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    • 专利摘要:
    The invention relates to methods of inducing an immune response against malaria, in particular methods of immunizing against malaria comprising the administration of: i) a circumsporozoite (CS) antigen, which is a CS or protein. one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a thrombospondin-related adhesion protein antigen (TRAP), which is a TRAP protein or one of its immunogenic or variant fragments.
    公开号:BE1022950B1
    申请号:E2015/5585
    申请日:2015-09-21
    公开日:2016-10-21
    发明作者:William Ripley Ballou Jr；Katie Ewer；Adrian Hill；Alfredo Nicosia；Thomas Rampling；Johan Vekemans
    申请人:Glaxosmithkline Biologicals S.A.；The Chancellor Masters And Scholars Of The University Of Oxford；
    IPC主号:
    专利说明:

    NEW METHODS OF INDUCING AN IMMUNE RESPONSE Technical field
    The present invention relates to methods of inducing an immune response against malaria, particularly methods of immunizing against malaria comprising administering: i) a circumsporozoite (CS) antigen, which is a CS or one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a thrombospondin-related adherence protein antigen (TRAP), which is a TRAP protein or an immunogenic or variant fragment thereof.
    Background of the invention
    Malaria is one of the major health problems in the world. During the 20th century, economic and social development, together with anti-malaria campaigns, resulted in the eradication of malaria in large parts of the world, reducing the affected area of the Earth's surface from 50% to 21% . Nevertheless, half of the world's population lives in areas where malaria is transmitted. It is estimated that 3.3 billion people are at risk of contracting malaria. For the year 2012, the World Health Organization reported a global estimate of 207 million malaria cases. The disease killed approximately 627,000 people, the vast majority of whom were children under five living in sub-Saharan Africa. One of the most acute forms of the disease is caused by the protozoan parasite Plasmodium falciparum which is responsible for most of the mortality attributable to malaria. The life cycle of the parasite is complex, requiring two hosts, the man and the mosquito to complete. Human infection is initiated by the inoculation of sporozoites by the saliva of an infected mosquito. Sporozoites migrate to the liver and there infect hepatocytes (hepatic stage) where they multiply and differentiate, via the intracellular exoerythrocytic stage, into the stage of merozoites that infect erythrocytes to initiate cyclic replication in the blood stage. asexual. The cycle is complemented by the differentiation of a number of merozoites in erythrocytes into sexually mature gametocytes that are ingested by the mosquito, in which they develop through a range of midgut stages to produce sporozoites that migrate to the salivary gland.
    The sporozoite stage has been identified as a potential target for an antimalarial vaccine. The main surface protein of the sporozoite is known as the circumsporozoite protein (CS protein). The RTS, S, CS-based malaria vaccine has been in development since 1987 and is currently the most advanced candidate malaria vaccine in the study. This vaccine specifically targets the pre-erythrocytic stage of P. falciparum. The RTS, S vaccine is adjuvanted with ASO1, a liposomal formulation containing QS21 and 3D-MPL. Recent data from a large-scale Phase III clinical trial, in which RTS, S was administered in three doses, at an interval of one month, showed that over 18 months of follow-up, the RTS, S almost halved the number of malaria cases in young children (aged 5 to 17 months at the first vaccination) and reduced by approximately one quarter the cases of malaria in infants (aged 6 to 12 weeks at first vaccination) (Otieno et al. (2013). Results were presented at the 6th Multilateral Initiative on Malaria (MIM) Pan-African Conference, Durban, and recently published (The RTS, S Clinical Trial Partnership, July 2014, PLoS Medicine, 7: el001685) Despite the success of this RTS, S vaccine, a new generation of malaria vaccines with an efficiency closer to 100% is still needed to reduce mortality and address elimination and possible eradication of malaria.
    Another antigen that is being developed for use in antimalarial vaccination is the thrombospondin-related adherence protein (TRAP) (also called anonymous thrombospondin-related protein), an antigen also expressed on sporozoites and at the hepatic infection. TRAP has been tested in clinical vaccine trials in the form of both a DNA vaccine and several viral vector vaccines (MVA, chimpanzee adenovirus) encoding a multi-chain fusion protein. epitopes containing additional B cell and CD8 + and CD4 + T cell epitopes from several malaria antigens, known as ME-TRAP (Gilbert et al., 1997 Nat Biotechnol 15: 1280; Moorthy et al. 2003 Vaccine 21: 1995, Ewer et al., 2013 Nat Commun 4: 2836. doi: 10.1038 / ncomms3836). WO 2008/122769 discloses recombinant simian adenovirus vectors defective for replication encoding TRAP or its variants and the use of these vectors in vaccination. It has been found that the ChAd63 chimeric adenovirus isolate (also known as AdCh63) encoding the ME-TRAP antigen is immunogenic and that the immunogenicity can be activated by the subsequent administration of a coding MVA vector. for the ME-TRAP. More recently, it has been found that booster-booster immunization with ChAd63 ME-TRAP and MVA ME-TRAP in healthy adults in Gambia and Kenya is safe and immunogenic (Ogwang et al., 2013 PLoS 8: e57726).
    The investigators explored the combination of RTS, S with other protein targets and vaccine platforms. For example, RTS, S has been combined with merozoite surface protein 1 or recombinant TRAP protein in an adjuvant (Cummings et al., 2002 Safety, Immunogenicity, and Efficacy of Candidate Malaria Vaccinia Containing CSP and MSP-1 Antigens. Presented at: 51st Annual Meeting of the ASTMH,
    Denver, CO, USA; Rester et al. Vaccine 2014, Jun 18). Unfortunately, although the combination of RTS, S + TRAP was safe and immunogenic (Walsh et al., 2004 Am J Trop Med Hyg 70: 499), no amplified protection was observed (Rester et al., 2014). , above). This suggests that it is not possible to rely on the immunological mechanisms, not fully understood, when combining separate vaccines that theoretically should provide superior protection compared to each single antigen (Regules et al., 2011 Expert Rev Vaccines 10: 589). In other words, it is not always possible to predict whether the combination of two partially protective antimalarial vaccines will lead to enhanced efficacy.
    In conclusion, although progress has been made in the area of malaria vaccine research and development, there is still a need for new, highly effective, broad-spectrum malaria immunization methods. safe and long-lasting. Summary of the invention
    Surprisingly, it has now been discovered that an immunization regimen comprising the administration of both a CS protein variant and a TRAP variant nucleotide vector has produced protection levels. very high. In addition, when late infections were included as a recognized measure of the effectiveness of a vaccine, the efficacy increased to 100%. To our knowledge, this is the first vaccine regimen that shows 100% efficacy in any malaria challenge study with appropriate sample size, for example> 10 vaccine recipients per group. Most surprisingly, the effectiveness of this combination approach was well maintained for six months after vaccination, so that when vaccine recipients who were protected in the first month after vaccination were stimulated again ( "Rechallenged") six months after the challenge, there was an unprecedented high level of sustainable efficacy, so that seven out of eight individuals who were re-challenged were protected six months after vaccination. Thus, in one embodiment, the method of the invention produces an efficacy six months after vaccination, which is greater than the efficacy achieved with an otherwise similar protocol that does not include immunization with a vector encoding a variant of TRAP, as a protocol as described in the examples herein, comprising only immunization with RTS, S / AS01B. Specifically, the sterilization efficacy rate at 6 months with the combined vaccine used here is 72% (82% initial sterilization efficiency x 7/8 during the new stimulation ("re-challenge")) , while the six-month sterilization efficacy rate reported by Rester et al. ((2009) J Infect Dis 200, 337-346) was only 22% (50% initial sterilization efficiency x 4/9 during the new stimulation ("re-challenge")). In another embodiment, the overall efficacy six months after vaccination (determined according to the example herein) is greater than 65%, as greater than 70%. In another embodiment, the sterilization efficacy six months after vaccination (determined according to the example herein) is greater than 50%.
    Therefore, in a first aspect of the invention, there is provided a method of inducing an immune response against malaria in a human subject comprising administering: i) a circumsporozoite (CS) plasmodial antigen, which is a CS protein or one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a thrombospondin-related adherence protein (TRAP) plasmodial antigen, which is a TRAP protein or an immunogenic fragment thereof or variants.
    In another aspect, there is provided one or more immunogenic compositions for use in a method of inducing an immune response against malaria in a human subject, wherein the method comprises administering: i) a plasmodial CS antigen which is a CS or protein. one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a plasmodium TRAP antigen, which is a TRAP protein or one of its immunogenic or variant fragments.
    In yet another aspect, the invention relates to the use of: i) a plasmodial CS antigen, which is a CS protein or one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a plasmodial TRAP antigen, which is a TRAP protein or an immunogenic or variant fragment thereof, in the manufacture of a medicament for inducing an immune response against malaria in a human subject, wherein i) and ii) are administered sequentially or simultaneously.
    In another aspect, the invention provides a composition, comprising: i) a plasmodial CS antigen, which is a CS protein or an immunogenic or variant fragment thereof and ii) a nucleotide vector encoding a plasmodial TRAP antigen, which is a TRAP protein or one of its immunogenic fragments or variants.
    In another aspect, the invention relates to a kit comprising: i) a plasmodial CS antigen, which is a CS protein or one of its immunogenic or variant fragments, optionally in combination with an adjuvant (for example, an AS01 adjuvant, such as AS01B) and ii) a nucleotide vector encoding a plasmodium TRAP antigen, which is a TRAP protein or an immunogenic or variant fragment thereof.
    Brief description of the figures
    Figure la: Kaplan-Meier curves: time to patence. Percentage of subjects remaining aparasitic over time as a result of challenge with sporozoites.
    Figure lb: Anti-CS antibody level.
    Figure 2a: Immunogenicity of TRAP from T cells measured by antibody-secreting (OR) secretory staining cells (SFC) versus anti-TRAP antibodies (abscissa).
    Figure 2b: Immunogenicity of CS from T lymphocytes (ordinate) versus anti-CS antibodies (abscissa).
    FIG. 2c: Immunogenicity of TRAP according to T lymphocytes (ordinate) as a function of anti-CS antibodies (abscissa).
    Figure 2d: Comparison of anti-TRAP antibody levels observed in various studies.
    Figure 2e: Anti-CS antibody level (ordered) versus days to parasitaemia (blood smear).
    Figure 2f: Anti-CS antibody level (ordinate) as a function of days to parasitaemia (20 parasites per ml by PCR).
    Figure 2g: Anti-CS (ordinate) antibody level as a function of days to parasitaemia (500 parasites per ml by PCR).
    Figure 3: Kaplan-Meier curves: time to patency. The percentage of subjects in groups 1 and 2 remaining unparasitic over time as a result of new stimulation ("re-challenge").
    Figure 4: The amino acid sequence of RTS, in which the first 194 amino acids are the polypeptide sequence of P. falciparum CS and the last 230 amino acids are the hepatitis B antigen S sequence.
    Figure 5: The amino acid sequence of ME-TRAP, with the 559 amino acid sequence of TRAP, starting at amino acid 232, underlined.
    Figure 6: Nucleotide sequence encoding ME-TRAP.
    Figure 7: Antibody response to CS measured by ELISA. The panel VAC59 presents the data from example 2. The panel VAC55 presents the data from example 1.
    Detailed Description Definitions
    When used herein, the term "immunogenic fragment" includes a fragment of a protein of any length provided that it retains immunogenic properties. For example, the fragment may comprise 5 or more consecutive amino acids, such as 10 or more consecutive amino acids, for example, 20 or more consecutive amino acids, such as 50 consecutive amino acids or more, for example 100 consecutive amino acids or more than one protein.
    A "variant" of a polypeptide may contain amino acid substitutions, preferably conservative substitutions (for example, 1 to 50, such as 1 to 25, especially 1 to 10, and especially 1 residue (s) of amino acid can / may be modified (s)) compared to the reference sequence. Suitably, such substitutions do not occur in the region of an epitope, and, therefore, do not have a significant impact on the immunogenic properties of the antigen. The term "conservative amino acid substitution" refers to the substitution (conceptually or otherwise) of an amino acid of a group by a different amino acid of the same group. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between the corresponding proteins of homologous organisms (Schulz, GE and RH Schinner, Principles of Protein Structure, Springer-Verlag ). According to these analyzes, it is possible to define groups of amino acids in which the amino acids within a group are preferentially exchanged with one another, and therefore resemble each other mainly in their impact on the overall structure of the protein (Schulz, GE and RH Schinner, Principles of Protein Structure, Springer-Verlag). An example of a set of amino acid groups defined in this way comprises: (i) a charged group, consisting of Glu and Asp, Lys, Arg and His, (ii) a positively charged group consisting of Lys, Arg and His, (iii) a negatively charged group, consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large nonpolar aliphatic group consisting of Val, Leu and Ile, (vii) a slightly polar group consisting of Met and Cys, (viii) a small residue group consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro, (ix) an aliphatic group, consisting of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group, consisting of Ser and Thr. Protein variants may also include those in which additional amino acids are inserted relative to the reference sequence, for example, such insertions may occur at 1 to 10 locations (such as 1 to 5 locations, suitably 1 or 2 locations, particularly 1 location) and may, for example, involve the addition of 50 amino acids or less at each location (such as 20 or less, especially 10 or less, especially 5 or less). Conveniently, such insertions do not occur in the region of an epitope and, therefore, do not have a significant impact on the immunogenic properties of the antigen. An example of insertions includes a short sequence of histidine residues (eg, 2 to 6 residues) to aid the expression and / or purification of the antigen in question. The variants also include proteins where other epitopes have been added to an antigen. For example, other epitopes derived from malarial antigens may have been added to a particular antigen. An example of such a variant is ME-TRAP, which is a variant of TRAP containing other epitopes from other plasmodial antigens, see below. Variants also include those in which amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, particularly 1 location) and may, for example, involve deletion of 50 amino acids or less at each location (such as 20 or less, especially 10 or less, especially at least 5). Suitably, such deletions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. It will be understood by those skilled in the art that a particular protein variant may include substitutions, deletions, and additions (or any combination thereof). The variants preferably have at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity (such as at least about 95%, at least about 98% or at least about 99%) with the associated reference sequence. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, and are described in Altschul et al., Nue. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215: 403,410 (1990), respectively.
    Other aspects and embodiments of the invention
    As described above, in a first aspect, the invention relates to a method of inducing an immune response against malaria in a human subject comprising administering: i) a circumsporozoite (CS) plasmodial antigen , which is a CS protein or one of its immunogenic or variant fragments and ii) a nucleotide vector encoding an antigen of the thrombospondin related thrombospondin related protein (TRAP), which is a TRAP protein or one of its immunogenic or variant fragments.
    In general, the purpose of the method of the invention is to induce a protective immune response, that is to say to immunize or vaccinate the subject to prevent infection and / or malarial disease. In one embodiment, the vaccine efficacy of the method of the invention is improved compared to a therapeutic regimen that. includes only the RTS, S. For example, the rate of protection and / or vaccine efficacy including time delays until patency (i.e. the condition of showing a detectable parasitic infection, as determined by the examples herein) may be improved by at least 10%, as 25% compared to an otherwise existing but partially effective therapeutic regimen that includes only RTS, S.
    In one embodiment, a protection rate and / or vaccine efficacy (including delays in infection) of over 80%, as well as over 90%, determined according to the examples herein is obtained.
    The circumsporozoite protein (CS) (see for example
    UniProt No. Q7K740 for the CS sequence of isolate 3D7) is present on sporozoites of all Plasmodium and is the most abundant surface protein of sporozoites. CS protein is involved in sporozoite motility and invasion during passage from the inoculation site into the circulation, from where they migrate to the liver and enter the hepatocytes.
    Many recombinant and synthetic constructs of CSs and variants have been tested for utility in malaria immunization. In one embodiment, the plasmodial CS antigen is a CS protein of Plasmodium falciparum or Plasmodium vivax or an immunogenic or variant fragment thereof.
    An appropriate variant of the CS protein may be a variant in which portions of the CS protein are in the form of a hybrid protein with the surface antigen S from the hepatitis B virus (HBs Ag). The CS variant antigen may be, for example, in the form of a hybrid protein comprising substantially the entire C-terminal portion of the CS protein, four or more tandem repeats of the immunodominant region of the CS protein, and HBs Ag. The hybrid protein may comprise a sequence that contains at least 160 amino acids and is substantially homologous to the C-terminal portion of the CS protein, but lacks the hydrophobic anchor sequence. The CS protein can be devoid of the last 12 amino acids from the C-terminus. In addition, it may contain 4 or more repeating units, for example 10 or more Asn-Ala-Asn-Pro tetrapeptide (NANP).
    The hybrid protein for use in the invention may be a protein which comprises a portion of the P. falciparum CS protein substantially corresponding to amino acids 207 to 395 of the CS protein derived from P. falciparum clone 3D7, derived from NF54 strain fused in reading frame via a linear linker at the N-terminus of HBsAg. The linker may comprise a portion of preS2 from HBsAg. CS constructs suitable for use in the present invention are disclosed in WO 93/10152, which are granted in the United States in US Pat. Nos. 5,928,902 and 6,169,171.
    A particular hybrid protein for use in the invention is the hybrid protein known as RTS (Figure 4) (SEQ ID NO: 1) (described in WO 93/10152 (where it is called RTS *) and in WO 98/05355) which consists of: - a methionine residue - three amino acid residues, Met Ala Pro - a sequence of 189 amino acids representing amino acids 207 to 395 of the CS protein of the strain 3D7 of P. falciparum - a glycine residue - four amino acid residues, Pro Val Thr Asn, representing the four carboxy-terminal residues of the hepatitis B virus preS2 protein (serotype adw), and - a sequence of 226 amino acids, encoded by nucleotides 1653 to 2330, and specifying hepatitis B virus protein S (serotype adw) RTS may be in the form of mixed particles of RTS, S. The particles of RTS, S include two polypeptides, RTS and S, which can be synthesized sim laterally and spontaneously from composite particulate structures (RTS, S).
    The RTS protein can be expressed in yeasts, for example S. cerevisiae. In such a host, RTS will be expressed and spontaneously assemble into lipoprotein particles. The recipient yeast strain may already carry in its genome several integrated copies of a Hepatitis B antigen S expression cassette. Therefore, the resulting strain synthesizes two polypeptides, S and RTS, which spontaneously assemble into mixed particles (RTS, S). These particles may have the CSP sequences of the hybrid on their surface. The RTS and S in these mixed particles may be present in a particular ratio, for example 1/4. RTS, S has been described, for example, in Vekemans et al. (2009) Vaccine 27: G67 and Regules et al. (2011) Expert Rev.
    Vaccines 10: 589.
    In another embodiment, the plasmodial CS antigen is derived from the P. vivax CS protein (i.e., CSV). Suitable variants of the P. vivax CS protein have been described. For example, WO 2008/009652, which is published in the US in US 20100150998, discloses suitable proteins for use in the present invention, for example, hybrid fusion proteins comprising: a. at least one repeating unit derived from the repetitive region of a P. vivax type I circumsporozoite protein, b. at least one repeating unit derived from the repetitive region of a P. vivax circumsporozoite type II protein, and c. surface antigen S derived from hepatitis B virus, or one of its fragments. SEQ ID NO: 17 of WO 2008/009652 discloses a specific hybrid fusion protein, named CVS-S. When coexpressed with hepatitis B virus-derived surface antigen S, P. vivax CSV-S, S particles, analogous to RTS, S particles of P. falciparum, are formed (WO 2008/009652). Such particles may also be used in the present invention.
    In another embodiment, the plasmodial CS antigen is a mixed particle comprising RTS and CSV-S, and optionally the unfused Hepatitis B antigen S. Such particles have been described in WO 2008/009650, which is published in the US in US 20100062028.
    Therefore, in one embodiment of the method or use of the invention, the plasmodial CS antigen is one or more of those selected from the group consisting of: a. RTS, b. CSV-S, c. RTS, S 'd. CSV-S, S and e. mixed particles comprising RTS and CSV-S, and optionally the unfused Hepatitis B virus S antigen. The plasmodial CS antigen is administered in an amount sufficient to generate an immune response in the human subject. In a preferred embodiment, the plasmodial CS antigen is RTS, S and the amount of RTS, S is between 25 and 75, as 50, micrograms per dose or between 12.5 and 37.5, as 25, micrograms. per dose or between 5 and 20, as 10 μg per dose.
    In a preferred embodiment, the plasmodial CS antigen is administered in combination with an adjuvant.
    In one embodiment, the adjuvant comprises a TLR agonist (Toll-type receptor). The use of TLR agonists in adjuvants is well known in the state of the art and has been described, for example, by Lahiri et al. (2008) Vaccine 26: 6777. TLRs that can be stimulated to achieve adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9. TLR2, TLR4, TLR7 and TLR8 agonists, particularly TLR4 agonists, are preferred.
    Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3-O-deacylated monophosphoryl lipid A (3D-MPL). U.S. Patent 4,436,727 discloses an MPL and its manufacture. U.S. Patent 4,912,094 and Reexamination Certificate B1,4912,094 disclose 3D-MPL and a method for its manufacture. Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a synthetic lipid A molecule (see, e.g., Fox et al (2012) Clin Immunol Vaccine 19: 1633). In another embodiment, the TLR4 agonist may be a TLR4 synthesis agonist such as a synthetic disaccharide molecule, structurally similar to MPL and 3D-MPL or it may be synthetic monosaccharide molecules. , such as aminoalkylglucosaminide phosphate (AGP) compounds disclosed, for example, in WO 9850399, WO0134617, WO0212258, WO3065806, WO04062599, WO06016997, WO0612425, WO03066065, and WO0190129. such molecules have been described in the scientific and patent literature as lipid A mimetics. The lipid A mimetics appropriately share some functional and / or structural activity with lipid A, and in one aspect they are recognized by TLR4 receptors. AGPs as described herein are sometimes referred to as lipid A mimetics in the state of the art. In a preferred embodiment, the TLR4 agonist is 3D-MPL. TLR4 agonists, such as 3-O-deacylated monophosphoryl lipid A (3D-MPL), and their use as adjuvants in vaccines have been described, for example, in WO 96/33739 and WO 2007/068907 and described by Alving et al. (2012) Curr Opin in Immunol 24: 310.
    In another embodiment, the adjuvant comprises an immunologically active saponin, such as QS21. Adjuvants comprising saponins have been described in the state of the art. Saponins are described in: Lacaille-Dubois and Wagner (1996) A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2: 363. Saponins are known as adjuvants in vaccines. For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), has been described by Dalsgaard et al. in 1974 ("Saponin Adjuvants", Archiv für die gesamte Virusforschung, Vol 44, Springer Verlag, Berlin, 243) as having an adjuvant activity. HPLC-purified fractions of Quil A that retain adjuvant activity have been isolated by HPLC without quil A-associated toxicity (Kensil (1991) J. Immunol 146: 431. Quil A moieties are also described in US Pat. U.S. Patent 5,057,540 and "Saponins as Vaccinia Adjuvants", Kensil, CR, Crit Rev. Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55.
    Two of these moieties, suitable for use in the present invention, are QS7 and QS21 (also known as QA-7 and QA-21). QS21 is a preferred immunologically active saponin moiety for use in the present invention. QS21 has been described by Kensil (2000) in O'Hagan: Vaccine Adjuvants: Preparation Methods and Research Protocol. Homana Press, Totowa, NJ, Chapter 15. Particulate adjuvant systems comprising Quil A moieties, such as QS21 and QS7, are described, for example, in WO 96/33739, WO 96/11711 and WO 2007. / 068907.
    In addition to the saponin component, the adjuvant preferably comprises a sterol. The presence of a sterol can further reduce the reactogenicity of compositions comprising saponins, see, for example, EP 0822831. Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. Cholesterol is particularly appropriate. Suitably, the immunologically active saponin fraction is QS21 and the ratio QS21 / sterol is 1/100 to 1/1 w / w, such as 1/10 to 1/1 w / w, for example 1/5 at 1/1 p / p.
    In a preferred embodiment of the invention, the adjuvant comprises 3D-MPL and QS21.
    In some embodiments, the adjuvant is in the form of an oil-in-water emulsion, for example, comprising squalene, alpha-tocopherol and a surfactant (see, for example, WO 95 / 17210) or in the form of a liposome. A liposomal presentation is preferred. Thus, in a preferred embodiment of the invention, the adjuvant comprises 3D-MPL and QS21 in a liposomal formulation.
    The term "liposome", when used herein, refers to unilamellar or multilamellar lipid structures enclosing an aqueous interior. Liposomes and liposomal formulations are well known in the state of the art. Liposomal presentations are described, for example, in WO 96/33739 and WO 2007/068907. Lipids that are capable of forming liposomes include all substances with fatty acid or fatty acid properties.
    The size of the liposomes can vary from 30 nm to several μm depending on the composition of phospholipids and the process used for their preparation. In particular embodiments of the invention, the size of the liposomes will be in the range of 50 nm to 500 nm and in other embodiments from 50 nm to 200 nm. Dynamic scattering of laser light is a method used to measure the size of liposomes well known to those skilled in the art.
    In a particularly suitable embodiment, the liposomes used in the invention comprise DOPC and a sterol, especially cholesterol. Thus, in a particular embodiment, the compositions of the invention comprise QS21 in any amount herein described in the form of a liposome, wherein said liposome comprises DOPC and a sterol, especially cholesterol.
    In one embodiment, the adjuvant comprises between 25 and 75, as 50 micrograms, of 3D-MPL per dose and between 25 and 75, as 50 micrograms of QS21 per dose. In another embodiment, the adjuvant comprises between 12.5 and 37.5, as 25 micrograms, of 3D-MPL per dose and between 12.5 and 37.5, as 25 micrograms of QS21 per dose. In another embodiment, the adjuvant comprises between 5 and 20, as 10 micrograms, of 3D-MPL per dose and between 5 and 20, as 10 micrograms of QS21 per dose.
    It is well known that for parenteral administration the solutions will have to be physiologically isotonic (i.e., they will have to have a pharmaceutically acceptable osmolality) to avoid cell deformation or lysis. An "isotonicity agent" is a compound that is physiologically tolerated and that imparts appropriate tonicity to a formulation (eg, immunogenic compositions of the invention) to prevent the net flow of water through cell membranes that are in contact with the formulation. Aqueous adjuvant compositions are known which contain 100 mM or more of sodium chloride, for example adjuvant system A (ASA) in WO 2005/112991 and WO 2008/142133 or liposomal adjuvants disclosed in WO 2007/068907.
    In some embodiments, the isotonicity agent used for the composition is a salt. However, in other embodiments, the composition comprises a nonionic isotonicity agent and the concentration of sodium chloride or the ionic strength in the composition is less than 100 mM, as less than 80 mM, for example less than 30 mM, as less than 10 mM or less than 5 mM. In a preferred embodiment, the nonionic isotonicity agent is a polyol, such as sorbitol. The concentration of sorbitol may range, for example, from about 3% to about 15% (w / v), such as from about 4% to about 10% (w / v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist, wherein the isotonicity agent is a salt or a polyol have been described in WO 2010/142685, see, for example, Examples 1 and 2 in WO 2010/142685.
    As described above, the methods and uses of the invention include administering a nucleotide vector encoding a thrombospondin-related adherence protein (TRAP) antigen, which is a TRAP protein or one of its immunogenic fragments or variants.
    In one embodiment of the method and / or the use of the invention, the plasmodial TRAP antigen is a Plasmodium falciparum TRAP antigen or Plasmodium vivax or an immunogenic or variant fragment thereof (see, for example Robson et al (1988) Nature 335: 79 for the TRAP sequence from the T9 / 96 isolate or the UniProt ID number Q94727 for the P. vivax TRAP). In another embodiment, the plasmodial TRAP antigen is ME-TRAP, a TRAP fusion protein comprising a sequence of several epitopes containing additional B cell epitopes, and CD8 + and CD4 + T cells from several malarial antigens. (SEQ ID NO: 2) (Figure 5) (Gilbert et al., 1997 Nat Biotechnol 15: 1280, Moorthy et al., 2003 Vaccine 21: 1995, WO 2008/122769). Figure 6 shows a vector insert encoding ME-TRAP (SEQ ID NO: 3).
    In one embodiment, the nucleotide vector encoding the plasmodial TRAP antigen is a simian adenovirus vector defective for replication. For example, a simian adenovirus vector which comprises a simian adenovirus genome in which it is stably integrated a transgene that encodes a plasmodial TRAP antigen operably linked to regulatory sequences that direct expression of the Plasmodial TRAP antigen in mammalian cells. In one embodiment, the simian adenoviral genome is the genome of a chimpanzee adenovirus vector. In another embodiment, the regulatory sequences that direct the expression of the transgene include a CMV promoter. For example, the regulatory sequences may include the HCMV IE1 gene promoter and a fragment of the 5 'untranslated region of the HCMV IE1 gene including intron A.
    In another embodiment, the simian adenovirus vector is ChAd63 which is derived from chimpanzee adenovirus isolate 63 (deposited with ECACC under accession number 05011210) (SEQ ID NO: 115 in US Patent Application 2011/0217332) or ChAd3, which is derived from Chimpanzee adenovirus isolate 3 (SEQ ID NO: 1 in WO 2005/071093) or ChAdOx1 (Antrobus et al., Molecular Therapy 22: 668-674, 2014; Dicks et al., PLoS One 2012 7 | e40385; WO 2012/172277). WO 2008/122769 discloses recombinant simian adenovirus vectors defective for replication encoding TRAP or variants thereof, including ChAd63 encoding the ME-TRAP antigen.
    The nucleotide vector encoding the plasmodial TRAP antigen is administered in an amount sufficient to generate an immune response in the human subject. In one embodiment, between 1 x 1010 and 1 x 1011, as 5 x 1010 viral particles are administered per dose.
    In some embodiments, the method or use of the invention comprises two administrations of a nucleotide vector encoding a plasmodial TRAP antigen. In such embodiments, the second (boost) administration may activate the immune response induced by the first administration (sensitization). In a preferred embodiment, the method or use comprises: i) administering an adenoviral vector that encodes a plasmodial TRAP antigen as described above, and ii) administering a non-vector. adenoviral which encodes a plasmodium TRAP antigen, which is a TRAP protein or one of its immunogenic or variant fragments.
    In one embodiment, said non-adenoviral vector is a recombinant poxvirus vector, such as vaccinia virus Ankara (MVA). MVA is a highly attenuated strain of vaccinia virus that has undergone multiple spontaneous, fully characterized deletions during more than 570 subcultures in chicken embryo fibroblast (CEF) cells. These included genes from the host range and genes encoding cytokine receptors. . The virus is unable to replicate effectively in humans and most cells of other mammals, but the abnormality of replication occurs at a late stage of virion assembly so that the expression of viral genes and recombinants is unchanged making MVA an effective expression vector in a single series unable to cause disseminated infection in mammals. The entire DNA sequence of MVA has been published (Antoine et al (1998) Virology 244: 365).
    In another embodiment, the adenoviral vector and the non-adenoviral vector both encode ME-TRAP. WO 2008/1227 69 discloses an MVA vector encoding ME-TRAP. In one embodiment, the non-adenoviral vector is MVA and between 1x108 and 1x109, such that 2x108 pfu is administered per dose.
    In some embodiments, the non-adenoviral vector encoding a plasmodial TRAP antigen, such as MVA encoding ME-TRAP is administered more than once, such as 2 or 3 times or more. Immunization regimes
    In some embodiments, the method of the invention or the use of the invention comprises two or more administrations, such as three, of the plasmodial CS antigen.
    In a preferred embodiment, wherein the method or use comprises three administrations of the plasmodial CS antigen, wherein the plasmodial CS antigen is the same in all three administrations and the plasmodial CS antigen is adjuvanted in the three administrations with an adjuvant comprising 3D-MPL and QS21 in a liposomal formulation. Most preferably, the plasmodial CS antigen is RTS, S.
    In one embodiment, the time interval between each administration of the plasmodial CS antigen is between 1 week and 1 year, for example between 2 weeks and 6 months, such as between 2 weeks and 6 weeks, for example 4 weeks. weeks. A three-dose accelerated regimen could be administration of the plasmodial CS antigen with 5- to 9-day intervals, such as administration at day 0, day 7, and day 14.
    In some embodiments, the dose of plasmodial CS antigen and the adjuvant dose are kept constant in all administrations.
    In other embodiments, however: a. the amount of plasmodial CS antigen is lower in the second administration, and / or one of the subsequent administrations, compared to the amount of plasmodial CS antigen in the first administration and / or b. the amount of adjuvant is lower in the second administration, and / or one of the subsequent administrations, compared to the amount of adjuvant in the first administration.
    For example, in one such embodiment, two or more administrations of the plasmodial CS antigen, such as RTS, S, are combined with an adjuvant comprising 3D-MPL and QS21 in a liposomal formulation, but the Adjuvant amount is lower in the second administration, or one of the subsequent administrations, compared to the amount of adjuvant in the first administration. For example, the lower amount is at least 10% lower, such as at least 25% lower, for example at least two times lower, as at least three times lower, for example at least four times lower, as at least five times lower, for example at least six times lower, as at least seven times lower, for example at least eight times lower, as at least nine times lower, for example at least ten times lower, as at least 15 times lower, by example at least 20 times lower.
    In another embodiment, the lower amount of adjuvant is between 2 and 50 times lower, as between 2 and 20 times lower, for example between 2 and 15 times lower, as between 2 and 10 times lower, by example between 3 and 7 times lower, as between 4 and 6 times lower.
    In one embodiment, the first administration comprises between 25 and 75, as 50 micrograms, of 3D-MPL and between 25 and 75, as 50 micrograms of QS21 in a liposomal formulation and one or more of the subsequent administrations comprise between 5 and 15 as 10 micrograms of 3D-MPL and between 5 and 15, as 10 micrograms of QS21 in a liposomal formulation.
    In another embodiment, the first administration comprises between 12.5 and 37.5, as 25 micrograms, of 3D-MPL and between 12.5 and 37.5, as 25 micrograms of QS21 in a liposomal formulation and one or several of the subsequent administrations comprise between 2.5 and 7.5, such as 5 micrograms of 3D-MPL and between 2.5 and 7.5, as 5 micrograms of QS21 in a liposomal formulation.
    In other embodiments, the amount of plasmodial CS antigen is lower in the second, or one of the subsequent administrations, compared to the amount of plasmodial CS antigen in the first administration. For example, the lower amount is at least 10% lower, such as at least 25% lower, for example at least two times lower, as at least three times lower, for example at least four times lower, as at least five times lower, for example at least six times lower, as at least seven times lower, for example at least eight times lower, as at least nine times lower, for example at least ten times lower, as at least 15 times lower, by example at least 20 times lower.
    In another embodiment, the lower amount is between 2 and 50 times lower, as between 2 and 20 times lower, for example between 2 and 15 times lower, as between 2 and 10 times lower, for example between 3 and 7 times lower, as between 4 and 6 times lower.
    In embodiments comprising administering an adenoviral vector that encodes a plasmodial TRAP antigen as described above, followed by administration of a non-adenoviral vector that encodes a plasmodial TRAP antigen, wherein TRAP plasmodial antigen is a TRAP protein or one of its immunogenic fragments or variants, the non-adenoviral vector can be administered, for example, at least two weeks, for example between 2 and 12 weeks, after the adenoviral vector, like 8 weeks after the adenoviral vector.
    In one embodiment, the method or use of the invention comprises the following administrations, possibly in the specified order: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation,
    b. Administration of ChAd63 coding for ME-TRAP c. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation d. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation e. Administration of MVA coding for ME-TRAP.
    In another embodiment here, the time interval between steps a. and B. is between 1 and 3 weeks, like 2 weeks, and in which the time interval between steps b. and c. is between 1 and 3 weeks, like 2 weeks, and in which the time interval between steps c. and D. is between 1 and 8 weeks, like 4 weeks, and in which the time interval between steps d. summer. is between 1 and 3 weeks, like 2 weeks.
    In other embodiments, administration of one or all of the TRAP coding vectors is carried out simultaneously, or substantially simultaneously, with administration of a plasmodial CS antigen. In this context, "essentially simultaneously" means an administration during the same visit to the clinic, that is to say the same day, as at 1 hour from each other, for example at 20 minutes, like 5 minutes from each other.
    In particular embodiments, the TRAP coding vectors are coadministered both substantially simultaneously and at the same site of the body as the plasmodial CS antigen (e.g., in one arm).
    In alternative embodiments, the invention specifically excludes the administration of any or all of the TRAP-encoding vectors simultaneously, or substantially simultaneously, with the administration of the plasmodial CS antigen. Again, in this context of exclusion, "essentially simultaneously" means an administration during the same visit to the clinic, that is to say, the same day, as 1 hour apart.
    In particular embodiments, the invention specifically excludes the coadministration of the TRAP encoding vector (s) and the plasmodial CS antigen at the same site on the body (e.g., in one arm) and essentially at the same time. . Thus, in such embodiments, administrations are performed at distinct sites of the body. Administration to a "distinct body site" when used herein in the context of administering two or more compositions to a subject means that the compositions are given at different sites, typically contralateral way or at different limbs or in all cases at a sufficient distance from each other so that any local effects of administrations do not interfere with each other. For example, the distance may be at least 0.5 cm, such as at least 1 cm, for example at least 5 cm.
    Thus, in some embodiments of the method and / or the use of the invention, the administration of one or all of the TRAP coding vectors is not performed substantially concurrently with the administration of a plasmodial CS antigen.
    In other embodiments, administration of one or all of the TRAP coding vectors is performed at a site distinct from the body as compared to those used for administration of the plasmodial CS antigen. .
    In other embodiments, any nucleotide vector encoding the plasmodial TRAP antigen is administered at a site distinct from the body, or given 1 day or more apart, from any administration of a plasmodial CS antigen. , as at more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, as 14 days or more apart.
    In other embodiments, if the method comprises two or more administrations of nucleotide vectors encoding a plasmodial TRAP antigen, the second and the other administrations are given at a site distinct from the body, or given at 1 day or more, any administration of a plasmodial CS antigen, as at more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, such as 14 days or more gap.
    In still other embodiments, if the method comprises administration with a non-adenoviral vector encoding a plasmodial TRAP antigen, said administration is given at a site distinct from the body, or given at 1 day or more of deviation, from any administration of a plasmodial CS antigen, as at more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, as 14 days or more apart .
    In still other embodiments, if the method comprises administration with an MVA vector encoding a plasmodial TRAP antigen, said administration is given at a site distinct from the body, or given at 1 day or more of deviation, from any administration of a plasmodial CS antigen, as at more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, such as 14 days or more apart .
    In one embodiment, the method or use of the invention comprises the following administrations, possibly in the specified order: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or substantially simultaneously, with
    administration of ChAd63 coding for ME-TRAP b. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or substantially simultaneously, with the administration of MVA coding for ME-TRAP, and c. Administration of RTS, S adjuvated with 3D-MPL and QS21 in a liposomal formulation simultaneously, or substantially simultaneously, with the administration of MVA encoding ME-TRAP.
    In another embodiment, the method or the use of. the invention comprises the following administrations, possibly in the specified order: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or substantially simultaneously, with
    administration of ChAd63 coding for ME-TRAP b. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation simultaneously, or substantially simultaneously, with the administration of MVA encoding ME-TRAP, and c. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation.
    In another embodiment, the method or the use of the invention comprises the following administrations, possibly in the specified order: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or substantially simultaneously, with
    administration of ChAd63 coding for ME-TRAP b. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, and c. Administration of RTS, S adjuvated with 3D-MPL and QS21 in a liposomal formulation simultaneously, or substantially simultaneously, with the administration of MVA encoding ME-TRAP.
    In one embodiment, the method or use of the invention comprises the following administrations, possibly in the specified order: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, simultaneously, or substantially simultaneously, with
    administration of ChAd63 coding for ME-TRAP b. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, and c. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation.
    In some embodiments, one or more vectors encoding TRAP are mixed with the plasmodial CS antigen prior to administration. '
    For example, the RTS, S and ME-TRAP coding vector (such as ChAd63 encoding ME-TRAP) can be provided together in a vial in lyophilized form and reconstituted with the adjuvant solution prior to administration. . Alternatively, the components of the composition may be provided in a liquid formulation and mixed prior to administration.
    Thus, in another aspect, the invention relates to a composition, such as a lyophilized or liquid composition, comprising: i) a plasmodial circumsporozoite (CS) antigen, which is a CS protein or an immunogenic or variant fragment thereof; and ii) a nucleotide vector encoding a plasmodial thrombospondin-related adherence protein (TRAP) antigen, which is a TRAP protein or an immunogenic or variant fragment thereof.
    In one embodiment, the CS antigen is the RTS, S, and the nucleotide vector encoding a plasmodial TRAP antigen is ChAd63 encoding ME-TRAP.
    The human subject to be treated using the method of the invention may be of any age. The method of the invention may be used in the context of an elimination program for malaria, in which case immunization essentially of the entire population, i.e. all age groups. , may be useful. However, in one embodiment, the human subject is over the age of 18 when the first composition is administered. In another embodiment, the human subject is less than five years old when the first composition is administered. In another embodiment, the subject is 6 to 12 weeks old or younger or 5 to 17 months old. Another particularly appropriate target population includes travelers to areas where malaria is endemic.
    The antigens can be administered by a variety of appropriate routes, including parenteral, intramuscular, intradermal, or subcutaneous administration.
    In another aspect, the invention relates to a kit comprising: i) a first container, such as a vial, comprising a circumsporozoite (CS) plasmodial antigen, which is a CS protein or one of its immunogenic or variant fragments and. ii) a second container, such as a vial, comprising a nucleotide vector encoding a plasmodial thrombospondin-related adherence protein (TRAP) antigen, which is a TRAP protein or an immunogenic or variant fragment thereof, and iii ) optionally, a third container separated with the adjuvant for the administration of the CS antigen and / or instructions for the use of the kit in the immunization of human subjects against malaria according to any of the methods described herein .
    The immunogenic compositions used in the invention can be made by mixing antigen or antigens and adjuvant. The antigen or antigens can be provided in a lyophilized form or in a liquid formulation. For each composition, there may be provided a kit comprising a first container comprising the antigen or antigens and a second container comprising the adjuvant. In another embodiment, both components are provided in a single formulation.
    Suitably, the immunogenic compositions according to the present invention have a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.5 ml, in particular a dose volume of about 0.5. ml, or 0.7 ml. The volume of the second immunogenic composition may be reduced, for example, from 0.05 ml to 0.5 ml, such as from 0.1 to 0.2 ml. The volumes of the compositions used may depend on the route of administration with smaller doses being given by the intradermal route.
    The teachings of all the references in this application, including the patent applications and the granted patents, are incorporated herein by reference in their entirety. The terms "comprising", "include" and "includes" herein are optionally substituted by the terms "consisting of", "consist of" and "consists of", respectively. The invention will be further described with reference to the following non-limiting examples.
    Example 1 - A sporozoite challenge phase I / IIa study to estimate the safety and protective efficacy of the combined RTS, S / AS01B + ChAd63 and MVA combination vaccine candidate vaccine regimen ME-TRAP and also RTS, S / AS01B alone
    A Phase I / IIa clinical trial was conducted in which the safety, immunogenicity, and protective efficacy of two candidate immunization regimens were tested.
    The first immunization regimen (group 1) consisted of 3 administrations of RTS, S / AS01B, a ChAd63 administration coding for ME-TRAP and a MVA administration coding for ME-TRAP. The second immunization regimen (group 2) consisted of 3 administrations of RTS, S / AS01B alone. In addition, unvaccinated controls (group 3) were included. The study was an open-label, partially randomized, multi-center Phase I / IIa study of a controlled human malaria infection (CHMI). The study population consisted of healthy adults aged 18 to 45 years old. Group size: Groups 1, 2 and 3 contained 17, 16 and 6 volunteers at the time of challenge, respectively.
    Vaccines from the RTS study, S / AS01B: RTS, S was produced in yeast (S. cerevisiae) essentially as described in WO 93/10152. Liposomal adjuvant 3D-MPL / QS21 (AS01) was produced essentially as described in Example II in WO 2007/068907. 50 micrograms (mcg) of RTS, S with sucrose as a cryoprotectant, presented as a freeze-dried pellet in a single dose vial was reconstituted with AS01B adjuvant in liquid form. AS01B adjuvant contained 50 mcg of MPL and 50 mcg of QS21 with liposomes. The volume for injection after reconstitution was 0.5 ml. Each dose of RTS, S / AS01B was given intramuscularly.
    ChAd63 ME-TRAP was manufactured as previously described (O'Hara et al., 2012 J Infect Dis 205: 772, Ewer et al., 2013 Nat Commun 4: 2836. doi: 10.1038 / ncomms3836). ChAd63 ME-TRAP was given intramuscularly at a dose of 5 x 1010 pv. MVA ME-TRAP was manufactured as previously described (McConkey et al., 2003 Nat Med 9: 729, O'Hara et al., 2012 J Infect Dis 205: 772, Ewer et al., 2013 Nat Commun 4: 2836 doi: 10.1038 / ncomms3836). MVA ME-TRAP was given intramuscularly at a dose of 2 x 108 pfu. Vaccination regimes
    The vaccination regimens that have been used are shown in the following table:
    CHMI
    CHMI by stimulation ("challenge") with sporozoites (mosquito bites) was performed at week 12. CHMI was performed as described in Ewer et al. 2013 Nat Commun 4: 2836. doi: 10.1038 / ncomms3836. After CHMI, twice daily and daily visits were scheduled to allow time measurement to a criterion of efficacy of 20 P. falciparum parasites / ml in peripheral blood by qPCR (performed as it is described in Andrews et al., 2005 Am J Trop Med Hyg 73: 191), 500 parasites / ml in peripheral blood by qPCR, and a blood stage infection defined by a set of symptoms, the result of smear and parasitaemia. .
    immunogenicity
    T-cell responses to ME-TRAP were measured by ELISpot and flow cytometric assays as described in O'Hara et al. 2012 J Infect Dis 205: 772 and Ewer et al. 2013 Nat Commun 4: 2836. doi: 10.1038 / ncomms3836. Antibody responses to CS were measured by ELISA as described in Rester et al. J Infect Dis 2009. Results
    Figure la represents the percentage of subjects remaining aparasitmic by blood smear and PCR over time following a challenge with sporozoites.
    Protection - sterilization efficiency
    Subjects who remained parasitic by blood smear and PCR after 20 days were considered protected.
    The results for the different groups were as follows:
    Group 1: 14 out of 17 volunteers were protected = 82.4%
    Group 2: 12 volunteers out of 16 were protected = 75% Group 3: 0 volunteers out of 6 were protected = 0%
    Statistical significance:
    Group 1 vs Group 2 against witnesses (log-rank) P = 1 x 10 "8
    Group 1 against controls: relative risk (log-rank) = 0.065 [0.003-0.19], P <10.7
    Group 2 against controls: relative risk (log-rank) = 0.122 [0.004-0.13], P <10-3
    Group 1 vs group 2: relative risk (log-rank) = 0.65 [0.15 -2.86], P = 0.57
    Efficacy of the vaccine (protection + significant delays over time until the occurrence of the malaria infection)
    In the control group (group 3), the 6 subjects became positive by microscopic examination of blood smear slides at day 11 (1 subject), 11.5 (1 subject), 12 (1 subject), 12.5 (1 subject ) and 13 (2 subjects), respectively. This gives an average of 12.17 +/- 0.8 days (standard deviation, SD). Vaccine recipients who are delayed more than the average plus two standard deviations from the control group are considered "delayed in time until patency" and show partial efficacy of the vaccine. In this trial, it would be a delay of more than 12.17 plus days (2 x 0.8) = 13.77 days.
    In group 1, the 3 subjects who became positive before day 21 became positive at day 14 (1 subject), 14.5 (1 subject) and 16 (1 subject). Thus, in the 3 subjects, there was a significant delay of this average (14.83 days) compared to the average of the group 3 (12.17 days) of 2.66 days. This delay of 2.66 days is more than 2 times the standard deviation of the control group (0.8 days) and therefore is considered statistically significant. The three subjects were delayed beyond day 13,77.
    In group 2, the 4 subjects who became positive before day 21 became positive at day 11.5 (1 subject), 12.5 (1 subject), 14 (1 subject) and 19 (1 subject). Thus, in 2 subjects, there was a significant delay compared to the group 3 average because 2 subjects were delayed beyond day 13,77.
    Thus, if subjects in whom a significant delay of infection is observed, are added to the sterile protected subjects, figure la, the vaccinal efficiencies become the following ones:
    Group 1: 17/17 have an efficiency = 100%
    Group 2: 14/16 exhibit efficacy = 87.5%
    Group 3: 0/6 has an efficiency = 0% as expected
    Statistical significance:
    Group 1 vs Group 2 against witnesses (chi-square): P <10-6
    Group 1 vs. Group 2 (chi-square): P = 0.066 (ie 0/17 vs. 2/16). A one-sided P-value is used because the previous hypothesis tested was that group 1 would be better protected than group 2.
    immunogenicity
    The most relevant relevant mechanism for the protective efficacy of the RTS, S vaccine is the induction of high titre antibodies. They were induced to a very similar level in groups 1 and 2 (Figure 1b) and these rates appeared similar to those in previous RTS trials, S / AS01 in the United States (Rester et al., 2008). Vaccine 26, 2191-2202, Rester et al (2009) J. Infect Dis 200, 337-346). Antibodies to TRAP were correlated with the immunogenicity of TRAP from T-cells in group 1 (Figure 2a); but the much lower level of T-cells induced against CS was not correlated with antibodies to CS
    (Figure 2b). Levels of antibodies to CS in individual vaccine recipients were not correlated with TRAP levels in group 1 (Figure 2c). Antibodies to TRAP in group 1 were found at a similar rate to those in a previous vaccine trial (Vac045, Hodgson et al., Submitted) using ME-TRAP in the same vectors used alone (Figure 2d). , suggesting no interference with the immunogenicity of TRAP according to the antibodies from the administration of RTS, S to the same individuals. Antibodies to CS were positively correlated with the three measures of vaccine efficacy (Figure 2e, 2f, 2g), ie at the time of diagnosis (Figure 2e), the time to 20 parasites per ml measured by PCR (Figure 2f) and the time up to 500 parasites per ml measured by PCR (Figure 2g). This supports a protective role of the high titre antibodies directed against CS induced by these vaccination regimens, consistent with previous studies. TRAP T cells measured by ELISPOT the day before challenge ("challenge") showed an average level of 1372 cells per million in group 1 vaccine recipients but were not correlated with efficiency (not shown), although the possibility of detecting such a correlation is very low.
    Main conclusion
    The highest percentage of sterile protection (82.4%) was obtained in group 1. To our knowledge, protection at 82.4% is the highest level of protection ever detected in any test group on a vaccine with a sample size> 10 in any study with malaria challenge.
    When delayed infections were included in vaccine efficacy, efficacy in group 1 increased to 100%. To our knowledge, this is the first vaccine group showing 100% efficacy in any challenge study with a group size of 10 or more.
    Data of the new stimulation ("re-challenge")
    The ability to provide sustained and sustained efficacy is a key feature of immunization. This may be a limiting characteristic of the overall effectiveness of a vaccine. For example, in young infants in the RTS phase III trial, S / AS01, the reduction in clinical malaria incidence per 6-month period was 47% (95% CI 39% to 54%). %) in months 1 to 6, 23% (95% CI 15% to 31%) in months 7 to 12, and 12% (95% CI 1% to 21%) in months 13 to 18 as a result of dose 3. This may be related to the reduced effectiveness of the same vaccine when used in US challenge studies in adults. The effectiveness of the vaccine in the first month after vaccination (usually at 3 weeks after the last dose) was 50% in the Rester et al. 2009. But during a new stimulation ("re-challenge") at 6 months, only 4 out of 9 (44%) were protected again. This can be calculated to represent approximately 22% overall sterilization efficacy at six months (50% x 44% = 22%). An earlier study with new stimulation ("re-challenge") with RTSS / AS02 (Stoute et al 1997 N Engl J Med 336 (2): 86, Stoute et al 1998 J Infect Dis 178 (4): 1139) found overall effectiveness at 6 months of 17%. Similarly, in a recent phase II study of a Sanaria-irradiated sporozoite vaccine, the efficacy of the vaccine in the first month seems high with 12/15 recipients of the vaccine showing no infection during stimulation ("challenge"). (Seder et al., 2013, Science 341, 1359-1365). Because of the failure of the infection in a provocation control, this represented a global estimate of the overall sterilization efficacy rate of 76% (Seder et al 2013 Science 341, 13591365, see legend of Figure 2). However, during the new stimulation ("re-challenge") of six of these protected individuals at 5 months, only 2 were again protected (Richie et al., Presented at the meeting ICOPA, Mexico City, August 2Q14) representing a overall sterilization efficacy rate of (76% x 33% = 25%) 25%.
    Of the 14 protected volunteers in Group 1, 8 were re-challenged. Of the 12 recipients of the protected group 2 vaccine, 6 were re-challenged. The new challenge ("re-challenge") followed the standard five-bite protocol (Ewer et al., Nat Communications 20134: 2836. doi: 10.1038 / ncomms3836) with P. falciparum and was initiated six months after the last vaccination. accompanied by five new volunteer witnesses.
    All control volunteers developed malaria with a mean time to patency of 12.4 days (Figure 3). Individual volunteers were diagnosed at days 11.5, 11.5, 12.5, 13, 13.5, respectively (standard deviation = 0.76 days). For group 1, 7 out of 8 vaccine recipients were protected (87.5%) and the remaining volunteer was diagnosed at day 17.5. For group 2, 5/6 were protected (83.3%) and the remaining volunteer was diagnosed at day 14.5. The sterilization efficacy of the global six-month vaccine can thus be calculated for group 1 as being 82.4% x 87.5% = 72.1%. The sterilization efficacy of the overall six-month vaccine can be calculated for group 2 as 75% x 83.3% = 62.5%.
    Comparison of sustainable protection rates
    To determine whether these improved rates of sustainable efficacy are significantly better than those previously reported, we compared group 1 and group 2 data with those reported by Rester et al. 2009 using the RTS, S / AS01 in an earlier test.
    Group 2 vs AS01 at Rester et al. 2009
    Group 2: -5 / (6x16 / 12) = 5/8 originally protected vaccine recipients AS01 Rester et al. : -4 / (9 x 36/18) = 4/18 originally protected vaccine recipients
    Chi square test (http://www.openepi.com), exact value half-P, bilateral, P = 0.07
    Group 1 versus AS01 at Rester et al. 2009
    Group 1: -7 / (8 x 17/14) = 7 / 9,71 recipients of protected vaccine AS01 Rester et al. : -4 / (9 x 36/18) = 4/18 originally protected vaccine recipients
    Chi square test, exact value mid-P, bilateral, P <0.02
    This analysis indicated that the RTS group, S / AS01 alone, was not significantly different from the use of the same vaccine regimen by Rester et al. However, group 1 is significantly improved compared to RTS, S / AS01 used by Rester et al. (2009).
    We conclude that the 6-month sterile protection rate for group 1 is the highest ever reported at 72% and this appears to be a very valuable improvement over RTS, S alone. We also note that at both the first challenge and the new challenge, each group 1 volunteer showed a significant efficacy of the vaccine: 17/17 at the first stimulation ( "Challenge") and 8/8 to the new stimulation ("rechallenge"). Again, to our knowledge, this sustainable efficacy rate is an unprecedented result in 30 years of Phase II trials of malaria vaccines.
    Example 2 - Safety, immunogenicity and efficacy of the combined RTS, S / AS01 malaria vaccine regimen administered simultaneously with ChAd-MVA viral vectors expressing ME-TRAP
    In another study, simultaneous administration of ChAd63 ME-TRAP and MVA ME-TRAP with RTS, S / AS01 was tested. The study was an open, partially randomized, multicentre, phase I / IIa study on a controlled human malaria infection (CHMI). The study population consisted of naïve healthy adults with malaria aged 18 to 45 years. Group size: Groups 1, 2, 3, 4 and 5 contained 8, 9, 10, 9 and 4 volunteers at the time of challenge, respectively.
    Vaccines from the study
    The vaccines in the study were the same as in Example 1 except that the third dose of RTS, S / AS01 given to two of the groups was 1 / 5th of the standard dose. The vaccines were administered intramuscularly in the deltoid muscle of the left arm. When two vaccines were administered simultaneously, they were carried out essentially at the same location and one administration immediately followed the other. Vaccination regimes
    The vaccination regimens that have been used are shown in the following table:
    CHMI and immunogenicity test
    CHMI and immunogenicity tests were performed as described in Example 1. Results
    participants
    Ten subjects were enrolled in group 1, group 2 and group 4. Eleven subjects were enrolled in group 3. A total of 5 subjects withdrew from the study after enrollment, but before stimulation ( "Challenge") (2 subjects in group 1, and 1 subject for each of groups 2, 3 and 4).
    Efficiency
    The numbers of subjects with sterile protection in each group are summarized below.
    Sterile protection at day 16/17:
    Group 1: 6/8 subjects (75%)
    Group 2: 8/9 subjects (88.9%)
    Group 3: 6/10 subjects (60%)
    Group 4: 5/9 subjects (55.6%),
    Groups 1 and 2 grouped: 14/17 (82.4%)
    Groups 3 and 4 grouped: 11/19 (57.9%)
    Group 5, mean time to diagnosis of malaria (+/- SD) = 11.63 days (+/- 0.22)
    Comparison of groups:
    The data show that, unlike the increase in efficiency observed in Example 1, the simultaneous administration of ChAd63 ME-TRAP and MVA ME-TRAP with RTS, S / AS01 did not result in an increase in compared to RTS, S / AS01 alone.
    Vaccine safety
    No safety concerns were raised during the trial. None of the defined rules for stopping or stopping the protocol has been enabled. A total of 2 serious adverse events (SIEs) occurred, but neither was associated with vaccination. No unexpected serious suspected adverse reactions (RISIS) have occurred. A higher frequency of adverse events (EI) was reported in groups 3 and 4, and the highest frequency of AEs in all groups was reported following the 2nd series of vaccinations. The frequency of secondary events in groups 3 and 4 were still largely comparable to previous trials in which RTS, S / AS01B and ChAd63 / MVA ME-TRAP were given alone. Pain, feverishness, fatigue, myalgia, discomfort were the El is no longer commonly reported.
    immunogenicity
    Ac titres to CS did not achieve activation after the 3rd dose of vaccine (day 56) in groups 3 and 4 and titres measured the day before CHMI (Pl) were approximately 2.5 times higher in groups 1 and 2 compared to groups 3 and 4. (Figure 7 (panel VAC59)). Significantly higher CS titers against CS were induced by RTS, S / AS01 alone (Example 2, Groups 1 and 2 (panel VAC59 in Figure 7) and Example 1, Group 2 (panel VAC55 in Figure 7)) or RTS, S / AS01 and staggered viral vectors (Example 1, Group 1 (panel VAC55 in Figure 7) compared to Example 2, Groups 3 and 4 (Panel VAC59, Figure 7).
    There was also only a very limited activation of the ACs directed against TRAP following the 3rd vaccination (day 56) in groups 3 and 4, compared to that observed in the study of example 1 (non presented). The TRAP T cell responses measured by an ELISPOT assay for interferon-D were not significantly different from those measured in group 1 subjects in the study of Example 1 who received RTS, S / AS01B with ChAd63 / MVA ME-TRAP, but with staggered vaccinations as opposed to simultaneous administration (data not shown).
    权利要求:
    Claims (46)
    [1]
    A method of inducing an immune response against malaria in a human subject comprising administering: i) a circumsporozoite (CS) plasmodial antigen, which is a CS protein or an immunogenic or variant fragment thereof and ii) a nucleotide vector encoding a plasmodial thrombospondin-related adherence protein (TRAP) antigen, which is a TRAP protein or an immunogenic or variant fragment thereof.
    [2]
    The method according to claim 1, wherein the plasmodial CS antigen is a CS protein of Plasmodium falciparum or Plasmodium vivax or an immunogenic or variant fragment thereof.
    [3]
    The method according to any one of the preceding claims, wherein the plasmodial CS antigen is selected from the group consisting of: a. RTS, b. CSV-S, c. RTS, S d. CSV-S, S and e. mixed particles comprising RTS and CSV-S, and optionally the unfused S antigen of hepatitis B.
    [4]
    A method according to any one of the preceding claims, wherein the plasmodial CS antigen is RTS, S and the amount of RTS, S is between 25 and 75, such as 50, micrograms per dose or between 12.5 and 37.5, as 25 micrograms per dose or between 5 and 20, as 10 micrograms per dose.
    [5]
    The method according to any one of the preceding claims, wherein the plasmodial CS antigen is administered in combination with an adjuvant.
    [6]
    The method of claim 5, wherein the adjuvant comprises a TLR agonist, such as a TLR4 agonist, for example 3D-MPL.
    [7]
    The method of claim 5 or 6, wherein the adjuvant comprises an immunologically active saponin, such as QS21, and optionally further comprises a sterol.
    [8]
    The method of any one of claims 5 to 7, wherein the adjuvant comprises 3D-MPL and QS21 in a liposomal formulation.
    [9]
    The method of claim 8, wherein the adjuvant comprises between 25 and 75, as 50 micrograms, of 3D-MPL per dose and between 25 and 75, as 50 micrograms of QS21 per dose.
    [10]
    The method of claim 8, wherein the adjuvant comprises between 12.5 and 37.5, as 25 micrograms, of 3D-MPL per dose and between 12.5 and 37.5, as 25 micrograms of QS21 per dose. .
    [11]
    The method of claim 8, wherein the adjuvant comprises between 5 and 20, as 10 micrograms, of 3D-MPL per dose and between 5 and 20, as 10 micrograms of QS21 per dose.
    [12]
    The method of any one of the preceding claims, the process comprising two or more administrations, such as three, administration of plasmodial CS antigen.
    [13]
    A method according to any one of the preceding claims, the process comprising three administrations of plasmodial CS antigen, the plasmodial CS antigen being the same in the three administrations and the plasmodial CS antigen being adjuvanted in the three administrations with a adjuvant comprising 3D-MPL and QS21 in a liposomal formulation.
    [14]
    14. The method of claim 13, wherein the time interval between each of the administrations is between 1 week and 1 year, for example between 2 weeks and 6 months, such as between 2 weeks and 6 weeks, for example 4 weeks.
    [15]
    The method according to any one of claims 12 to 14, wherein the dose of plasmodial CS antigen and the adjuvant dose are kept constant in all administrations.
    [16]
    The method of any one of claims 12 to 14, wherein: a. the amount of plasmodial CS antigen is lower in the second administration, and / or one of the subsequent administrations, compared to the amount of plasmodial CS antigen in the first administration and / or b. the amount of adjuvant is lower in the second administration, and / or one of the subsequent administrations, compared to the amount of adjuvant in the first administration.
    [17]
    The method according to any one of the preceding claims, wherein the plasmodial TRAP antigen is a Plasmodium falciparum TRAP antigen or Plasmodium vivax or an immunogenic or variant fragment thereof.
    [18]
    The method of any one of the preceding claims, wherein the plasmodial TRAP antigen is ME-TRAP.
    [19]
    The method of any of the preceding claims, wherein the nucleotide vector encoding the plasmodial TRAP antigen is a recombinant simian adenovirus vector defective for replication.
    [20]
    The method of claim 19, wherein the simian adenovirus vector is ChAd63, AdCh68, AdC3, AdC6, ChAdOx1 or AdC7.
    [21]
    21. The method of claim 20, wherein between 1 x 1010 and 1 x 1011, as 5 x 1010 viral particles are administered per dose.
    [22]
    The method of any one of claims 1 to 18, wherein the nucleotide vector encoding the plasmodial TRAP antigen is a non-adenoviral vector, such as the Ankara modified vaccinia vector (MVA).
    [23]
    A method according to any one of the preceding claims, which comprises i) administering an adenoviral vector which encodes a plasmodial TRAP antigen as described in any one of claims 19 to 21, and ii) the administration of a non-adenoviral vector which codes for a plasmodial TRAP antigen, the plasmodial TRAP antigen being a TRAP protein or one of its immunogenic or variant fragments.
    [24]
    The method of claim 23, wherein the non-adenoviral vector is a recombinant poxvirus vector, such as an MVA or a plasmid DNA vector.
    [25]
    The method of any one of claims 23 to 24, wherein the adenoviral vector and the non-adenoviral vector both encode ME-TRAP.
    [26]
    The method of claim 25, wherein the non-adenoviral vector is MVA and between 1 x 108 and 1 x 109, as 2 x 108 pfu are administered per dose.
    [27]
    27. A method according to any one of claims 23 to 26, wherein the non-adenoviral vector is administered at least two weeks, for example between 2 and 12 weeks, after the adenoviral vector, as in which the non-adenoviral vector is administered. weeks after the adenoviral vector.
    [28]
    The method according to any of the preceding claims, wherein the time interval between the first administration of a plasmodial CS antigen and the first administration of a nucleotide vector encoding the plasmodial TRAP antigen is between 1 day and 4 weeks, as between 1 week and 3 weeks, as 2 weeks.
    [29]
    The method of any one of the preceding claims, comprising the following administrations: a. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation, b. Administration of ChAd63 coding for ME-TRAP c. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation d. Administration of RTS, S adjuvanted with 3D-MPL and QS21 in a liposomal formulation e. Administration of MVA coding for ME-TRAP.
    [30]
    30. The method of claim 29, wherein the administrations are performed in the specified order.
    [31]
    31. The method of claim 30, wherein the time interval between steps a. and B. is between 1 and 3 weeks, like 2 weeks, and in which the time interval between steps b. and c. is between 1 and 3 weeks, like 2 weeks, and in which the time interval between steps c. and D. is between 1 and 8 weeks, like 4 weeks, and in which the time interval between steps d. summer. is between 1 and 3 weeks, like 2 weeks.
    [32]
    The method of any one of claims 1 to 27, wherein the administration of one or all of the TRAP coding vectors is carried out substantially simultaneously with the administration of a plasmodial CS antigen.
    [33]
    The method of any one of claims 1 to 31, wherein the administration of one or all of the TRAP coding vectors is not performed substantially concurrently with the administration of a plasmodial CS antigen.
    [34]
    34. The method according to any of claims 1 to 33, wherein the administration of one or all of the TRAP coding vectors is performed at a site distinct from the body as compared to that used for the treatment. administration of the plasmodial CS antigen.
    [35]
    The method according to one of claims 1 to 33, wherein any nucleotide vector encoding the plasmodial TRAP antigen is administered at a site distinct from the body, or given at least 1 day apart, from any administration of a plasmodial CS antigen, as at more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, as at 14 days or more apart.
    [36]
    36. Method according to one of claims 1 to 33, wherein if the method comprises two or more administrations of nucleotide vectors encoding a plasmodium TRAP antigen, the second and the other administrations are given at a site distinct from the body, or given at least 1 day apart from any administration of a plasmodial CS antigen, such as more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart as at 14 days or more away.
    [37]
    37. The method according to one of claims 1 to 33, wherein if the method comprises an administration with a non-adenoviral vector coding for a plasmodial TRAP antigen, said administration is given at a site distinct from the body, or given at 1 one or more days apart from any administration of a plasmodial CS antigen, such as more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, such as at 14 days or more away.
    [38]
    38. The method according to one of claims 1 to 33, wherein if the method comprises an administration with an MVA vector coding for a plasmodial TRAP antigen, said administration is given at a site distinct from the body, or given at 1 one or more days apart from any administration of a plasmodial CS antigen, such as more than 2 days apart, for example at more than 3, 4, 5, 6, or 7 days apart, such as at 14 days or more away.
    [39]
    39. A method according to any one of the preceding claims, wherein the human subject is over 18 years old when the first administration is performed.
    [40]
    40. The method of any one of claims 1 to 38, wherein the human subject is less than five years old when the first administration is performed.
    [41]
    41. An immunogenic composition for use in a method of inducing an immune response against malaria in a human subject, the method comprising administering i) a circumsporozoite (CS) plasmodial antigen, which is a CS or one of its immunogenic or variant fragments and ii) a nucleotide vector encoding a plasmodial thrombospondin-related adherence protein (TRAP) antigen, which is a TRAP protein or an immunogenic or variant fragment thereof.
    [42]
    42. Immunogenic composition according to claim 41, comprising one or more of the other characteristics as cited in claims 2 to 40.
    [43]
    43. Use of i) a plasmodial circumsporozoite (CS) antigen, which is a CS protein or an immunogenic or variant fragment thereof and ii) a nucleotide vector encoding a thrombospondin-related adherence protein antigen (TRAP) ) plasmodial, which is a TRAP protein or one of its immunogenic or variant fragments, in the manufacture of a medicament for inducing an immune response against malaria in a human subject, wherein i) and ii) are administered sequentially or simultaneously.
    [44]
    44. Use according to claim 43 comprising one or more of the other characteristics as recited in claims 2 to 40.
    [45]
    45. A composition comprising i) a plasmodial circumsporozoite (CS) antigen, which is a CS protein or an immunogenic or variant fragment thereof and ii) a nucleotide vector encoding a thrombospondin-related adherence protein antigen (TRAP) ) Plasmodial, which is a TRAP protein or one of its immunogenic fragments or variants.
    [46]
    46. Kit comprising i) a first container comprising a plasmodial circumsporozoite (CS) antigen, which is a CS protein or one of its immunogenic or variant fragments and ii) a second container comprising a nucleotide vector encoding a protein antigen of thrombospondin-related (TRAP), which is a TRAP protein or one of its immunogenic or variant fragments, and iii) optionally, instructions for the use of the kit in the immunization of human subjects against malaria.
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    COHEN0|Patent 2613057 Summary
    同族专利:
    公开号 | 公开日
    BE1022950A1|2016-10-21|
    GB201416773D0|2014-11-05|
    WO2016046113A1|2016-03-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2006029887A2|2004-09-16|2006-03-23|Glaxosmithkline Biologicals Sa|Vaccines comprising plasmodium antigens|
    WO2009021931A1|2007-08-13|2009-02-19|Glaxosmithkline Biologicals S.A.|Vaccines|
    CN109803676A|2016-04-12|2019-05-24|牛津大学创新有限公司|Just exempt to target|
    GB201608821D0|2016-05-19|2016-07-06|Isis Innovation|Vaccines|
    法律状态:
    2018-06-28| FG| Patent granted|Effective date: 20161021 |
    2018-06-28| MM| Lapsed because of non-payment of the annual fee|Effective date: 20170930 |
    优先权:
    申请号 | 申请日 | 专利标题
    GB1416773.8|2014-09-23|
    GBGB1416773.8A|GB201416773D0|2014-09-23|2014-09-23|Novel Methods For Including An Imune Response|
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