgcctcccgc ttccacgcgg ggcgggagct gcggcgcggc ctggaccgaa agcgggtgct 10980gagggacgag gatttcgagg cggacgagct gacggggatc agccccgcgc gcgcgcacgt 11040ggccgcggcc aacctggtca cggcgtacga gcagaccgtg aaggaggaga gcaacttcca 11100aaaatccttc aacaaccacg tgcgcacgct gatcgcgcgc gaggaggtga ccctgggcct 11160gatgcacctg tgggacctgc tggaggccat cgtgcagaac cccacgagca agccgctgac 11220ggcgcagctg tttctggtgg tgcagcacag tcgggacaac gagacgttca gggaggcgct 11280gctgaatatc accgagcccg agggccgctg gctcctggac ctggtgaaca ttctgcagag 11340catcgtggtg caggagcgcg ggctgccgct gtccgagaag ctggcggcta tcaacttctc 11400ggtgctgagc ctgggcaagt actacgctag gaagatctac aagaccccgt acgtgcccat 11460agacaaggag gtgaagatcg acgggtttta catgcgcatg accctgaaag tgctgaccct 11520gagcgacgat ctgggggtgt accgcaacga caggatgcac cgcgcggtga gcgccagccg 11580ccggcgcgag ctgagcgacc aggagctgat gcacagcctg cagcgggccc tgaccggggc 11640cgggaccgag ggggagagct actttgacat gggcgcggac ctgcgctggc agcccagccg 11700ccgggccttg gaagctgccg gcggttcccc ctacgtggag gaggtggacg atgaggagga 11760ggagggcgag tacctggaag actgatggcg cgaccgtatt tttgctagat gcagcaacag 11820ccaccgcctc ctgatcccgc gatgcgggcg gcgctgcaga gccagccgtc cggcattaac 11880tcctcggacg attggaccca ggccatgcaa cgcatcatgg cgctgacgac ccgcaatccc 11940gaagccttta gacagcagcc tcaggccaac cggctctcgg ccatcctgga ggccgtggtg 12000ccctcgcgct cgaaccccac gcacgagaag gtgctggcca tcgtgaacgc gctggtggag 12060aacaaggcca tccgcggcga cgaggccggg ctggtgtaca acgcgctgct ggagcgcgtg 12120gcccgctaca acagcaccaa cgtgcagacg aacctggacc gcatggtgac cgacgtgcgc 12180gaggcggtgt cgcagcgcga gcggttccac cgcgagtcga acctgggctc catggtggcg 12240ctgaacgcct tcctgagcac gcagcccgcc aacgtgcccc ggggccagga ggactacacc 12300aacttcatca gcgcgctgcg gctgatggtg gccgaggtgc cccagagcga ggtgtaccag 12360tcggggccgg actacttctt ccagaccagt cgccagggct tgcagaccgt gaacctgagc 12420caggctttca agaacttgca gggactgtgg ggcgtgcagg ccccggtcgg ggaccgcgcg 12480acggtgtcga gcctgctgac gccgaactcg cgcctgctgc tgctgctggt ggcgcccttc 12540acggacagcg gcagcgtgag ccgcgactcg tacctgggct

acctgcttaa cctgtaccgc 12600gaggccatcg ggcaggcgca cgtggacgag cagacctacc aggagatcac ccacgtgagc 12660cgcgcgctgg gccaggagga cccgggcaac ctggaggcca ccctgaactt cctgctgacc 12720aaccggtcgc agaagatccc gccccagtac gcgctgagca ccgaggagga gcgcatcctg 12780cgctacgtgc agcagagcgt ggggctgttc ctgatgcagg agggggccac gcccagcgcc 12840gcgctcgaca tgaccgcgcg caacatggag cccagcatgt acgctcgcaa ccgcccgttc 12900atcaataagc tgatggacta cttgcatcgg gcggccgcca tgaactcgga ctactttacc 12960aacgccatct tgaacccgca ctggctcccg ccgcccgggt tctacacggg cgagtacgac 13020atgcccgacc ccaacgacgg gttcctgtgg gacgacgtgg acagcagcgt gttctcgccg 13080cgccccgcca ccaccgtgtg gaagaaagag ggcggggacc ggcggccgtc ctcggcgctg 13140tccggtcgcg cgggtgctgc cgcggcggtg cctgaggccg ccagcccctt cccgagcctg 13200cccttttcgc tgaacagcgt gcgcagcagc gagctgggtc ggctgacgcg gccgcgcctg 13260ctgggcgagg aggagtacct gaacgactcc ttgttgaggc ccgagcgcga gaagaacttc 13320cccaataacg ggatagagag cctggtggac aagatgagcc gctggaagac gtacgcgcac 13380gagcacaggg acgagccccg agctagcagc agcgcaggca cccgtagacg ccagcgacac 13440gacaggcagc ggggtctggt gtgggacgat gaggattccg ccgacgacag cagcgtgttg 13500gacttgggtg ggagtggtgg tggtaacccg ttcgctcact tgcgcccccg tatcgggcgc 13560ctgatgtaag aatctgaaaa aataaaaaac ggtactcacc aaggccatgg cgaccagcgt 13620gcgttcttct ctgttgtttg tagtagtatg atgaggcgcg tgtacccgga gggtcctcct 13680ccctcgtacg agagcgtgat gcagcaggcg gtggcggcgg cgatgcagcc cccgctggag 13740gcgccttacg tgcccccgcg gtacctggcg cctacggagg ggcggaacag cattcgttac 13800tcggagctgg cacccttgta cgataccacc cggttgtacc tggtggacaa caagtcggcg 13860gacatcgcct cgctgaacta ccagaacgac cacagcaact tcctgaccac cgtggtgcag 13920aacaacgatt tcacccccac ggaggccagc acccagacca tcaactttga cgagcgctcg 13980cggtggggcg gccagctgaa aaccatcatg cacaccaaca tgcccaacgt gaacgagttc 14040atgtacagca acaagttcaa ggcgcgggtg atggtctcgc gcaagacccc caatggggtc 14100gcggtggatg agaattatga tggtagtcag gacgagctga cttacgagtg ggtggagttt 14160gagctgcccg agggcaactt ctcggtgacc atgaccatcg atctgatgaa caacgccatc 14220atcgacaact acttggcggt ggggcgtcag aacggggtgc tggagagcga catcggcgtg 14280aagttcgaca cgcgcaactt ccggctgggc tgggaccccg tgaccgagct ggtgatgccg 14340ggcgtgtaca ccaacgaggc cttccacccc gacatcgtcc tgctgcccgg ctgcggcgtg 14400gacttcaccg agagccgcct cagcaacctg ctgggcatcc gcaagcggca gcccttccag 14460gagggcttcc agatcctgta cgaggacctg gaggggggca acatccccgc gctcttggat 14520gtcgaagcct atgagaaaag caaggaggag gccgccgcag cggcgaccgc agccgtggcc 14580accgcctcta ccgaggtgcg gggcgataat tttgctagcg ccgcggcagt ggccgaggcg 14640gctgaaaccg aaagtaagat agtcatccag ccggtggaga aggacagcaa ggacaggagc 14700tacaacgtgc tcgcggacaa gaaaaacacc gcctaccgca gctggtacct ggcctacaac 14760tacggcgacc ccgagaaggg cgtgcgctcc tggacgctgc tcaccacctc ggacgtcacc 14820tgcggcgtgg agcaagtcta ctggtcgctg cccgacatga tgcaagaccc ggtcaccttc 14880cgctccacgc gtcaagttag caactacccg gtggtgggcg ccgagctcct gcccgtctac 14940tccaagagct tcttcaacga gcaggccgtc tactcgcagc agctgcgcgc cttcacctcg 15000ctcacgcacg tcttcaaccg cttccccgag aaccagatcc tcgtccgccc gcccgcgccc 15060accattacca ccgtcagtga aaacgttcct gctctcacag atcacgggac cctgccgctg 15120cgcagcagta tccggggagt ccagcgcgtg accgtcactg acgccagacg ccgcacctgc 15180ccctacgtct acaaggccct gggcgtagtc gcgccgcgcg tcctctcgag ccgcaccttc 15240taaaaaatgt ccattctcat ctcgcccagt aataacaccg gttggggcct gcgcgcgccc 15300agcaagatgt acggaggcgc tcgccaacgc tccacgcaac accccgtgcg cgtgcgcggg 15360cacttccgcg ctccctgggg cgccctcaag ggccgcgtgc gctcgcgcac caccgtcgac 15420gacgtgatcg accaggtggt ggccgacgcg cgcaactaca cgcccgccgc cgcgcccgcc 15480tccaccgtgg acgccgtcat cgacagcgtg gtggccgatg cgcgccggta cgcccgcgcc 15540aagagccggc ggcggcgcat cgcccggcgg caccggagca cccccgccat gcgcgcggcg 15600cgagccttgc tgcgcagggc caggcgcacg ggacgcaggg ccatgctcag ggcggccaga 15660cgcgcggcct ccggcagcag cagcgccggc aggacccgca gacgcgcggc cacggcggcg 15720gcggcggcca tcgccagcat gtcccgcccg cggcgcggca acgtgtactg ggtgcgcgac 15780gccgccaccg gtgtgcgcgt gcccgtgcgc acccgccccc ctcgcacttg aagatgctga 15840cttcgcgatg ttgatgtgtc ccagcggcga ggaggatgtc caagcgcaaa tacaaggaag 15900agatgctcca ggtcatcgcg cctgagatct acggccccgc ggtgaaggag gaaagaaagc 15960cccgcaaact gaagcgggtc aaaaaggaca aaaaggagga ggaagatgtg gacggactgg 16020tggagtttgt gcgcgagttc gccccccggc ggcgcgtgca gtggcgcggg cggaaagtga 16080aaccggtgct gcggcccggc accacggtgg tcttcacgcc cggcgagcgt tccggctccg 16140cctccaagcg ctcctacgac gaggtgtacg gggacgagga catcctcgag caggcggtcg 16200agcgtctggg cgagtttgct tacggcaagc gcagccgccc cgcgcccttg aaagaggagg 16260cggtgtccat cccgctggac cacggcaacc ccacgccgag cctgaagccg gtgaccctgc 16320agcaggtgct gccgagcgcg gcgccgcgcc ggggcttcaa gcgcgagggc ggcgaggatc 16380tgtacccgac catgcagctg atggtgccca agcgccagaa gctggaggac gtgctggagc 16440acatgaaggt ggaccccgag gtgcagcccg aggtcaaggt gcggcccatc aagcaggtgg 16500ccccgggcct gggcgtgcag accgtggaca tcaagatccc cacggagccc atggaaacgc 16560agaccgagcc cgtgaagccc agcaccagca ccatggaggt gcagacggat ccctggatgc 16620cggcgccggc ttccaccact cgccgaagac gcaagtacgg cgcggccagc ctgctgatgc 16680ccaactacgc gctgcatcct tccatcatcc ccacgccggg ctaccgcggc acgcgcttct 16740accgcggcta caccagcagc cgccgcaaga ccaccacccg ccgccgccgt cgtcgcaccc 16800gccgcagcag caccgcgact tccgccgccg ccctggtgcg gagagtgtac cgcagcgggc 16860gcgagcctct gaccctgccg cgcgcgcgct accacccgag catcgccatt taactctgcc 16920gtcgcctcct acttgcagat atggccctca catgccgcct ccgcgtcccc attacgggct 16980accgaggaag aaagccgcgc cgtagaaggc tgacggggaa cgggctgcgt cgccatcacc 17040accggcggcg gcgcgccatc agcaagcggt tggggggagg cttcctgccc gcgctgatcc 17100ccatcatcgc cgcggcgatc ggggcgatcc ccggcatagc ttccgtggcg gtgcaggcct 17160ctcagcgcca ctgagacaca gcttggaaaa tttgtaataa aaaaatggac tgacgctcct 17220ggtcctgtga tgtgtgtttt tagatggaag acatcaattt ttcgtccctg gcaccgcgac 17280acggcacgcg gccgtttatg ggcacctgga gcgacatcgg caacagccaa ctgaacgggg 17340gcgccttcaa ttggagcagt ctctggagcg ggcttaagaa tttcgggtcc acgctcaaaa 17400cctatggcaa caaggcgtgg aacagcagca cagggcaggc gctgagggaa aagctgaaag 17460agcagaactt ccagcagaag gtggtcgatg gcctggcctc gggcatcaac ggggtggtgg 17520acctggccaa ccaggccgtg cagaaacaga tcaacagccg cctggacgcg gtcccgcccg 17580cggggtccgt ggagatgccc caggtggagg aggagctgcc tcccctggac aagcgcggcg 17640acaagcgacc gcgtcccgac gcggaggaga cgctgctgac gcacacggac gagccgcccc 17700cgtacgagga ggcggtgaaa ctgggtctgc ccaccacgcg gcccgtggcg cctctggcca 17760ccggggtgct gaaacccagc agcagcagcc agcccgcgac cctggacttg cctccgcctg 17820cttcccgccc ctccacagtg gctaagcccc tgccgccggt ggccgtcgcg tcgcgcgccc 17880cccgaggccg cccccaggcg aactggcaga gcactctgaa cagcatcgtg ggtctgggag 17940tgcagagtgt gaagcgccgc cgctgctatt aaaagacact gtagcgctta acttgcttgt 18000ctgtgtgtat atgtatgtcc gccgaccaga aggaggaaga ggcgcgtcgc cgagttgcaa 18060gatggccacc ccatcgatgc tgccccagtg ggcgtacatg cacatcgccg gacaggacgc 18120ttcggagtac ctgagtccgg gtctggtgca gttcgcccgc gccacagaca cctacttcag 18180tctggggaac aagtttagga accccacggt ggcgcccacg cacgatgtga ccaccgaccg 18240cagccagcgg ctgacgctgc gcttcgtgcc cgtggaccgc gaggacaaca cctactcgta 18300caaagtgcgc tacacgctgg ccgtgggcga caaccgcgtg ctggacatgg ccagcaccta 18360ctttgacatc cgcggcgtgc tggatcgggg gcccagcttc aaaccctact ccggcaccgc 18420ctacaacagc ctggctccca agggagcgcc caacacttgc cagtggacat ataaagctgg 18480tgatactgat acagaaaaaa cctatacata tggaaatgca cctgtgcaag gcattagcat 18540tacaaaggat ggtattcaac ttggaactga cagcgatggt caggcaatct atgcagacga 18600aacttatcaa ccagagcctc aagtgggtga tgctgaatgg catgacatca ctggtactga 18660tgaaaaatat ggaggcagag ctcttaagcc tgacaccaaa atgaagcctt gctatggttc 18720ttttgccaag cctaccaata aagaaggagg ccaggcaaat gtgaaaaccg aaacaggcgg 18780taccaaagaa tatgacattg acatggcatt cttcgataat cgaagtgcag ctgccgccgg 18840cctagcccca gaaattgttt tgtatactga gaatgtggat ctggaaactc cagataccca 18900tattgtatac aaggcaggta cagatgacag tagctcttct atcaatttgg gtcagcagtc 18960catgcccaac agacccaact acattggctt cagagacaac tttatcggtc tgatgtacta 19020caacagcact ggcaatatgg gtgtactggc tggacaggcc tcccagctga atgctgtggt 19080ggacttgcag gacagaaaca ccgaactgtc ctaccagctc ttgcttgact ctctgggtga 19140cagaaccagg tatttcagta tgtggaatca ggcggtggac agttatgacc ccgatgtgcg 19200cattattgaa aatcacggtg tggaggatga acttcctaac tattgcttcc ccctggatgc 19260tgtgggtaga actgatactt accagggaat taaggccaat ggtgataatc aaaccacctg 19320gaccaaagat gatactgtta atgatgctaa tgaattgggc aagggcaatc ctttcgccat 19380ggagatcaac atccaggcca acctgtggcg gaacttcctc tacgcgaacg tggcgctgta 19440cctgcccgac tcctacaagt acacgccggc caacatcacg ctgcccacca acaccaacac 19500ctacgattac atgaacggcc gcgtggtggc gccctcgctg gtggacgcct acatcaacat 19560cggggcgcgc tggtcgctgg accccatgga caacgtcaac cccttcaacc accaccgcaa 19620cgcgggcctg cgataccgct ccatgctcct gggcaacggg cgctacgtgc ccttccacat 19680ccaggtgccc caaaagtttt tcgccatcaa gagcctcctg ctcctgcccg ggtcctacac 19740ctacgagtgg aacttccgca aggacgtcaa catgatcctg cagagctccc tcggcaacga 19800cctgcgcacg gacggggcct ccatcgcctt caccagcatc aacctctacg ccaccttctt 19860ccccatggcg cacaacaccg cctccacgct cgaggccatg ctgcgcaacg acaccaacga 19920ccagtccttc aacgactacc tctcggcggc caacatgctc taccccatcc cggccaacgc 19980caccaacgtg cccatctcca tcccctcgcg caactgggcc gccttccgcg gctggtcctt 20040cacgcgcctc aagacccgcg agacgccctc gctcggctcc gggttcgacc cctacttcgt 20100ctactcgggc tccatcccct acctcgacgg caccttctac ctcaaccaca ccttcaagaa 20160ggtctccatc accttcgact cctccgtcag ctggcccggc aacgaccgcc tcctgacgcc 20220caacgagttc gaaatcaagc gcaccgtcga cggagagggg tacaacgtgg cccagtgcaa 20280catgaccaag gactggttcc tggtccagat gctggcccac tacaacatcg gctaccaggg 20340cttctacgtg cccgagggct acaaggaccg catgtactcc ttcttccgca acttccagcc 20400catgagccgc caggtcgtgg acgaggtcaa ctacaaggac taccaggccg tcaccctggc 20460ctaccagcac aacaactcgg gcttcgtcgg ctacctcgcg cccaccatgc gccagggcca 20520gccctacccc gccaactacc cctacccgct catcggcaag agcgccgtcg ccagcgtcac 20580ccagaaaaag ttcctctgcg accgggtcat gtggcgcatc cccttctcca gcaacttcat 20640gtccatgggc gcgctcaccg acctcggcca gaacatgctc tacgccaact ccgcccacgc 20700gctagacatg aatttcgaag tcgaccccat ggatgagtcc acccttctct atgttgtctt 20760cgaagtcttc gacgtcgtcc gagtgcacca gccccaccgc ggcgtcatcg aggccgtcta 20820cctgcgcacg cccttctcgg ccggcaacgc caccacctaa gcctcttgct tcttgcaaga 20880tgacggcctg cgcgggctcc ggcgagcagg agctcagggc catcctccgc gacctgggct 20940gcgggccctg cttcctgggc accttcgaca agcgcttccc gggattcatg gccccgcaca 21000agctggcctg cgccatcgtc aacacggccg gccgcgagac cgggggcgag cactggctgg 21060ccttcgcctg gaacccgcgc tcccacacct gctacctctt cgaccccttc gggttctcgg 21120acgagcgcct caagcagatc taccagttcg agtacgaggg cctgctgcgt cgcagcgccc 21180tggccaccga ggaccgctgc gtcaccctgg aaaagtccac ccagaccgtg cagggtccgc 21240gctcggccgc ctgcgggctc ttctgctgca tgttcctgca cgccttcgtg cactggcccg 21300accgccccat ggacaagaac cccaccatga acttgctgac gggggtgccc aacggcatgc 21360tccagtcgcc ccaggtggaa cccaccctgc gccgcaacca ggaggcgctc taccgcttcc 21420tcaacgccca ctccgcctac tttcgctccc accgcgcgcg catcgagaag gccaccgcct 21480tcgaccgcat gaatcaagac atgtaatccg gtgtgtgtat gtgaatgctt tattcatcat 21540aataaacagc acatgtttat gccaccttct ctgaggctct gactttattt agaaatcgaa 21600ggggttctgc cggctctcgg catggcccgc gggcagggat acgttgcgga actggtactt 21660gggcagccac ttgaactcgg ggatcagcag cttcggcacg gggaggtcgg ggaacgagtc 21720gctccacagc ttgcgcgtga gttgcagggc gcccagcagg tcgggcgcgg agatcttgaa 21780atcgcagttg ggacccgcgt tctgcgcgcg agagttacgg tacacggggt tgcagcactg 21840gaacaccatc agggccgggt gcttcacgct cgccagcacc gtcgcgtcgg tgatgccctc 21900cacgtccaga tcctcggcgt tggccatccc gaagggggtc atcttgcagg tctgccgccc 21960catgctgggc acgcagccgg gcttgtggtt gcaatcgcag tgcaggggga tcagcatcat 22020ctgggcctgc tcggagctca tgcccgggta catggccttc atgaaagcct ccagctggcg 22080gaaggcctgc tgcgccttgc cgccctcggt gaagaagacc ccgcaggact tgctagagaa 22140ctggttggtg gcgcagccag cgtcgtgcac gcagcagcgc gcgtcgttgt tggccagctg 22200caccacgctg cgcccccagc ggttctgggt gatcttggcc cggtcggggt tctccttcag 22260cgcgcgctgc ccgttctcgc tcgccacatc catctcgatc gtgtgctcct tctggatcat 22320cacggtcccg tgcaggcacc gcagcttgcc ctcggcctcg gtgcacccgt gcagccacag 22380cgcgcagccg gtgctctccc agttcttgtg ggcgatctgg gagtgcgagt gcacgaagcc 22440ctgcaggaag cggcccatca tcgtggtcag ggtcttgttg ctggtgaagg tcagcggaat 22500gccgcggtgc tcctcgttca catacaggtg gcagatacgg cggtacacct cgccctgctc 22560gggcatcagc tggaaggcgg acttcaggtc gctctccacg cggtaccggt ccatcagcag 22620cgtcatcact tccatgccct tctcccaggc cgaaacgatc ggcaggctca gggggttctt 22680caccgttgtc atcttagtcg ccgccgccga agtcaggggg tcgttctcgt ccagggtctc 22740aaacactcgc ttgccgtcct tctcggtgat gcgcacgggg ggaaagctga agcccacggc 22800cgccagctcc tcctcggcct gcctttcgtc ctcgctgtcc tggctgatgt cttgcaaagg 22860cacatgcttg gtcttgcggg gtttcttttt gggcggcaga ggcggcggcg gagacgtgct 22920gggcgagcgc gagttctcgc tcaccacgac tatttcttct ccttggccgt cgtccgagac 22980cacgcggcgg taggcatgcc tcttctgggg cagaggcgga ggcgacgggc tctcgcggtt 23040cggcgggcgg ctggcagagc cccttccgcg ttcgggggtg cgctcctggc ggcgctgctc 23100tgactgactt cctccgcggc cggccattgt gttctcctag ggagcaagca tggagactca 23160gccatcgtcg ccaacatcgc catctgcccc cgccgccgcc gacgagaacc agcagcagca 23220gaatgaaagc ttaaccgccc cgccgcccag ccccacctcc gacgccgcag ccccagacat 23280gcaagagatg gaggaatcca tcgagattga cctgggctac gtgacgcccg cggagcacga 23340ggaggagctg gcagcgcgct tttcagcccc ggaagagaac caccaagagc agccagagca 23400ggaagcagag agcgagcaga accaggctgg gctcgagcat ggcgactacc tgagcggggc 23460agaggacgtg ctcatcaagc atctggcccg ccaatgcatc atcgtcaagg acgcgctgct 23520cgaccgcgcc gaggtgcccc tcagcgtggc ggagctcagc cgcgcctacg agcgcaacct 23580cttctcgccg cgcgtgcccc ccaagcgcca gcccaacggc acctgcgagc ccaacccgcg 23640cctcaacttc tacccggtct tcgcggtgcc cgaggccctg gccacctacc acctcttttt 23700caagaaccaa aggatccccg tctcctgccg cgccaaccgc acccgcgccg acgccctgct 23760caacctgggc cccggcgccc gcctacctga tatcgcctcc ttggaagagg ttcccaagat 23820cttcgagggt ctgggcagcg acgagactcg ggccgcgaac gctctgcaag gaagcggaga 23880ggagcatgag caccacagcg ccctggtgga gttggaaggc gacaacgcgc gcctggcggt 23940cctcaagcgc acggtcgagc tgacccactt cgcctacccg gcgctcaacc tgccccccaa 24000ggtcatgagc gccgtcatgg accaggtgct catcaagcgc gcctcgcccc tctcggagga 24060ggagatgcag gaccccgaga gctcggacga gggcaagccc gtggtcagcg acgagcagct 24120ggcgcgctgg ctgggagcga gtagcacccc ccagagcctg gaagagcggc gcaagctcat 24180gatggccgtg gtcctggtga ccgtggagct ggagtgtctg cgccgcttct tcgccgacgc 24240ggagaccctg cgcaaggtcg aggagaacct gcactacctc ttcagacacg ggttcgtgcg 24300ccaggcctgc aagatctcca acgtggagct gaccaacctg gtctcctaca tgggcatcct 24360gcacgagaac cgcctggggc agaacgtgct gcacaccacc ctgcgcgggg aggcccgccg 24420cgactacatc cgcgactgcg tctacctgta cctctgccac acctggcaga cgggcatggg 24480cgtgtggcag cagtgcctgg aggagcagaa cctgaaagag ctctgcaagc tcctgcagaa 24540gaacctcaag gccctgtgga ccgggttcga cgagcgcacc accgccgcgg acctggccga 24600cctcatcttc cccgagcgcc tgcggctgac gctgcgcaac gggctgcccg actttatgag 24660ccaaagcatg ttgcaaaact ttcgctcttt catcctcgaa cgctccggga tcctgcccgc 24720cacctgctcc gcgctgccct cggacttcgt gccgctgacc ttccgcgagt gccccccgcc 24780gctctggagc cactgctacc tgctgcgcct ggccaactac ctggcctacc actcggacgt 24840gatcgaggac gtcagcggcg agggcctgct cgagtgccac tgccgctgca acctctgcac 24900gccgcaccgc tccctggcct gcaaccccca gctgctgagc gagacccaga tcatcggcac 24960cttcgagttg caaggccccg gcgagggcaa ggggggtctg aaactcaccc cggggctgtg 25020gacctcggcc tacttgcgca agttcgtgcc cgaggactac catcccttcg agatcaggtt 25080ctacgaggac caatcccagc cgcccaaggc cgagctgtcg gcctgcgtca tcacccaggg 25140ggccatcctg gcccaattgc aagccatcca gaaatcccgc caagaatttc tgctgaaaaa 25200gggccacggg gtctacttgg acccccagac cggagaggag ctcaacccca gcttccccca 25260ggatgccccg aggaagcagc aagaagctga aagtggagct gccgccgccg ccggaggatt 25320tggaggaaga ctgggagagc agtcaggcag aggaggagga gatggaagac tgggacagca 25380ctcaggcaga ggaggacagc ctgcaagaca gtctggagga ggaagacgag gtggaggagg 25440cagaggaaga agcagccgcc gccagaccgt cgtcctcggc ggaggaggag aaagcaagca 25500gcacggatac catctccgct ccgggtcggg gtcgcggcgg ccgggcccac agtagatggg 25560acgagaccgg gcgcttcccg aaccccacca cccagaccgg taagaaggag cggcagggat 25620acaagtcctg gcgggggcac aaaaacgcca tcgtctcctg cttgcaagcc tgcgggggca 25680acatctcctt cacccggcgc tacctgctct tccaccgcgg ggtgaacttc ccccgcaaca 25740tcttgcatta ctaccgtcac ctccacagcc cctactactg tttccaagaa gaggcagaaa 25800cccagcagca gcagcagcag cagaaaacca gcggcagcag ctagaaaatc cacagcggcg 25860gcaggtggac tgaggatcgc ggcgaacgag ccggcgcaga cccgggagct gaggaaccgg 25920atctttccca ccctctatgc catcttccag cagagtcggg ggcaagagca ggaactgaaa 25980gtcaagaacc gttctctgcg ctcgctcacc cgcagttgtc tgtatcacaa gagcgaagac 26040caacttcagc gcactctcga ggacgccgag gctctcttca acaagtactg cgcgctcact 26100cttaaagagt agcccgcgcc cgcccacaca cggaaaaagg cgggaattac gtcaccacct 26160gcgcccttcg cccgaccatc atcatgagca aagagattcc cacgccttac atgtggagct 26220accagcccca gatgggcctg gccgccggcg ccgcccagga ctactccacc cgcatgaact 26280ggctcagtgc cgggcccgcg atgatctcac gggtgaatga catccgcgcc caccgaaacc 26340agatactcct agaacagtca gcgatcaccg ccacgccccg ccatcacctt aatccgcgta 26400attggcccgc cgccctggtg taccaggaaa ttccccagcc cacgaccgta ctacttccgc 26460gagacgccca ggccgaagtc cagctgacta actcaggtgt ccagctggcc ggcggcgccg 26520ccctgtgtcg tcaccgcccc gctcagggta taaagcggct ggtgatccga ggcagaggca 26580cacagctcaa cgacgaggtg gtgagctctt cgctgggtct gcgacctgac ggagtcttcc 26640aactcgccgg atcggggaga tcttccttca cgcctcgtca ggccgtcctg actttggaga 26700gttcgtcctc gcagccccgc tcgggtggca tcggcactct ccagttcgtg gaggagttca 26760ctccctcggt ctacttcaac cccttctccg gctcccccgg ccactacccg gacgagttca 26820tcccgaactt cgacgccatc agcgagtcgg tggacggcta cgattgaatg tcccatggtg 26880gcgcggctga cctagctcgg cttcgacacc tggaccactg ccgccgcttc cgctgcttcg 26940ctcgggatct cgccgagttt gcctactttg agctgcccga ggagcaccct cagggcccgg 27000cccacggagt gcggatcgtc gtcgaagggg gtctcgactc ccacctgctt cggatcttca 27060gccagcgtcc gatcctggcc gagcgcgagc aaggacagac ccttctgacc ctgtactgca 27120tctgcaacca ccccggcctg catgaaagtc tttgttgtct gctgtgtact gagtataata 27180aaagctgaga tcagcgacta ctccggactt ccgtgtgttc ctgctatcaa ccagtccctg 27240ttcttcaccg ggaacgagac cgagctccag ctccagtgta agccccacaa gaagtacctc 27300acctggctgt tccagggctc tccgatcgcc gttgtcaacc actgcgacaa cgacggagtc 27360ctgctgagcg gccctgccaa ccttactttt tccacccgca gaagcaagct ccagctcttc 27420caacccttcc tccccgggac ctatcagtgc gtctcgggac cctgccatca caccttccac 27480ctgatcccga ataccacagc gtcgctcccc gctactaaca accaaactac ccaccaacgc 27540caccgtcgcg acctttcctc tgggtctaat accactaccg gaggtgagct ccgaggtcga 27600ccaacctctg ggatttacta cggcccctgg gaggtggtag

ggttaatagc gctaggccta 27660gttgcgggtg ggcttttggc tctctgctac ctatacctcc cttgctgttc gtacttagtg 27720gtgctgtgtt gctggtttaa gaaatgggga agatcaccct agtgagctgc ggtgtgctgg 27780tggcggtggt gctttcgatt gtgggactgg gcggcgcggc tgtagtgaag gagaaggccg 27840atccctgctt gcatttcaat cccgacaaat gccagctgag ttttcagccc gatggcaatc 27900ggtgcgcggt gctgatcaag tgcggatggg aatgcgagaa cgtgagaatc gagtacaata 27960acaagactcg gaacaatact ctcgcgtccg tgtggcagcc cggggacccc gagtggtaca 28020ccgtctctgt ccccggtgct gacggctccc cgcgcaccgt gaataatact ttcatttttg 28080cgcacatgtg cgacacggtc atgtggatga gcaagcagta cgatatgtgg ccccccacga 28140aggagaacat cgtggtcttc tccatcgctt acagcgtgtg cacggcgcta atcaccgcta 28200tcgtgtgcct gagcattcac atgctcatcg ctattcgccc cagaaataat gccgaaaaag 28260aaaaacagcc ataacacgtt ttttcacaca cctttttcag accatggcct ctgttaaatt 28320tttgctttta tttgccagtc tcattgccgt cattcatgga atgagtaatg agaaaattac 28380tatttacact ggcactaatc acacattgaa aggtccagaa aaagccacag aagtttcatg 28440gtattgttat tttaatgaat cagatgtatc tactgaactc tgtggaaaca ataacaaaaa 28500aaatgagagc attactctca tcaagtttca atgtggatct gacttaaccc taattaacat 28560cactagagac tatgtaggta tgtattatgg aactacagca ggcatttcgg acatggaatt 28620ttatcaagtt tctgtgtctg aacccaccac gcctagaatg accacaacca caaaaactac 28680acctgttacc actatacagc tcactaccaa tggctttctt gccatgcttc aagtggctga 28740aaatagcacc agcattcaac ccaccccacc cagtgaggaa attcccagat ccatgattgg 28800cattattgtt gctgtagtgg tgtgcatgtt gatcatcgcc ttgtgcatgg tgtactatgc 28860cttctgctac agaaagcaca gactgaacga caagctggaa cacttactaa gtgttgaatt 28920ttaatttttt agaaccatga agatcctagg ccttttagtt ttttctatca ttacctctgc 28980tctatgcaat tctgacaatg aggacgttac tgtcgttgtc ggatcaaatt atacactaaa 29040aggtccagca aaaggtatgc tttcgtggta ttgttggttc ggaactgacg agcaacagac 29100agaactttgc aatgctcaaa aaggcaaaac ctcaaattct aaaatctcta attatcaatg 29160caatggcact gacttagtat tgctcaatgt cacgaaagca tatgctggca gttacacctg 29220ccctggagat gatgccgaca atatgatttt ttacaaagtg gaagtggttg atcccactac 29280tccaccgccc accaccacaa ctactcatac cacacacaca gaacaaacac cagaggcagc 29340agaagcagag ttggccttcc aggttcacgg agattccttt gctgtcaata cccctacacc 29400cgatcagcgg tgtccggggc tgctcgtcag cggcattgtc ggtgtgcttt cgggattagc 29460agtcataatc atctgcatgt tcatttttgc ttgctgctat agaaggcttt accgacaaaa 29520atcagaccca ctgctgaacc tctatgttta attttttcca gagccatgaa ggcagttagc 29580gctctagttt tttgttcttt gattggcatt gtttttagtg ctgggttttt gaaaaatctt 29640accatttatg aaggtgagaa tgccactcta gtgggcatca gtggtcaaaa tgtcagctgg 29700ctaaaatacc atctagatgg gtggaaagac atttgcgatt ggaatgtcac tgtgtataca 29760tgtaatggag ttaacctcac cattactaat gccacccaag atcagaatgg taggtttaag 29820ggccagagtt tcactagaaa taatgggtat gaatcccata acatgtttat ctatgacgtc 29880actgtcatca gaaatgagac tgccaccacc acacagatgc ccactacaca cagttctacc 29940actactacca tgcaaaccac acagacaacc actacatcaa ctcagcatat gaccaccact 30000acagcagcaa agccaagtag tgcagcgcct cagccccagg ctttggcttt gaaagctgca 30060caacctagta caactactag gaccaatgag cagactactg aatttttgtc cactgtcgag 30120agccacacca cagctacctc cagtgccttc tctagcaccg ccaatctctc ctcgctttcc 30180tctacaccaa tcagtcccgc tactactccc accccagctc ttctccccac tcccctgaag 30240caaactgagg acagcggcat gcaatggcag atcaccctgc tcattgtgat cgggttggtc 30300atcctggccg tgttgctcta ctacatcttc tgccgccgca ttcccaacgc gcaccgcaaa 30360ccggcctaca agcccatcgt tatcgggcag ccggagccgc ttcaggtgga agggggtcta 30420aggaatcttc tcttctcttt tacagtatgg tgattgaact atgattccta gacaattctt 30480gatcactatt cttatctgcc tcctccaagt ctgtgccacc ctcgctctgg tggccaacgc 30540cagtccagac tgtattgggc ccttcgcctc ctacgtgctc tttgccttca tcacctgcat 30600ctgctgctgt agcatagtct gcctgcttat caccttcttc cagttcattg actggatctt 30660tgtgcgcatc gcctacctgc gccaccaccc ccagtaccgc gaccagcgag tggcgcggct 30720gctcaggctc ctctgataag catgcgggct ctgctacttc tcgcgcttct gctgttagtg 30780ctcccccgcc ccgtcgaccc ccggtccccc actcagtccc ccgaagaggt ccgcaaatgc 30840aaattccaag aaccctggaa attcctcaaa tgctaccgcc aaaaatcaga catgcttccc 30900agctggatca tgatcattgg gatcgtgaac attctggcct gcaccctcat ctcctttgtg 30960atttacccct gctttgactt tggttggaac tcgccagagg cgctctatct cccgcctgaa 31020cctgacacac caccacagca acctcaggca cacgcactac caccaccaca gcctaggcca 31080caatacatgc ccatattaga ctatgaggcc gagccacagc gacccatgct ccccgctatt 31140agttacttca atctaaccgg cggagatgac tgacccactg gccaacaaca acgtcaacga 31200ccttctcctg gacatggacg gccgcgcctc ggagcagcga ctcgcccaac ttcgcattcg 31260ccagcagcag gagagagccg tcaaggagct gcaggacggc atagccatcc accagtgcaa 31320gaaaggcatc ttctgcctgg tgaaacaggc caagatctcc tacgaggtca ccccgaccga 31380ccatcgcctc tcctacgagc tcctgcagca gcgccagaag ttcacctgcc tggtcggagt 31440caaccccatc gtcatcaccc agcagtcggg cgataccaag gggtgcatcc actgctcctg 31500cgactccccc gactgcgtcc acactctgat caagaccctc tgcggcctcc gcgacctcct 31560ccccatgaac taatcacccc cttatccagt gaaataaata tcatattgat gatgatttaa 31620ataaaaaata atcatttgat ttgaaataaa gatacaatca tattgatgat ttgagtttta 31680aaaaataaag aatcacttac ttgaaatctg ataccaggtc tctgtccatg ttttctgcca 31740acaccacctc actcccctct tcccagctct ggtactgcag accccggcgg gctgcaaact 31800tcctccacac gctgaagggg atgtcaaatt cctcctgtcc ctcaatcttc attttatctt 31860ctatcagatg tccaaaaagc gcgtccgggt ggatgatgac ttcgaccccg tctaccccta 31920cgatgcagac aacgcaccga ccgtgccctt catcaacccc cccttcgtct cttcagatgg 31980attccaagag aagcccctgg gggtgctgtc cctgcgactg gctgaccccg tcaccaccaa 32040gaacggggaa atcaccctca agctgggaga gggggtggac ctcgactcct cgggaaaact 32100catctccaac acggccacca aggccgccgc ccctctcagt ttttccaaca acaccatttc 32160ccttaacatg gatacccctc tttataccaa agatggaaaa ttatccttac aagtttctcc 32220accgttaaac atattaaaat caaccattct gaacacatta gctgtagctt atggatcagg 32280tttaggactg agtggtggca ctgctcttgc agtacagttg gcctctccac tcacttttga 32340tgaaaaagga aatattaaaa ttaacctagc cagtggtcca ttaacagttg atgcaagtcg 32400acttagtatc aactgcaaaa gaggggtcac tgtcactacc tcaggagatg caattgaaag 32460caacataagc tggcctaaag gtataagatt tgaaggtaat ggcatagctg caaacattgg 32520cagaggattg gaatttggaa ccactagtac agagactgat gtcacagatg catacccaat 32580tcaagttaaa ttgggtactg gccttacctt tgacagtaca ggcgccattg ttgcttggaa 32640caaagaggat gataaactta cattatggac cacagccgac ccctcgccaa attgcaaaat 32700atactctgaa aaagatgcca aactcacact ttgcttgaca aagtgtggaa gtcaaattct 32760gggtactgtg actgtattgg cagtgaataa tggaagtctc aacccaatca caaacacagt 32820aagcactgca ctcgtctccc tcaagtttga tgcaagtgga gttttgctaa gcagctccac 32880attagacaaa gaatattgga acttcagaaa gggagatgtt acacctgctg agccctatac 32940taatgctata ggttttatgc ctaacataaa ggcctatcct aaaaacacat ctgcagcttc 33000aaaaagccat attgtcagtc aagtttatct caatggggat gaggccaaac cactgatgct 33060gattattact tttaatgaaa ctgaggatgc aacttgcacc tacagtatca cttttcaatg 33120gaaatgggat agtactaagt acacaggtga aacacttgct accagctcct tcaccttctc 33180ctacatcgcc caagaatgaa cactgtatcc caccctgcat gccaaccctt cccaccccac 33240tctgtctatg gaaaaaactc tgaagcacaa aataaaataa agttcaagtg ttttattgat 33300tcaacagttt tacaggattc gagcagttat ttttcctcca ccctcccagg acatggaata 33360caccaccctc tccccccgca cagccttgaa catctgaatg ccattggtga tggacatgct 33420tttggtctcc acgttccaca cagtttcaga gcgagccagt ctcgggtcgg tcagggagat 33480gaaaccctcc gggcactccc gcatctgcac ctcacagctc aacagctgag gattgtcctc 33540ggtggtcggg atcacggtta tctggaagaa gcagaagagc ggcggtggga atcatagtcc 33600gcgaacggga tcggccggtg gtgtcgcatc aggccccgca gcagtcgctg ccgccgccgc 33660tccgtcaagc tgctgctcag ggggtccggg tccagggact ccctcagcat gatgcccacg 33720gccctcagca tcagtcgtct ggtgcggcgg gcgcagcagc gcatgcggat ctcgctcagg 33780tcgctgcagt acgtgcaaca caggaccacc aggttgttca acagtccata gttcaacacg 33840ctccagccga aactcatcgc gggaaggatg ctacccacgt ggccgtcgta ccagatcctc 33900aggtaaatca agtggcgctc cctccagaac acgctgccca cgtacatgat ctccttgggc 33960atgtggcggt tcaccacctc ccggtaccac atcaccctct ggttgaacat gcagccccgg 34020atgatcctgc ggaaccacag ggccagcacc gccccgcccg ccatgcagcg aagagacccc 34080gggtcccggc aatggcaatg gaggacccac cgctcgtacc cgtggatcat ctgggagctg 34140aacaagtcta tgttggcaca gcacaggcat atgctcatgc atctcttcag cactctcagc 34200tcctcggggg tcaaaaccat atcccagggc acggggaact cttgcaggac agcgaacccc 34260gcagaacagg gcaatcctcg cacataactt acattgtgca tggacagggt atcgcaatca 34320ggcagcaccg ggtgatcctc caccagagaa gcgcgggtct cggtctcctc acagcgtggt 34380aagggggccg gccgatacgg gtgatggcgg gacgcggctg atcgtgttcg cgaccgtgtc 34440atgatgcagt tgctttcgga cattttcgta cttgctgtag cagaacctgg tccgggcgct 34500gcacaccgat cgccggcggc ggtcccggcg cttggaacgc tcggtgttga aattgtaaaa 34560cagccactct ctcagaccgt gcagcagatc tagggcctca ggagtgatga agatcccatc 34620atgcctgata gctctgatca catcgaccac cgtggaatgg gccagaccca gccagatgat 34680gcaattttgt tgggtttcgg tgacggcggg ggagggaaga acaggaagaa ccatgattaa 34740cttttaatcc aaacggtctc ggagcacttc aaaatgaagg tcgcggagat ggcacctctc 34800gcccccgctg tgttggtgga aaataacagc caggtcaaag gtgatacggt tctcgagatg 34860ttccacggtg gcttccagca aagcctccac gcgcacatcc agaaacaaga caatagcgaa 34920agcgggaggg ttctctaatt cctcaatcat catgttacac tcctgcacca tccccagata 34980attttcattt ttccagcctt gaatgattcg aactagttcc tgaggtaaat ccaagccagc 35040catgataaag agctcgcgca gagcgccctc caccggcatt cttaagcaca ccctcataat 35100tccaagatat tctgctcctg gttcacctgc agcagattga caagcggaat atcaaaatct 35160ctgccgcgat ccctaagctc ctccctcagc aataactgta agtactcttt catatcctct 35220ccgaaatttt tagccatagg accaccagga ataagattag ggcaagccac agtacagata 35280aaccgaagtc ctccccagtg agcattgcca aatgcaagac tgctataagc atgctggcta 35340gacccggtga tatcttccag ataactggac agaaaatcac ccaggcaatt tttaagaaaa 35400tcaacaaaag aaaaatcctc caggtgcacg tttagagcct cgggaacaac gatgaagtaa 35460atgcaagcgg tgcgttccag catggttagt tagctgatct gtaaaaaaca aaaaataaaa 35520cattaaacca tgctagcctg gcgaacaggt gggtaaatcg ttctctccag caccaggcag 35580gccacggggt ctccggcgcg accctcgtaa aaattgtcgc tatgattgaa aaccatcaca 35640gagagacgtt cccggtggcc ggcgtgaatg attcgacaag atgaatacac ccccggaaca 35700ttggcgtccg cgagtgaaaa aaagcgcccg aggaagcaat aaggcactac aatgctcagt 35760ctcaagtcca gcaaagcgat gccatgcgga tgaagcacaa aatcctcagg tgcgtacaaa 35820atgtaattac tcccctcctg cacaggcagc gaagcccccg atccctccag atacacatac 35880aaagcctcag cgtccatagc ttaccgagca gcagcacaca acaggcgcaa gagtcagaga 35940aaggctgagc tctaacctgt ccacccgctc tctgctcaat atatagccca gatctacact 36000gacgtaaagg ccaaagtcta aaaatacccg ccaaataatc acacacgccc agcacacgcc 36060cagaaaccgg tgacacactc aaaaaaatac gcgcacttcc tcaaacgccc aaactgccgt 36120catttccggg ttcccacgct acgtcatcgg aattcgactt tcaaattccg tcgaccgtta 36180aaaacgtcac ccgccccgcc cctaacggtc gcccgtctct cggccaatca ccttcctccc 36240tccccaaatt caaacagctc atttgcatat taacgcgcac caaaagtttg aggtatatta 36300ttgatgatga tcttaattaa tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 36360taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 36420ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 36480gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 36540gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 36600cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 36660ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 36720tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 36780gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 36840tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 36900ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 36960ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 37020ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 37080accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 37140tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 37200cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 37260taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 37320caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 37380gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 37440gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 37500ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 37560attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 37620gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 37680tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 37740agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 37800gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 37860actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 37920tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 37980attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 38040tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 38100tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 38160aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 38220tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 38280cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 38340acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaatttta attaaccc 38398

Claims

1. A replication deficient simian adenoviral vector C7 encoding a protein comprising CS protein from P. falciparum or an immunogenic fragment thereof.

2. The adenoviral vector of claim 1, wherein the protein encoded comprises the sequence of Seq ID No: 7.

3. The adenoviral vector of claim 1, where the encoded protein encoded comprises the sequence of Seq ID No: 8.

4. The adenoviral vector any one of claim 1, wherein the encoded protein has the sequence of Seq ID No: 1.

5. The adenoviral vector of claim 1, wherein the encoded protein has the sequence of Seq ID No: 3.

6. A pharmaceutical composition comprising the adenoviral vector of claim 1 and an excipient.

7. An immunogenic composition for the treatment or prophylaxis of malaria comprising the adenoviral vector of claim 1 and an adjuvant.

8. The immunogenic composition of claim 7, which further comprises a protein.

9. The immunogenic composition of claim 8, wherein the protein is RTS,S.

10. An immunogenic composition comprising (1) a replication deficient simian adenoviral vector C7 encoding a protein comprising CS protein from P. falciparum or an immunogenic fragment thereof, (2) a malaria antigen, and (3) an adjuvant.

11. A kit comprising (1) a replication deficient simian adenoviral vector C7 encoding a protein comprising CS protein from P. falciparum or an immunogenic fragment thereof, (2) a malaria antigen, and (3) an adjuvant.

12. The immunogenic composition of claim 10, wherein the protein encoded by the adenoviral vector C7 has the sequence of Seq ID No: 1.

13. The immunogenic composition of claim 10, wherein the malaria antigen is RTS,S.

14. The immunogenic composition of claim 10, wherein the adjuvant comprises 3D-MPL.

15. The immunogenic composition of claim 10, wherein the adjuvant comprises a saponin.

16. The immunogenic composition of claim 15, wherein the saponin is QS21.

17. The immunogenic composition of claim 10, wherein the adjuvant comprises 3D-MPL and QS21.

18. The immunogenic composition of claim 10, wherein the composition is an oil-in-water emulsion.

19. The immunogenic composition of claim 10, wherein the composition is a liposomal formulation.

20. The immunogenic composition of claim 10 comprising (1) a replication deficient simian adenoviral vector C7 encoding a protein comprising CS protein from P. falciparum having the sequence of Seq ID No: 1, (2) malaria antigen RTS,S, and (3) an adjuvant comprising 3D-MPL and QS21.

21. A process for the preparation of a the adenoviral vector of claim 1 comprising the steps of: a. propagating the adenoviral vector on a suitable cell line, and b. recovering the adenoviral vector.

22. A process for the preparation of a composition of the immunogenic composition of claim 10 comprising the step of admixing the adenoviral vector of claim 1 with at least one excipient/carrier.

23. (canceled)

24. (canceled)

25. (canceled)

26. A method of treatment or prophylaxis of malaria in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the immunogenic composition of claim 10.

27. A method for the treatment or prophylaxis of malaria in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the adenoviral vector of claim 1.

28. The method of claim 27, wherein the adenoviral vector is administered to the subject in a prime-boost regime.