JP2011505796A - vaccine - Google Patents

Abstract

The present invention relates to a replication-deficient simian adenovirus vector C7 that encodes a protein comprising CS protein from Plasmodium falciparum or a fragment thereof (eg, those shown in SEQ ID NO: 1 or SEQ ID NO: 3). The present invention also relates to a method for producing the viral vector and the use of the viral vector in the treatment / prevention of malaria infection. Also described are compositions, vaccines and kits comprising the viral vectors. In one embodiment, the present invention uses a synthetic C7 viral vector. The C7 viral vectors of the invention can be co-administered or co-formulated with a malaria antigen such as RTS, S, optionally in the presence of an adjuvant such as 3D-MPL and / or an saponin (eg QS21).
[Selection] Figure 16

Description

  The present invention relates to monkey-derived adenovirus vectors, and particularly to monkey-derived adenovirus vectors encoding novel malaria antigens derived from the plasma sporozoite protein of Plasmodium falciparum. The invention further relates to a method for producing said viral vector and its use in the treatment / prevention of malaria infection.

  Malaria is one of the world's major health problems, with between 2 and 4 million people dying from the disease each year.

  One of the most acute forms of the disease is caused by the parasitic protozoan Plasmodium falciparum (P. falciparum), which is responsible for most of the deaths caused by malaria.

  The life cycle of P. falciparum is complex and requires two hosts, human and mosquito to complete. Infection to humans is initiated by inoculation of sporozoites into the bloodstream by the bite of infected mosquitoes. Sporozoites migrate to the liver, where they infect hepatocytes, where the sporozoites differentiate into the merozoite stage via the extra-erythrocytic intracellular stage, which infects the red blood cells (RBC) and circulates in the asexual blood stage. To start. This circulation is completed by the differentiation of a large number of merozoites into sexual stage gamete cells in the RBC, which are ingested by mosquitoes, and in the mosquito body, the cells are in a series in the midgut. It develops through this stage and becomes a sporozoite, which moves to the salivary glands.

  The sporozoite stage of P. falciparum is seen as a potential target for malaria vaccines. Inactivated (irradiated) sporozoites have been shown to induce protection against experimental human malaria (Am. J, Trop. Med. Hyg 24: 297-402, 1975). However, in practice and in business planning, it is impossible to produce a vaccine against malaria in the general population based on this method using irradiated sporozoites.

  The main surface protein of sporozoites is known as circumsporozoite protein (CS protein). It is thought to be involved in the motility and invasiveness of the sporozoites during the transfer of the sporozoites from the initial site of mosquito inoculation into the circulation (in which it moves to the liver).

  Plasmodia species CS proteins are characterized by a central repeat domain (repeat region) adjacent to non-repeating amino (N-terminal) and carboxy (C-terminal) fragments.

  At present, the most advanced malaria vaccine in the clinic is based on lipoprotein particles called RTS, S (also known as virus-like particles). This particle contains a portion of the CS protein of P. falciparum that substantially corresponds to amino acids 207-395 of the CS protein of P. falciparum (strain NF54 [3D7]) fused to the N-terminus of the S antigen from hepatitis B. contains. The S antigen may comprise a portion of pre-S2.

  RTS, S particles are usually delivered with a powerful adjuvant.

  Nevertheless, malaria vaccines utilizing recombinant adenovirus vectors have been presented. For example, WO 2004/055187 describes certain viral vectors, including specific adeno5 (Ad5) and adeno35 (Ad35) vectors (both derived from human adenoviruses) that encode CS proteins.

  There are over 40 different human adenovirus serotypes that differ in 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 diarrhea in infants.

  Immunity against adenovirus infection is thought to last a lifetime after infection. In particular, it is believed that pre-existing immunity against Ad5 and Ad35 can result in neutralization of therapeutic adenoviral vectors based on human adenovirus. This can reduce the therapeutic effectiveness of the vector. This is because the vector is prevented from entering the cell and generating the relevant antigen in vivo.

International Publication No. 2004/055187 Pamphlet International Publication No. 2003/046124 Pamphlet

Am. J, Trop. Med. Hyg 24: 297-402, 1975

  The present invention relates to a replication-deficient monkey (simian) adenoviral vector C7 (also referred to as Pan7 or CV-33) encoding a protein comprising CS protein from P. falciparum or a fragment thereof, such as those shown in SEQ ID NO: 1 or SEQ ID NO: 3. Providing a vaccine for the prevention and / or treatment of malaria, including

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1: Protein / antigen amino acid sequence derived from the CS protein of P. falciparum (referred to herein as Ade2).
SEQ ID NO: 2: nucleic acid sequence encoding the protein of SEQ ID NO: 1 (referred to herein as the Ade2 gene).
SEQ ID NO: 3: Alternative amino acid sequence of a protein / antigen derived from the CS protein of P. falciparum (referred to herein as Ade1).
SEQ ID NO: 4: nucleic acid sequence encoding the protein of SEQ ID NO: 3 (referred to herein as the Ade1 gene).
SEQ ID NO: 5: Capsid protein sequence from Chimp Adeno 7 (SEQ ID NO: 17 of WO 03/046124).
SEQ ID NO: 6: Amino acid sequence from Chimp Adeno 7 (SEQ ID NO: 20 of WO 03/046124).
SEQ ID NO: 7: amino acid sequence from P. falciparum CS protein.
SEQ ID NO: 8: amino acid sequence from P. falciparum CS protein.
SEQ ID NO: 9: Amino acid sequence from P. falciparum CS protein.
SEQ ID NO: 10: amino acid sequence from P. falciparum CS protein.
SEQ ID NO: 11: nucleotide sequence of CpG 1826.
SEQ ID NO: 12: nucleotide sequence of CpG 1758.
SEQ ID NO: 13: Nucleotide sequence of CpG.
SEQ ID NO: 14: Nucleotide sequence of CpG 2006.
SEQ ID NO: 15: nucleotide sequence of CpG 1668.
SEQ ID NO: 16: nucleotide sequence of CpG 5456.
SEQ ID NO: 17: Shows the nucleotide sequence of an expression cassette that replaces the Ade2 expression cassette, cloned into a C7 adenoviral vector.
SEQ ID NO: 18: Shows the nucleotide sequence of the Ade2 expression cassette cloned into a C7 adenoviral vector.
SEQ ID NO: 19: This shows the complete nucleotide sequence of the synthetic recombinant vector C7-Ade2.

The plasmid map of pCR2.1-Ade2 is shown. The plasmid map of pShuttle6-Ade2 is shown. The plasmid map of pC7000-CMV Ade2 is shown. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and C7 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and C7 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and C7 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and C7 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade2 and Ad5 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade2 and Ad5 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade2 and Ad5 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade2 and Ad5 Ade2 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and Ad5 Ade1 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and Ad5 Ade1 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and Ad5 Ade1 in C57B1 / 6 mice. 2 shows a comparison of CS-specific T cell responses induced by C7 Ade1 and Ad5 Ade1 in C57B1 / 6 mice. Figure 2 shows anti-CS antibody response measured by ELISA in C57B1 / 6 mice. FIG. 4 shows the kinetics of CS-specific CD8 T cell responses induced by C7-Ade2 in CB6F1 mice. FIG. 4 shows the kinetics of CS-specific CD8 T cell responses induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows the kinetics of CS-specific CD4 T cell responses induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows the kinetics of CS-specific CD4 T cell responses induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD8 T cell response induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD8 T cell response induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD4 T cell response induced by C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD4 T cell response induced by C7-Ade2 in CB6F1 mice. FIG. 5 shows the kinetics of CS-specific CD8 T cell responses induced by C7-Ade2 in initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 5 shows the kinetics of CS-specific CD8 T cell responses induced by C7-Ade2 in initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows the kinetics of CS-specific CD4 T cell responses induced by C7-Ade2 upon initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows the kinetics of CS-specific CD4 T cell responses induced by C7-Ade2 upon initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows the kinetics of HBs-specific CD8 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows the kinetics of HBs-specific CD4 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS-specific CD8 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS-specific CD8 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of HBs-specific CD8 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 5 shows the cytokine profile of CS-specific CD4 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 5 shows the cytokine profile of CS-specific CD4 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 5 shows cytokine profiles of HBs-specific CD4 T cell responses induced by C7-Ade2 in primary / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows antibody responses induced by C7-Ade2 in initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. FIG. 6 shows antibody responses induced by C7-Ade2 in initial / boost stimulation or co-formulation using RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows antibody responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. FIG. 6 shows antibody responses induced by co-formulation of C7-Ade2 + RTS, S / AS01B in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD8 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows cytokine profiles of CS- and HBs-specific CD4 T cell responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Shown are antibody responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Shown are antibody responses induced by C7-Ade2, RTS, S and AS01B responses in CB6F1 mice. Figure 2 shows the kinetics of CS-specific CD8 T cell responses induced by synthetic C7-Ade2 in CB6F1 mice. Figure 2 shows the kinetics of CS-specific CD8 T cell responses induced by synthetic C7-Ade2 in CB6F1 mice. Figure 2 shows the kinetics of CS-specific CD4 T cell responses induced by synthetic C7-Ade2 in CB6F1 mice. Figure 2 shows the kinetics of CS-specific CD4 T cell responses induced by synthetic C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD8 T cell response induced by synthetic C7-Ade2 in CB6F1 mice. FIG. 6 shows a cytokine profile of CS-specific CD8 T cell response induced by synthetic C7-Ade2 in CB6F1 mice. FIG. 5 shows a cytokine profile of CS-specific CD4 T cell response induced by synthetic C7-Ade2 in CB6F1 mice. FIG. 5 shows a cytokine profile of CS-specific CD4 T cell response induced by synthetic C7-Ade2 in CB6F1 mice.

  The sequence and production of C7 is described in WO 2003/046124. WO 2003/046124, SEQ ID NO: 6 (penton sequence), 9 (nucleic acid sequence), 10 and 11 (hexon sequence), and 12 (fiber protein) are incorporated herein by reference. The deposit number for C7 is [ATCC VR-593].

  Vectors can be grouped into several families, and there are assumptions that adenoviral vectors within a given family may have similar characteristics, but the characteristics and characteristics of any given adenoviral vector Is often unique.

  The use of C7 is considered particularly advantageous. This is because when a protein-encoding gene is inserted, it is apparently more stable than other known vectors such as C6 described in WO 2003/046124. In other words, C7 is considered less prone to reconfiguration. Of course, it is very important that any adenoviral vector used in a vaccine is stable because pharmaceuticals need to be shown to be well-characterized, stable and safe before they can be sold. is important.

  Existing immunity against C7 appears to be very low, so the risk of neutralization of the viral vector after the first administration to the patient is low.

  Furthermore, it is believed that there are one or more other properties of C7 that can make it particularly suitable for administration to humans and / or generating a favorable immune response in vivo.

  In one embodiment, the present invention uses a synthetic C7 viral vector that may be particularly suitable for obtaining regulatory approval for human administration.

  In one embodiment, the last 12-14 amino acids of the malaria antigen component from the CS protein have been removed.

  In one embodiment, the malaria antigen encoded by the adenoviral vector has been modified to remove a potential glycosylation site. For example, as shown at about position 379 of SEQ ID NO: 1, the amino acid alanine can be used in place of serine.

（配列番号7）
（これは、場合によっては、アミノ酸約81-99に位置する）。 In one embodiment of the invention, the protein / antigen used comprises the following amino acids:

(SEQ ID NO: 7)
(This is optionally located at about amino acids 81-99).

In one embodiment, the encoded protein / antigen is an amino acid:
AIGL (SEQ ID NO: 8)
For example at the C-terminus.

（配列番号9）。 In one embodiment, the present invention uses a protein comprising the following amino acids:

(SEQ ID NO: 9).

（配列番号10）。 In one embodiment, the present invention uses a protein comprising the following amino acids:

(SEQ ID NO: 10).

  In another embodiment, the protein / antigen used comprises the sequence of SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 9.

  In another embodiment, the protein / antigen used comprises the sequence of SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 10.

  In one embodiment, the encoded protein / antigen is SEQ ID NO: 1 or 3.

  The protein sequence shown in SEQ ID NO: 1 is novel and constitutes one embodiment of the present invention.

  The polynucleotide encoding the protein sequence of SEQ ID NO: 1, in particular the polynucleotide sequence of SEQ ID NO: 2 also constitutes one embodiment of the present invention. This polynucleotide sequence (SEQ ID NO: 2) has already been codon optimized for expression in humans.

  In some cases, the polynucleotide sequence encoding the protein of SEQ ID NO: 1 may be codon optimized.

  The invention also extends to a vector / plasmid / host used in the production of a novel hybrid fusion protein of SEQ ID NO: 1 or used in the production of a viral vector of the invention.

  If protein production and isolation is required, a suitable plasmid can be used to insert the sequence encoding 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 generally contains an integrated marker to aid selection, eg, a gene encoding antibiotic resistance or LEU2 or HIS auxotrophy.

  The host cell can be prokaryotic or eukaryotic, but is preferably a yeast, such as Saccharomyces (eg, Saccharomyces cerevisiae, eg, name RIT DC5 cir (o), ATCC database with depositor Smith Kline-RIT (accession number) 20820) DC5) and non-Saccharomyces yeasts. These include Schizosaccharomyces (e.g. Schizosaccharomyces pombe), Kluyveromyces (e.g. Kluyveromyces lactis), Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarrowia (e.g. Yarrowia lipolyticaces (e.g. Yarrowia lipolyticaces) ) Is included.

  In one embodiment, the present invention provides the use of the vector of the present invention or the protein of SEQ ID NO: 1 for the treatment or prevention of malaria.

  In one embodiment, the present invention provides a pharmaceutical formulation comprising a viral vector of the present invention and an excipient, eg, an isotonic carrier suitable for injection. Suitable excipients are described in more detail below.

In one embodiment, the formulation is
The adenoviral vector of the present invention,
A malaria antigen, such as lipoprotein particles, in particular RTS, S, and optionally an adjuvant, such as an adjuvant comprising saponin and / or 3D-MPL.

  If the vector encodes the sequence of SEQ ID NO: 1, the vector is particularly suitable for use in therapeutic regimes using a 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. It is believed that the use of the vector in a plan to use RTS, S can effectively enhance the efficacy of RTS, S.

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

RTS, S
RTS, S can be produced as described in WO 93/10152 (eg from P. falciparum NF54 / 3D7 strain). The nucleotide sequence of the RTS expression cassette and putative translation product is described in FIG. 9 of WO 93/10152 (in which it is referred to as RTS ^* ).

  In the context of the present specification, an excipient means an ingredient in a pharmaceutical formulation that itself has no therapeutic effect. Diluents or carriers are included in the definition of excipients. Suitable carriers include PBS, saline (saline) and the like. Adjuvants are also included in the definition of excipients. This is because adjuvants can show physiological effects in vivo, but this effect is global and not a specific therapeutic effect in the absence of therapeutic ingredients.

Adjuvants Individual adjuvants include metal salts, oil-in-water emulsions, Toll-like receptor agonists (especially Toll-like receptor 2 agonists, Toll-like receptor 3 agonists, Toll-like receptor 4 agonists, Toll-like receptor 7 agonists, Toll -Like receptor 8 agonist and Toll-like receptor 9 agonist), saponin or an adjuvant selected from the group thereof.

  In one embodiment, the adjuvant is a Toll-like receptor (TLR) 4 ligand, such as an agonist, such as a lipid A derivative, in particular monophosphoryl lipid A, more particularly 3-deacylated monophosphoryl lipid A (3D-MPL). It is.

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

  3D-MPL is sold by Corixa under the trademark MPL® and primarily promotes the response of CD4 + T cells with an IFN-g (Th1) phenotype. It can be manufactured according to the method 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, small particle 3D-MPL is used in the compositions of the present invention. Small particle 3-MPL has a particle size such that it can be sterile filtered through a 0.22 μm filter. Such preparations are described in WO 94/21292. Synthetic derivatives of lipid A are known and are believed to be TLR4 agonists, including but not limited to:

OM174 (2-deoxy-6-O- [2-deoxy-2-[(R) -3-dodecanoyloxytetra-decanoylamino] -4-o-phosphono-β-D-glucopyranosyl] -2- [ (R) -3-hydroxytetradecanoylamino] -α-D-glucopyranosyl dihydrogen phosphate) (WO 95/14026),
OM 294 DP (3S, 9R) -3-[(R) -Dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R)-[(R) -3-hydroxytetradecanoylamino Decane-1,10-diol, 1,10-bis (dihydrogenophosphate) (WO99 / 64301 and WO 00/0462),
OM 197 MP-Ac DP (3S-, 9R) -3-[(R) -Dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9-[(R) -3-hydroxytetradecanoyl Amino] decane-1,10-diol, 1-dihydrogenophosphate 10- (6-aminohexanoate) (WO 01/46127).

  Typically, when 3D-MPL is used, the antigen and 3D-MPL are delivered as an oil-in-water emulsion or multiple oil-in-water emulsions. The uptake of 3D-MPL is advantageous because it is a stimulator of effector T cell responses.

  Other TLR4 ligands that can be used include alkyl glucosaminide phosphates (AGP), such as those disclosed in WO 9850399 or US 6303347 (a method for producing AGP is also disclosed), or disclosed in US 6764840 And pharmaceutically acceptable salts of AGP. Some AGPs are TLR4 agonists and some are TLR4 antagonists. Both are considered useful as adjuvants.

  Another immunostimulant used 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. In 1974, Dalsgaard et al. (“Saponin adjuvants”, Archiv. Fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254 ) Was first described as having adjuvant activity. Purified fragments of Quil A, such as QS7 and QS21 (also known as QA7 and QA21), which retain adjuvant activity without the toxicity associated with Quil A, have been isolated by HPLC. QS21 is a natural saponin derived from the bark of Quillaja saponaria Molina that induces CD8 + cytotoxic T cells (CTL), Th1 cells and a prevalent IgG2a antibody response.

  Specific formulations of QS21 that further contain sterols have been described (WO 96/33739). The ratio of QS21: sterol is typically on the order of 1: 100 to 1: 1 (weight to weight). In general, there is an excess of sterol and the ratio of QS21: sterol is at least 1: 2 w / w. Typically, for human administration, QS21 and sterol are present in the vaccine in the range of about 1 μg to about 100 μg, such as about 10 μg to about 50 μg per dose.

  Liposomal formulations generally contain a neutral lipid, such as phosphatidylcholine (which is usually amorphous at room temperature), such as egg yolk phosphatidylcholine, dioleoylphosphatidylcholine or dilaurylphosphatidylcholine. The liposome may also contain a charged lipid that enhances the stability of the liposome-QS21 structure in the case of liposomes composed of saturated lipids. In these cases, the amount of charged lipid is often 1-20% w / w, for example 5-10%. The ratio of sterol to phospholipid is 1-50% (mol / mol), for example 20-25%.

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

  Saponins can be in the form of micelles, mixed micelles (generally but not exclusively with bile salts), or ISCOM matrices (EP 0 109 942), liposomes or related colloidal structures such as insects In the form of a helical (spiral) or cyclic multimeric complex or lipid / layered structure, and a lamellar (when formulated with cholesterol and lipid), or an oil-in-water emulsion (eg in WO 95/17210) It can be in form.

  Usually, the saponin is provided in the form of a liposomal formulation, ISCOM or an oil-in-water emulsion.

  Immunostimulatory oligonucleotides can also be used. Specific examples of oligonucleotides used in the adjuvants or vaccines of the present invention include CpG-containing oligonucleotides, generally two or more dinucleotide CpG motifs separated by at least 3, more preferably at least 6 or more nucleotides. Are included. A CpG motif is a cytosine nucleotide followed by a guanine nucleotide. CpG oligonucleotides are typically deoxynucleotides. In one embodiment, there is a phosphorodithioate, or more preferably a phosphorothioate linkage, between nucleotides in the oligonucleotide, although phosphodiester and other internucleotide linkages are also within the scope of the present invention. is there. Oligonucleotides having mixed internucleotide linkages are also included within the scope of the present invention. Methods for the production of phosphorothioate oligonucleotides or phosphorodithioates are described in US 5,666,153, US 5,278,302 and WO 95/26204.

該配列はホスホロチオアート修飾ヌクレオチド間連結を含有しうる。 Specific examples of oligonucleotides include the following:

The sequence may contain phosphorothioate modified internucleotide linkages.

  Other CpG oligonucleotides may contain one or more of the above sequences with minor deletions or additions.

  The CpG oligonucleotide can be synthesized by any method known in the art (see, eg, EP 468520). Conveniently, such oligonucleotides can be synthesized using an automated synthesizer.

  Specific examples of TLR2 agonists include peptidoglycan or lipoprotein.

  Imidazoquinolines such as Imiquimod and Resiquimod are known TLR7 agonists. Single-stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), and double-stranded RNA and poly IC (polyinosin-polycytidylic acid, a commercially available synthetic mimic of viral RNA) are typical examples of TLR3 agonists . 3D-MPL is an example of a TLR4 agonist, and CpG is an example of a TLR9 agonist.

  Immunostimulants can be included alternatively or additionally. In one embodiment, the immunostimulator is 3-deacylated monophosphoryl lipid A (3D-MPL).

  In one embodiment, the adjuvant comprises 3D-MPL.

  In one embodiment, the adjuvant comprises QS21.

  In one embodiment, the adjuvant comprises CpG.

  In one embodiment, the adjuvant is formulated as an oil-in-water emulsion.

  In one embodiment, the adjuvant is formulated as a liposome.

  Adjuvant combinations include 3D-MPL and QS21 (EP 0 671 948 B1) oil-in-water emulsions, or liposome formulations containing 3D-MPL and QS21 or 3D-MPL formulated with other carriers (EP 0 689 454 B1 ) Is included. Another preferred adjuvant system comprises a combination of 3D-MPL, QS21 and CpG oligonucleotides described in US 6558670 and US 6544518.

The production of pharmaceutical vaccines is generally described in New Trends and Developments in Vaccines, Voller et al., University Park Press, Baltimore, Maryland, USA, 1978. Encapsulation within liposomes is described, for example, in Fullerton, US Pat. No. 4,235,877.

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

  In one embodiment, the present invention provides a C7 adenoviral vector of the present invention and a malaria antigen, such as a novel antigen of RTS, S or SEQ ID NO: 1 or a virus-like particle thereof, and an excipient, optionally an adjuvant. Compositions comprising in the presence are provided.

  Immunogenicity in the context of the present specification is intended to mean the ability to elicit an immune response, where the response is specific for the malaria component in the relevant formulation. This response may require the presence of appropriate adjuvants and / or booster stimuli. For example, in order to obtain an appropriate immunogenic response, a booster that includes a dose similar to or less than the original dose may be required.

  The composition / pharmaceutical formulation of the invention can comprise a mixture of one or more other antigens, such as antigens derived from P. falciparium and / or P. vivax, wherein the antigen comprises It is selected from DBP, PvTRAP, PvMSP2, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PvAMA1 and RBP or fragments thereof.

  Other specific examples of 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 other Plasmodium Their analogs (analogs) in the genus species are included.

  The present invention also relates to the use of C7 for encoding malaria antigens, for example, for the treatment and / or prevention of malaria, or for the manufacture of medicaments thereto, particularly as described herein. Regarding use.

  The present invention also includes a method of treatment comprising administering a therapeutically effective amount of one or more embodiments of the present invention. In some cases, a C7 viral vector of the invention is co-administered with a malaria antigen, such as RTS, S or an antigen of SEQ ID NO: 1, optionally in the presence of an adjuvant comprising saponins such as 3D-MPL and / or QS21. Or it can be co-blended.

  A C7 vector can also be co-administered or co-formulated with another adenoviral vector of different serotype and / or origin encoding the same or different antigens.

  The invention extends to the use of any of the embodiments described herein in a prime-boost method, eg, in this case the prime dose at time zero (and the subsequent prime within eg 3 months). Antigen booster) and booster is administered at about 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the last initial challenge, and in some cases, one year after the first boost Later, additional booster antigens are administered.

  Advantageously, one or more embodiments of the present invention comprising the combination vaccines described above are specific humoral (ie antibody responses) and / or cellular immune responses (eg CD8 + and / or CD4 +), eg antibody responses and Stimulate a CD8 + and / or CD4 + response, in particular a CD8 + and antibody response, i.e. a response specific to the CS protein and / or S antigen, as appropriate.

  This type of balanced immune response may be required to obtain so-called sterile protection against malaria infection.

  Furthermore, the combined antibody response can be enhanced compared to the antibody response to the adjuvanted protein-only method.

In one embodiment, the present invention provides a supplemental component of the first-boost stimulation method,
A C7 adenoviral vector encoding the same or different malaria antigen,
Another viral vector encoding a malaria antigen, for example a CS protein from P. falciparum, for example a human adenoviral vector, for example Ad5 or Ad35, or a simian adenoviral vector of a different serotype (ie other than C7), and / or Provide the use of C7 as primary or booster in the first-boost stimulation method using malaria antigens such as RTS, S and adjuvants such as adjuvants including saponin and / or 3D-MPL.

  The present invention also provides any of the embodiments described herein for the manufacture of a medicament for the treatment and / or prevention of malaria infection.

The amount of 3D-MPL used is generally small, but depending on the vaccine formulation, it is 1-1000 μg / dose, for example in the range of 1-500 μg / dose, for example 1-100 μg / dose, for example 50 or 25 μg / dose. Can be a range.

  The amount of CpG or immunostimulatory oligonucleotide in the adjuvants or vaccines of the invention is generally small, but depending on the vaccine formulation, 1-1000 μg / dose, such as in the range of 1-500 μg / dose, such as 1-100 μg / dose. Range.

  The amount of saponin used in the adjuvant of the present invention is 1-1000 μg / dose, for example 1-500 μg / dose, for example in the range 1-250 μg / dose, in particular 1-100 μg / dose, for example in the range 50 or 25 μg / dose. sell.

  When administering the protein, the dose is for example 1 to 500 μg, for example 10 to 100 μg, in particular 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 μg / dose sell.

When administering an adenoviral vector, the dose can be, for example, 10 ³ to 10 ¹⁶ vpu, such as 10 ⁶ to 10 ¹⁰ vpu.

  When a combination is used, the amount used for each component of the combination may correspond to the amount that the component is administered alone.

  The invention also extends to kits containing the elements used in the combination of the invention.

  The present invention further relates to a method for producing the adenoviral vector of the present invention and a preparation containing the same.

  The present invention also relates to a method for producing the protein of SEQ ID NO: 1.

  In the context of this specification, comprising shall be construed as comprising.

  The invention extends to embodiments consisting of or consisting essentially of related elements, including certain elements, corresponding to the embodiments described herein.

  The discussion in the background section of this specification is described for the purpose of placing the invention within its technical background. It should not be construed as an admission of what is known in the art, and in particular, no admission of what is equivalent to common general knowledge.

(Example)
The following examples are presented to illustrate the methodologies that can be used in the present invention.

  A synthetic gene was produced by Medigenomix. The gene was cloned into the pCR2.1-TOPO-TA cloning vector (Invitrogen; see Figure 1). This vector was digested with NotI and BamHI to produce a recombinant shuttle plasmid vector (-Ade2). A map of the shuttle plasmid is shown in FIG.

Methodology and Expression Cassette Description for Viral Rescue Expression cassettes are cytomegalovirus (CMV) early promoter and first exon, intron from plasmid pCI (purchased from Promega), DNA encoding Ade2, and rabbit globin polyadenylation Contains signal. The complete cassette is flanked by recognition sites for restriction enzymes I-CeuI and PI-SceI, respectively. The expression cassette was excised from the shuttle plasmid using I-CeuI and PI-SceI and the SA1V-24 E1-deleted genome (ie C7) -pC7000pkGFP (Roy et al., Hum Gene Ther. (2004) 5: 519 Was introduced into the plasmid molecular clone described in FIG. The plasmid molecular clone DNA was linearized by digestion with the restriction enzyme PacI and transfected into HEK293 cells to rescue the recombinant adenovirus. The adenovirus was propagated, amplified and purified using standard techniques.

  The sequence of the expression cassette (Ade2) from I-CeuI to the PI-Sce recognition site is shown in SEQ ID NO: 18.

C57Bl / 6 in C57Bl / 6 mice at a dose range of 10e10, 10e9, 10e8 virions of C7 chimpadenovirus expressing either Ade1 or Ade2 immunogenic constructs of C7-Ade1 and C7-Ade2 in C57Bl / 6 mice Mice were immunized once intramuscularly. As a positive control, several mice were immunized with human adenovirus 5 (10e9 and 10e8 doses) expressing either construct Ade1 or Ade2. As negative controls, several mice were immunized with empty C7 and empty Ad5 viral vectors.

  Peripheral blood is collected and pooled on days 14, 28, 34 and 49 after immunization, including the sequence of interest, ie, the N-terminal region (N-terminal) or C-terminal region (C-terminal) of the CS protein Flow cytometry measures Ag-specific CD4 and CD8 T cell responses that produce IL-2 and / or IFN-gamma after overnight in vitro restimulation with a pool of 15-mer peptides did. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

  The results show that both constructs induced CS-specific CD4 and CD8 T cell responses under the experimental conditions described above (FIGS. 4-15). Of note, C-terminal specific CD8 T cell responses were detected only in mice immunized with adenovirus containing the Ade2 insert (FIGS. 4 and 8). In particular, these C-terminal specific CD8 T cell responses were similar in mice immunized with 10e10vp C7 Ade2 or 10e9vp Ad5 Ade2 (FIG. 8).

  Anti-CS antibody responses were also measured by ELISA on sera collected 48 days after immunization. In particular, the total Ig response to the R32LR polypeptide (ie, including the central portion of P. falciparum CSP) was measured (Mettens et al., Vaccine 2008). The results show that a single immunization with C7 Ade1 or C7 Ade2 induces a low level of R32LR specific antibody response. The intensity of this response correlates with the number of virus particles used for immunization (dose range effect).

Immunogenicity of C7-Ade2 in CB6F1 mice
CB6F1 mice were immunized once intramuscularly (5 pools of mice / group) at a dose range of 10e10, 10e9, 10e8 virions of C7 chimpadenovirus expressing Ade2 construct. Peripheral blood was collected and pooled on days 21, 28, and 35 after immunization, and the 15-mer containing the sequence of interest, ie, the N-terminal region (N-terminal) or C-terminal region (C-terminal) of CS protein After overnight in vitro restimulation with a pool of peptides, CSC-terminal and CSN-terminal specific CD4 and CD8 T cell responses producing IL-2 and / or IFN-gamma were measured by flow cytometry. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

  The results show that in this mouse strain, a single immunization with 10e9vp or 10e10vp C7 Ade2 induces C-terminal and N-terminal specific CD4 and CD8 T cell responses (FIGS. 17-20). . In particular, the intensity of the average observed response is at least equivalent if not higher when the 10e9vp dose is used. While the CS-specific CD8 T cell response is primarily N-terminal specific, the CS-specific CD4 T cell response equally targets the N-terminal and C-terminal regions of the CS protein.

  Cytokine profiles of CS-specific CD4 and CD8 T cell responses were also determined. It was similar across all time points tested. The profiles shown 28 days after immunization are shown in FIGS. 21-24 and are representative of other time points tested. Briefly, the CS-specific CD8 T cell response is largely mediated by CD8 T cells that produce only IFNg (FIGS. 21 and 22). The CS-specific CD4 T cell response is also caused by IFNg-producing CD4 T cells and, to a lesser extent, CD2 T cells that produce only IL-2 or both IL-2 and IFNg (FIG. 23). And 24).

Immunogenicity of C7-Ade2 in CB6F1 mice using RTS, S / AS01B or C7-Ade2 in co- formation The immunogenicity of C7 chimpadenovirus expressing Ade2 construct in co-formulation (combo) was tested (4 pools of mice / group). AS01B is an adjuvant system containing 3D-MPL and QS21 formulated in liposomes. Mice were immunized intramuscularly on days 0, 14 and 28 as follows.

here,
A = 10e9 vp C7 Ade2,
P = 5μg RTS, S / 50μl AS01B,
C = 10e10 vp C7 Ade2 + 5 μg RTS, S / 50 μl AS01B.

  Peripheral blood was collected, pooled and sequenced on day 21 (7d pII), 35 (7d pIII), 49 (21d pIII), 63 (35d pIII), 77 (49d pIII) after immunization CS C-terminus, CS N-terminus producing IL-2 and / or IFN-gamma after overnight in vitro restimulation with a pool of 15-mer peptides including (CS N-terminus, CS C-terminus or HBs) And HBs-specific CD4 and CD8 T cell responses were measured by flow cytometry. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

The results show that: That is,
APP, PPA and CCC all induce an N-terminal specific CD8 T cell response. At 7d pII, an N-terminal specific CD8 T cell response is observed in the CCC group and then in the APP group. At subsequent time points tested (21d pIII, 35d pIII and 49d pIII), these responses are of similar intensity in the APP, PPA and CCC groups and are maintained as such (FIG. 25).
APP, PPA and CCC induce a C-terminal specific CD8 T cell response that still persists 49 days after the third immunization (FIG. 26).
• N-terminal specific CD4 T cell responses have been detected primarily in mice immunized with APP or CCC, with higher intensity such responses detected in the APP group (FIG. 27).
• All groups show C-terminal specific CD4 T cell responses. However, APP and CCC induce higher levels of similar C-terminal specific CD4 T cell responses, which are about 2-3 times higher than those induced by PPA and PPP at all time points tested ( Figure 28).
HBs-specific CD4 and CD8 T cell responses are higher in animals immunized with PPP than when immunized with APP, PPA or CCC (FIGS. 29 and 30).
• The CCC treatment plan is the only one with simultaneous induction of CS and HBs-specific CD4 and CD8 T cell responses.

  Cytokine profiles of CS and HBs-specific CD4 and CD8 T cell responses were also measured and were similar across all time points tested. Representative examples of all time points tested are shown below from the 21d pIII time points (FIGS. 31-36). Briefly, the Ag-specific CD8 T cell response is largely mediated by CD8 T cells that produce IFNg (FIGS. 31-33). In contrast, the Ag-specific CD4 T cell response is between CD4 T cells producing IFNg, IFNg and IL-2, and to a lesser extent, CD4 T cells producing IL-2. Provided by the mixture (Figures 34-36).

  In addition, Ag-specific antibody responses were measured by ELISA on sera collected 14 and 42 days after the third immunization. In particular, total Ig responses to R32LR polypeptides (ie, including the central portion of P. falciparum CSP) and to HBs were measured (Mettens et al., Vaccine 2008). All immunization methods elicited R32LR and HBs specific antibody responses that persisted until the last time point tested (ie, 42 days after the third immunization) (FIGS. 37 and 38).

Immunogenicity of C7-Ade2 + RTS, S / AS01B co-formulation in CB6F1 mice In this experiment, the immunogenicity of chimpadenovirus C7 Ade2 co-formulation (Combo) with RTS, S / AS01B Compared. In particular, the immune responses elicited by one, two or three injections of the combo in CB6F1 mice were compared (4 pools of mice / group). Also, different intervals between the two injections of the combo (ie, 14 and 21 days) were evaluated. Finally, a group of mice immunized with APP was used as a control in the experiment. The experimental design is summarized below.

here,
A = C7 Ade2 with 10e9 vp,
P = 5μg RTS, S / 50μl AS01B,
C = 10e10 vp C7 Ade2 + 5 μg RTS, S / 50 μl AS01B.

  Peripheral blood is collected and pooled on days 35, 42, 49, 63 and 98, and the sequence of interest (ie, CS protein N-terminal region (N-terminal), C-terminal region (C-terminal) or HBs CS C-terminal, CS N-terminal and HBs-specific CD4 and CD8 T cell responses producing IL-2 and / or IFN-gamma after overnight in vitro restimulation with a pool of 15-mer peptides including Was measured by flow cytometry. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

  The results show that under these experimental conditions, two immunizations of the combo were required to simultaneously induce CS C-terminal, CS N-terminal and HBs-specific CD4 and CD8 T cell responses . The interval between the two immunizations of the combo did not appear to have a significant effect on the level of Ag-specific T cell response detected. In mice immunized 3 times with the combo, there was a trend towards higher HBs-specific CD4 and CD8 T cell responses. The kinetics of Ag-specific T cell responses are shown in Figures 39-44.

  Cytokine profiles of CS and HBs-specific CD4 and CD8 T cell responses were also measured and were similar across all time points tested. Representative examples from all time points tested are shown below from day 42 of the study (FIGS. 42-47). Briefly, the Ag-specific CD8 T cell response is largely mediated by CD8 T cells that produce IFNg (FIGS. 45-47). In contrast, the Ag-specific CD4 T cell response is between CD4 T cells producing IFNg, IFNg and IL-2, and to a lesser extent, CD4 T cells producing IL-2. Provided by the mixture (Figures 48-50).

  Ag-specific antibody responses were also measured by ELISA on sera collected on days 56 and 99 of the study. In particular, total Ig responses to R32LR polypeptides (ie, including the central portion of P. falciparum CSP) and to HBs were measured (Mettens et al., Vaccine 2008). All immunization methods elicited R32LR and HBs specific antibody responses that persisted until the last time point tested (ie, 42 days after the third immunization). Within each group, these responses were of similar intensity at both time points tested. In addition, when the group immunized with the combo was compared, a higher response tendency was observed in the group immunized with the 3-dose combo (FIGS. 51 and 52).

Assess the need for each component of the combo (ie C7-Ade2, RTS, S and AS01B) to elicit CS and HBs T cell and Ab responses simultaneously in CB6F1 mice. In this experiment, CS and HBs specific The need for each component of the combo (ie, C7-Ade2, RTS, S and AS01B) to elicit CD4 and CD8 T cell responses simultaneously was assessed. In this experiment, CB6F1 mice (6 pool mice / group) were immunized twice intramuscularly (Day 0 and Day 14) with the combo or its components as shown below.

here,
A = C7 Ade2 with 10e9 vp,
P = 5μg RTS, S / 50μl AS01B,
C = 10e9 vp C7 Ade2 + 5μg RTS, S / 50μl AS01B,
C7 empty = 10e9 vp C7 empty vector (no insert),
RTS, S = 5 μg RTS, S,
AS01B = GSK proprietary adjuvant system 1B,
Buffer = AS01B buffer (no immunostimulant).

  Peripheral blood is collected and pooled on days 14, 28, 70, 91 and 112, and the sequence of interest (ie CS protein N-terminal region (N-terminal), C-terminal region (C-terminal) or HBs CS C-terminal, CS N-terminal and HBs-specific CD4 and CD8 T cell responses producing IL-2 and / or IFN-gamma after overnight in vitro restimulation with a pool of 15-mer peptides including Was measured by flow cytometry. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

  Ag-specific antibody responses were also measured by ELISA on sera collected on days 42 and 84 of the study. In particular, total Ig responses to R32LR polypeptides (ie, including the central portion of P. falciparum CSP) and to HBs were measured (Mettens et al., Vaccine 2008).

  The results show that each component of the combo is required to elicit CS (N-terminal and C-terminal) CD4 and CD8 T cell responses (Figures 53-58) and R32LR and HBs antibody responses (Figures 65 and 66) simultaneously It is shown that.

  Mean cytokine profiles of CS and HBs specific CD4 and CD8 T cell responses were also measured at each time point in the study. These are shown in FIGS. Briefly, Ag-specific CD8 T cell responses are largely mediated by CD8 T cells that produce IFNg (FIGS. 59-61). In contrast, the Ag-specific CD4 T cell response is between CD4 T cells producing IFNg, IFNg and IL-2, and to a lesser extent, CD4 T cells producing IL-2. Provided by the mixture (Figures 62-64).

Immunogenicity of synthetic C7 Ade2 in CB6F1 mice
A synthetic C7 chimpadenovirus expressing the Ade2 construct was made available and its immunogenicity in mice compared to that of the original C7 Ade2. In this experiment, CB6F1 mice (6 pool mice / group) were immunized with 10e9 vp original C7 Ade2 or its synthetic counterpart. Peripheral blood collected 21 days, 28 days and 35 days after immunization, pooled, and 15-mer containing the sequence of interest (ie, N-terminal region (N-terminal), C-terminal region (C-terminal) of CS protein) After overnight in vitro restimulation with a pool of peptides, CSC-terminal and CSN-terminal CD4 and CD8 T cell responses producing IL-2 and / or IFN-gamma were measured by flow cytometry. As a negative control, some cells were cultured overnight in culture medium (unstimulated). The Ag-specific response was calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide stimulated cells.

  The results show that both C7 adenoviruses elicited similar levels of N-terminal and C-terminal specific CD4 and CD8 T cell responses. In particular, regardless of the viral vector used (original or synthetic), the Ag-specific CD8 T cell response was primarily N-terminal specific (FIGS. 67 and 68). N-terminal and C-terminal specific CD4 T cell responses were of similar intensity regardless of the viral vector used (FIGS. 69 and 70). Average cytokine profiles of CS-specific CD4 and CD8 T cell responses were also measured and are shown in FIGS. Briefly, the Ag-specific CD8 T cell response is largely mediated by CD8 T cells that produce IFNg (FIGS. 71-72). In contrast, the Ag-specific CD4 T cell response is between CD4 T cells producing IFNg, IFNg and IL-2, and to a lesser extent, CD4 T cells producing IL-2. Provided by the mixture (Figures 73-74).

配列番号2
ヌクレオチド配列

配列番号3

配列番号4

配列番号5
チンプアデノ 7（WO 03/046124の配列番号17）

配列番号6
チンプアデノ 7（WO 03/046124の配列番号20）

配列番号17
配列番号3のタンパク質の発現カセットの配列。タンパク質コード領域は下線で示されている。

配列番号18
Ade2発現カセットの配列を以下に示す。タンパク質コード領域は下線で示されている。

SEQ ID NO: 1
Amino acid sequence

SEQ ID NO: 2
Nucleotide sequence

SEQ ID NO: 3

SEQ ID NO: 4

SEQ ID NO: 5
Thimpadeno 7 (SEQ ID NO: 17 of WO 03/046124)

SEQ ID NO: 6
Thimpadeno 7 (SEQ ID NO: 20 of WO 03/046124)

SEQ ID NO: 17
The sequence of the expression cassette of the protein of SEQ ID NO: 3. The protein coding region is underlined.

SEQ ID NO: 18
The sequence of the Ade2 expression cassette is shown below. The protein coding region is underlined.

Claims (26)

  Replication-deficient simian adenovirus vector C7 encoding a protein comprising CS protein from P. falciparum or a fragment thereof.   2. The viral vector of claim 1, wherein the encoded protein comprises the sequence of SEQ ID NO: 7.   3. The viral vector according to claim 1 or claim 2, wherein the encoded protein comprises the sequence of SEQ ID NO: 8.   The viral vector according to any one of claims 1 to 3, wherein the encoded protein has the sequence of SEQ ID NO: 1.   3. The viral vector according to claim 1 or claim 2, wherein the encoded protein has the sequence of SEQ ID NO: 3.   A composition comprising the viral vector according to any one of claims 1 to 5 and an excipient.   A vaccine composition for the treatment or prevention of malaria, comprising the viral vector according to any one of claims 1 to 5 and an adjuvant.   8. The vaccine of claim 7, further comprising a protein.   9. A vaccine according to claim 8, wherein the protein is RTS, S.   (1) A vaccine composition comprising a replication-deficient simian adenovirus vector C7 encoding a protein comprising the CS protein from P. falciparum or a fragment thereof, (2) a malaria antigen, and (3) an adjuvant.   (1) A kit comprising a replication-deficient simian adenovirus vector C7 encoding a protein comprising CS protein from P. falciparum or a fragment thereof, (2) malaria antigen, and (3) an adjuvant.   12. The vaccine or kit according to claim 10 or claim 11, wherein the protein encoded by the adenoviral vector C7 has the sequence of SEQ ID NO: 1.   The vaccine or kit according to any one of claims 7 to 12, wherein the malaria antigen is RTS, S.   The vaccine or kit according to any one of claims 7 to 13, wherein the adjuvant comprises 3D-MPL.   14. A vaccine or kit according to any one of claims 7 to 13, wherein the adjuvant comprises saponin.   16. A vaccine or kit according to claim 15, wherein the saponin is QS21.   The vaccine or kit according to any one of claims 7 to 13, wherein the adjuvant comprises 3D-MPL and QS21.   18. A vaccine or kit according to any one of claims 7 to 17, wherein the formulation is provided as an oil-in-water emulsion.   The vaccine according to any one of claims 7 to 10 or 12 to 17, wherein the vaccine is a liposome preparation.   (1) a replication-deficient simian adenovirus vector C7 encoding a protein comprising the CS protein from P. falciparum having the sequence of SEQ ID NO: 1, (2) malaria antigen RTS, S, and (3) 3D-MPL and QS21 11. A vaccine composition according to claim 10, comprising an adjuvant comprising A method for producing a viral vector according to any one of claims 1 to 5,
a. Propagating the vector on a suitable cell line,
b. A production method comprising a step of recovering the vector.   The composition of claim 6 or the composition of claims 7-10 or 12-20, comprising mixing the viral vector of any one of claims 1-5 with at least one excipient / carrier. A method for producing the vaccine according to any one of the above.   The viral vector according to any one of claims 1 to 5, for the treatment or prevention of malaria.   6. A viral vector according to any one of claims 1 to 5 for priming or boosting in a first-boost stimulation method for the treatment or prevention of malaria.   Use of the viral vector according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment or prevention of malaria. a. The viral vector according to any one of claims 1 to 5,
b. The composition of claim 6, or
c. 21. A method of treatment comprising administering a therapeutically effective amount of the vaccine of any one of claims 7-10 or 12-20.

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