WO2019145399A1 - Vaccins pour la prophylaxie d'infections par s. aureus - Google Patents

Vaccins pour la prophylaxie d'infections par s. aureus Download PDF

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WO2019145399A1
WO2019145399A1 PCT/EP2019/051710 EP2019051710W WO2019145399A1 WO 2019145399 A1 WO2019145399 A1 WO 2019145399A1 EP 2019051710 W EP2019051710 W EP 2019051710W WO 2019145399 A1 WO2019145399 A1 WO 2019145399A1
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exactly
nucleic acid
polypeptide
amino acid
cell
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PCT/EP2019/051710
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English (en)
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Niels Iversen MØLLER
Andreas Holm MATTSSON
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Evaxion Biotech Aps
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • the present invention relates to the field of antimicrobial prophylaxis and therapy.
  • the present invention relates to novel vaccine compositions where the immunogens are derived from Staphylococcus aureus.
  • the invention further relates to vectors,
  • Vaccination is considered to be a very effective method of preventing infectious diseases in human and veterinary health care. Vaccination is the administration of effective amounts of antigenic material (the vaccine) to produce immunity to a disease/disease-causing pathogenic agent. Vaccines have contributed to the eradication of smallpox, the near eradication of polio, and the control of a variety of diseases, including rubella, measles, mumps, chickenpox, typhoid fever.
  • vaccines were based on killed or live attenuated, microorganisms, or parts purified from them.
  • Subunit vaccines are considered as a modern upgrade of these types of vaccine, as the subunit vaccines contain one or more protective antigens, which are more or less the weak spot of the pathogen.
  • protective antigens which are more or less the weak spot of the pathogen.
  • An antigen is said to be protective if it is able to induce protection from subsequent challenge by a disease-causing infectious agent in an appropriate animal model following immunization .
  • the empirical approach to subunit vaccine development which includes several steps, begins with pathogen cultivation, followed by purification into components, and then testing of antigens for protection. Apart from being time and labour consuming, this approach has several limitations that can lead to failure. It is not possible to develop vaccines using this approach for microorganisms, which cannot easily be cultured and only allows for the identification of the antigens, which can be obtained in sufficient quantities.
  • the empirical approach has a tendency to focus on the most abundant proteins, which in some cases are not immuno-protective. In other cases, the antigen expressed during in vivo infection is not expressed during in vitro cultivation.
  • antigen discovery by use of the empirical approach demands an extreme amount of proteins in order to discover the protective antigens, which are like finding needles in the haystack. This renders it a very expensive approach, and it limits the vaccine development around diseases, which is caused by pathogens with a large genome or disease areas, which perform badly in a cost-effective perspective.
  • PCT/EP2017/068694 are disclosed a number of polypeptides, nucleic acids, vectors, and compositions that are useful as vaccine agents.
  • the present invention provides antigenic material in the form of polypeptides, nucleic acids and vaccine vectors (including cells and virus) that can induce protective immunity against S. aureus infection.
  • This antigenic material is useful as (vaccine) immunogens per se but also in combination with any one of the immunogens disclosed in international patent application publications WO 2012/136653, WO 2015/082536, WO 2017/144523, and in PCT application no. PCT/EP2017/068694.
  • the present invention relates to a polypeptide comprising or consisting of
  • polypeptide being antigenic in a mammal.
  • a 2 nd aspect of the invention relates to an isolated nucleic acid fragment, which comprises or consists of a nucleotide sequence encoding a polypeptide according to the first of the invention as well as any embodiments of these aspects..
  • a 3 rd aspect of the invention relates to a vector comprising the nucleic acid of the 2 nd aspect of the invention or of any embodiments of the 3 rd aspect, such as a cloning vector or an expression vector.
  • a 4 th aspect of the invention relates to a cell which is transformed so as to carry the vector of 1) the 3 rd aspect of the present invention or 2) any embodiments of the 3 rd aspect. Also part of this aspect is a cell line derived from such a transformed cell of the present invention.
  • a 5 th aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a
  • polypeptide of the 1 st aspect of the invention as well as any embodiments of this aspect, a nucleic acid fragment of the 2 nd aspect of the invention or the embodiments of the 2 nd aspect, a vector of the 3 rd aspect of the invention or of any embodiments thereof, or a cell of the 4 th aspect of the invention and any embodiments of the 4 th aspect, and a pharmaceutically acceptable carrier, vehicle or diluent
  • a 6 th aspects of the invention relates to a method for inducing immunity in an animal by administering at least once an immunogenically effective amount of a polypeptide of the first or second aspect of the invention as well as of embodiments this aspect, a nucleic acid fragment of the 2 nd aspect of the invention as well as any embodiment of the 2 nd aspect, a vector of the 3 rd aspect of the invention as well as any embodiment of the 3 rd aspect, a cell of the 4 th aspect of the invention as well as any embodiment thereof, or a pharmaceutical composition of the 5 th aspect of the invention as well as any embodiment thereof, so as to induce adaptive immunity against S. aureus in the animal .
  • the present invention also relates to the polypeptides of the invention, the nucleic acid or vector of the invention, the cells of the invention, or the pharmaceutical compositions of the invention for use as a pharmaceutical, in particular for use in the treatment, prophylaxis or amelioration of infection with S. aureus.
  • Fig . 1 shows survival plots after challenge infection in mice immunized with immunogens of the invention in a peritonitis model . Dotted lines indicates control, full lines indicates immunogen.
  • polypeptide is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i .e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked .
  • the polypeptide (s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups.
  • sequence means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a reference amino acid sequence or nucleic acid sequence, respectively
  • amino acid sequence is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins.
  • adjuvant or "immunological adjuvant” has its usual meaning in the art of vaccine technology, i .e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen.
  • vaccination with the adjuvant alone does not provide an immune response against the immunogen
  • vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • An “assembly of amino acids” means two or more amino acids bound together by physical or chemical means.
  • the "3D conformation” is the 3 dimensional structure of a biomolecule such as a protein.
  • the 3D conformation is also termed “the tertiary structure” and denotes the relative locations in 3 dimensional space of the amino acid residues forming the polypeptide.
  • An immunogenic carrier is a molecule or moiety to which an immunogen or a hapten can be coupled in order to enhance or enable the elicitation of an immune response against the immunogen/hapten.
  • Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen/hapten, which is not sufficiently immunogenic in its own right - typically, the immunogenic carrier is capable of eliciting a strong cellular immune response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen antibody producing cells and cytotoxic cells.
  • the large carrier molecules have to a certain extent been substituted by so-called promiscuous epitopes, i.e. shorter peptides that are recognized by a large fraction of MHC-haplotypes in a population, and which elicit antigen specific cellular immune responses.
  • An "immunogen” is a substance of matter which is capable of inducing an adaptive immune response in a host, whose immune system is exposed to the immunogen.
  • immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system but which are not necessarily capable of inducing immunity - an antigen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against the antigen.
  • a "hapten” is a (typically) small molecule, which can neither induce nor elicit an immune response, but if conjugated to an immunogenic carrier, a specific adaptive immune response can be induced against a hapten upon exposure of the immune system with the hapten carrier conjugate.
  • An “adaptive immune response” is an immune response in response to exposure to an antigen or immunogen, where the immune response is specific for antigenic determinants of the antigen/immunogen - examples of adaptive immune responses are induction of antigen specific antibody production or antigen specific induction/activation of cellular immune responses.
  • a "protective, adaptive immune response” is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen or a pathology-related agent that includes the antigen.
  • prophylactic vaccination aims at establishing a protective adaptive immune response against one or several pathogens.
  • Stimulation of the immune system means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect.
  • a number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system.
  • the result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
  • a "T-helper lymphocyte response” is an immune response elicited on the basis of a peptide, which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell and which stimulates T-helper lymphocytes in an animal species as a consequence of T-cell receptor recognition of the complex between the peptide and the MHC Class II molecule presenting the peptide.
  • Hybridization under “stringent conditions” is herein defined as hybridization performed under conditions by which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide.
  • target sequences can be identified which are 100% complementary to a probe (homologous probing) .
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) .
  • Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution .
  • stringent wash temperature conditions are selected to be about 5°C to about 2°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow
  • Tm temperature of the midpoint of transition
  • an antibody refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.
  • An “antibody combining site” is the three- dimensional binding space with an internal surface shape and charge distribution
  • Antibody includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
  • Specific binding denotes binding between two substances which goes beyond binding of either substance to randomly chosen substances and also goes beyond simple association between substances that tend to aggregate because they share the same overall
  • vector is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed .
  • the term further denotes certain biological vehicles useful for the same purpose, e.g. viral and bacterial vectors - both these infectious agents are capable of introducing a heterologous nucleic acid sequence into a host and effect subsequence expression of a nucleic acid in the host.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed.
  • chimeric polypeptide is a polypeptide, which is constituted by amino acid stretches derived from at least two different proteins, where these at least two stretches are fused to each other, optionally via a linker. By nature, a chimeric polypeptide does not occur in nature.
  • Typical linkers are flexible, meaning that they allow the joint polypeptides in a fusion construct to have a high degree of movement.
  • Such flexible linkers are often rich in small, non-polar amino acid residues (such as glycine residues) but will often incorporate small polar amino acid residues such as serine or threonine residues, too.
  • Such linkers are known as GS linkers.
  • a “linker” is an amino acid sequence, which is introduced between two other amino acid sequences in order to separate them spatially. Linkers are widely used in recombinant biotechnology and are reviewed in Chen X et al. (2013), Advanced drug delivery reviews 65(10) : 1357-1369. doi : 10.1016/j.addr.2012.09.039. A linker may be "rigid”, meaning that it does substantially not allow the two amino acid sequences that it connects to move freely relative to each other. Likewise, a “flexible” linker allows the two sequences connected via the linker to move substantially freely relative to each other. In the fusion proteins, which are part of the present invention, both types of linkers are useful. Linkers of interest are listed in the following table:
  • the at least or exactly 5 contiguous amino acids referred to in option b) in the definition of the first aspect of the invention constitute at least or exactly or at most 6, such as at least or exactly or at most 7, at least or exactly or at most 8, at least or exactly or at most 9, at least or exactly or at most 10, at least or exactly or at most 11, at least or exactly or at most 12, at least or exactly or at most 13, at least or exactly or at most 14, at least or exactly or at most 15, at least or exactly or at most 16, at least or exactly or at most 17, at least or exactly or at most 18, at least or exactly or at most 19, at least or exactly or at most 20, at least or exactly or at most 21, at least or exactly or at most 22, at least or exactly or at most 23, at least or exactly or at most 24, at least or exactly or at most 25, at least or exactly or at most 26, at least or exactly or at most 27 at least or exactly or at most 28, at least or exactly or at most 29, at least or exactly or at most 30, at least or exactly or at most 31, at least or exactly or at most 32, at least or exactly or
  • the number of the contiguous amino acid residues from SEQ ID NO: 1 is at least or exactly or at most 365-n, where n is any integer between 1 and 360; that is, the at least 5 contiguous amino acids can be at least any number between 5 and the length of the reference sequence minus one, in increments of one.
  • the polypeptide of the invention also has a sequence identity with the amino acid sequence of a) defined above of at least 61%, such as at least 63%, at least 63% at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81,%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.
  • polypeptide of the invention in some embodiments also has a sequence identity with the amino acid sequence of b) defined above of at least 61%, such as at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81,%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.
  • the polypeptide of the invention is also one that has at least or exactly or at most 5 contiguous amino acid residues defined for option b) above and also has its N-terminal amino acid residue corresponding to any one of amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
  • the polypeptide as disclosed in any of the embodiments above may be fused to an amino acid or amino acid sequence. Non-limiting examples of such fusion partners are
  • heterologous amino acid sequence that comprises or constitutes an immunogenic carrier molecule or one or more separate T-helper epitopes
  • polypeptide disclosed above is fused to a heterologous amino acid sequence, this fusion may be direct or via a suitable peptide linker, cf. the discussion of linkers above.
  • the polypeptide of the invention is in certain embodiments also covalently linked (i.e. fused or conjugated) to an immunogenic carrier molecule; or, phrased otherwise, the polypeptide of the invention also includes such an immunogenic carrier molecule in addition to the polypeptides of the present invention.
  • the immunogenic carrier molecule is a typically polypeptide that induces T-helper lymphocyte responses in a majority of humans, such as immunogenic carrier proteins selected from the group consisting of keyhole limpet hemocyanin or a fragment thereof, tetanus toxoid or a fragment thereof, diphtheria toxoid or a fragment thereof. Other suitable carrier molecules are discussed infra.
  • One further fusion partner, which is preferably incorporated is a "His tag", i.e. a stretch of amino acids, which is rich or only consists of histidinyl residues so as to facilitate protein purification.
  • the polypeptide of the invention is capable of inducing an adaptive immune response against the polypeptide in a mammal, in particular in a human being.
  • the adaptive immune response is a protective adaptive immune response against infection with S. aureus, in particular multi-resistant S. aureus.
  • the polypeptide may in these cases induce a humeral and/or a cellular immune response.
  • SEQ ID NO: 1 includes antigenic determinants (epitopes) that are as such recognized by antibodies and/or when bound to MHC molecules by T-cell receptors.
  • B-cell epitopes i.e. antibody binding epitopes
  • mutated versions of the polypeptides of the invention e.g. version where each single non-alanine residue in SEQ ID NO: 1 are point mutated to alanine - this method also assists in identifying complex assembled B-cell epitopes; this is the case when binding of the same antibody is modified by exchanging amino acids in different areas of the full-length polypeptide.
  • the nucleic acid fragment of the invention referred to above preferably is a DNA fragment or an RNA fragment.
  • Exemplary DNA fragments are provided as SEQ ID NO: 2 (DNA encoding SEQ ID NOs: 1, i.e. an exemplary polypeptide of the first aspect of the invention) and as SEQ ID NO: 3 (RNA encoding SEQ ID NO: 1). Also the complimentary sequences are embraced by the present invention.
  • polypeptides can be encoded by a plethora of nucleic acid sequences due to the degeneracy of the genetic code, the skilled person will understand that none single nucleic acid sequence is particularly preferred as long as it encodes a polypeptide of the present invention. Rather, the skilled person will design suitable coding sequences that are codon optimised with respect to e.g. the expression system wherein recombinant production of the polypeptide is to take place. However, as a suitable starting point, the native DNA and RNA sequences are provided as SEQ ID NOs: 2 and 3.
  • nucleic acids that encode the polypeptide of the invention can have a sequence identity with the nucleotide sequence SEQ ID NO: 1 or 2 of at least 60%, such as at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.
  • 3 rd aspect - vectors of the invention of at least 60%, such as at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.
  • nucleic acid fragments of the invention may be used for both production, carrier and vaccine purposes - the latter will require that the sequences are included in expression vectors that may lead to production of immunogenic proteins in the mammal receiving the vector.
  • the nucleic acid is comprised in a vector capable of expressing the nucleic acid in man upon administration.
  • Such a vector of the invention often comprises in operable linkage and in the 5'-3' direction, an expression control region comprising an enhancer/promoter for driving expression of the nucleic acid, an optional signal peptide coding sequence, a nucleotide sequence of the invention, and optionally a terminator.
  • an expression vector useful for effecting production in cells of the polypeptide of the invention. Since the polypeptides of the invention are protozoan of origin, recombinant production has to be effected in host cells that can express the coding nucleic acid. Bacterial host cells may be used. However, if the vector is to drive expression in eukaryotic cell (as would be the case for a DNA or RNA vaccine vector), the expression control region should be adapted to this particular use.
  • the expression control region drives expression in a prokaryotic cell such as a bacterium, e.g. in E. coti, or in a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell.
  • a prokaryotic cell such as a bacterium, e.g. in E. coti
  • a eukaryotic cell such as a plant cell, an insect cell, or a mammalian cell.
  • the expression control region has to be able to drive expression in a mammalian, preferably human, cell.
  • the vector is capable of integrating the nucleic acid into the genome of a host cell - this is particularly useful if the vector is use in the production of stably transformed cells, where the progeny will also include the genetic information introduced via the vector.
  • vectors incapable of being integrated into the genome of a piscine host cell are useful in e.g. nucleic acid vaccination.
  • proteins can be produced at low cost in plants using an Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest.
  • Agrobacterium transfection system to genetically modify plants to express genes that encode the protein of interest.
  • One commercially available platform are those provided by i Bio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and i Bio, Inc (9 Innovatoin Way, Suite 100, Newark, DE 19711, USA) and disclosed in e.g. EP 2 853 599, EP 1 769 068, and EP 2 192 172.
  • the vector is an Agrobacterium vector or other vector suitable for transfection of plants.
  • the vector is typically selected from the group consisting of a virus, such as a virus which is non-pathogenic in mammals and in particular in humans, a bacterium such as a bacterium which is non-pathogenic in mammals such as humans, a plasmid, a minichromosome, and a cosmid.
  • viral vectors are viral vectors (in particular those useful as vaccine agents in humans). These may be selected from the group consisting of a retrovirus vector, such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector.
  • a retrovirus vector such as a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a pox virus vector.
  • pox virus vectors are preferred, in particular vaccinia virus vectors.
  • a particularly preferred vaccinia virus vector is a modified vaccinia Ankara (MVA) vector.
  • Polypeptides of the invention may as indicated be encoded by a nucleic acid molecule comprised in a vector.
  • a nucleic acid sequence can be "heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced, which includes a sequence homologous to a sequence in the cell but in a position within the host cell where it is ordinarily not found.
  • Vectors include naked DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • a vector of the present invention may encode polypeptide sequences such as a "tag" or immunogenicity enhancing peptide (e.g. an immunogenic carrier or a fusion partner that stimulates the immune system, such as a cytokine or active fragment thereof).
  • Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
  • GST glutathione S-transferase
  • Vectors of the invention may be used in a host cell to produce a polypeptide of the invention that may subsequently be purified for administration or the vector may be purified for direct administration for expression of the protein (as is the case when administering a nucleic acid vaccine).
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers
  • a “promoter” is a control sequence.
  • the promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled . It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i .e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction in connection with the compositions disclosed herein.
  • promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the cell type or organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression.
  • the promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.
  • inducible elements which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T Cell Receptor, HLA DQa and/or DQ , b- Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRa, b- Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, a-Fetoprotein, y-Globin, b-Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), al-Antitrypain, H2B (TH2B) Histone, Mouse and/or Type I Collagen, Glucose-Regulated Protein
  • Inducible Elements include MT II - Phorbol Ester (TFA)/Heavy metals; MMTV (mouse mammary tumor virus) - Glucocorticoids; b-Interferon - poly(rl)x/poly(rc); Adenovirus 5 E2 - EIA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; a-2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2k ⁇ > - Interferon; HSP70 - E1A/SV40 Large T Antigen; Proliferin - Phorbol Ester/TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormone a Gene - Thyroid
  • dectin-1 and dectin-2 promoters are also contemplated as useful in the present invention. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest.
  • the particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell.
  • a piscine cell is targeted (as is the case in nucleic acid vaccination)
  • a promoter might include either a bacterial, piscine or viral promoter as long as the promoter is effective in piscine cells.
  • the human cytomegalovirus (CMV) immediate early gene promoter the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention.
  • CMV human cytomegalovirus
  • SV40 early promoter the SV40 early promoter
  • Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention.
  • the use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well.
  • a desirable promoter for use with the vector is one that is not down- regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/polypeptide of the current invention in humans to elicit an immune response.
  • cytokines Non-limiting examples of these are CMV IE and RSV LTR.
  • a promoter that is up-regulated in the presence of cytokines is employed.
  • the MHC I promoter increases expression in the presence of IFN-y.
  • Tissue specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells and macrophages.
  • the mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters in man and it is contemplated that corresponding piscine promoters will be effective.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous
  • translational control signals including the ATG initiation codon, may need to be provided.
  • initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • the exogenous translational control signals and initiation codons can be either natural or synthetic and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES internal ribosome entry sites
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites.
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 3. Multiple Cloning Sites
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression.
  • the vectors or constructs of the present invention will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
  • the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.
  • RNA molecules modified with this poly(A) tail appear to more stable and are translated more efficiently.
  • terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the RNA.
  • Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. 6. Polyadenylation Signals
  • RNA fragments of the invention include a polyadenylation signal and that the RNA fragments include a poly(A) tail.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "on"), which is a specific nucleic acid sequence at which replication is initiated.
  • an autonomously replicating sequence can be employed if the host cell is yeast.
  • cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector.
  • a marker When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP for colorimetric analysis.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • Transformed cells of the invention are useful as organisms for producing the polypeptide of the invention, but also as simple "containers" of nucleic acids and vectors of the invention. Also, the transformed cells find use as immunization and vaccine agents.
  • Certain transformed cells of the invention are capable of replicating the nucleic acid fragment of the 2 nd aspect of the invention.
  • Preferred transformed cells of the invention are capable of expressing the nucleic acid fragment.
  • the transformed cell according is prokaryotic, such as a bacterium, but generally both prokaryotic cells and eukaryotic cells may be used.
  • Suitable prokaryotic cells are bacterial cells selected from the group consisting of Escherichia (such as E. coli ), Bacillus (e.g. Bacillus subtiHs), Salmonella, and Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG). Generally, if prokaryotic cells are to be used as vaccine agents, they should be non-pathogenic and/or attenuated.
  • Eukaryotic cells can be in the form of yeasts (such as baker's yeast, Saccharomyces cerevisiae) and protozoans.
  • the transformed eukaryotic cells are derived from a multicellular organism such as a fungus (typically filamentous), an insect cell, a plant cell, or a mammalian cell.
  • the transformed cell of the invention is is stably transformed by having the nucleic acid defined above for option i) stably integrated into its genome, and in certain embodiments it is also preferred that the transformed cell secretes or carries on its surface the polypeptide of the invention, since this facilitates recovery of the polypeptides produced.
  • a particular version of this embodiment is one where the transformed cell is a bacterium and secretion of the polypeptide of the invention is into the periplasmic space.
  • stably transformed cells are preferred - these i.a. allows that cell lines comprised of transformed cells as defined herein may be established - such cell lines are particularly preferred aspects of the invention.
  • Suitable cells for recombinant nucleic acid expression of the nucleic acid fragments of the present invention are prokaryotes and eukaryotes.
  • prokaryotic cells include E. coli ; members of the Staphylococcus genus, such as S. epidermidis ; members of the
  • Lactobacillus genus such as L. plantarunrr, members of the Lactococcus genus, such as L. lactis ; members of the Bacillus genus, such as B. subtilis ; members of the Corynebacterium genus such as C. glutamicuirr, and members of the Pseudomonas genus such as Ps.
  • eukaryotic cells include mammalian cells; insect cells; yeast cells such as members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastohs), members of the Hansenula genus (e.g. H. polymorpha ), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis ) and members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (e.g. P. pastohs), members of the Hansenula genus (e.g. H. polymorpha ), members of the Kluyveromyces genus (e.g. K. lactis or K. fragilis ) and members of the Saccharomyces genus (e.g. S. cerevisiae) , members of the Pichia genus (
  • Schizosaccharomyces genus e.g. S. pombe.
  • the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which includes any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors or viruses.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials
  • a plasmid or cosmid can be introduced into a prokaryote host cell for replication of many vectors or expression of encoded proteins.
  • Bacterial cells used as host cells for vector replication and/or expression include Staphylococcus strains, DH5a, JMI 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE(R) Competent Cells and SOLOP ACK(TM) Gold Cells (STRATAGENE®, La Jolla, CA).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastohs.
  • eukaryotic host cells for replication and/or expression of a vector examples include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM Baculovirus expression system from CLONTECH®
  • a heterologous nucleic acid segment such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM Baculovirus expression system from CLONTECH®
  • other examples of expression systems include STRATAGENE®'s COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • INVITROGEN® which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • nucleic acid delivery to effect expression of compositions of the present invention are believed to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859), including microinjection (U.S. Patent 5,789,215); by electroporation (U.S. Patent No.
  • Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055); or by PEG mediated transformation of protoplasts (U.S. Patents 4,684,611 and 4,952,500); by desiccation/inhibition mediated DNA uptake.
  • organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently
  • RNA vaccines Recently, the development of RNA vaccines has shown great promise. Hence technology for RNA vaccine delivery and expression are within the ambit of the present application.
  • compositions of the invention vaccines
  • compositions in particular vaccines, according to the invention are prophylactic but may also be used therapeutically.
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • the pharmaceutical compositions such as vaccines include merely one single antigen, immunogen, polypeptide, protein, nucleic acid or vector of the invention, but in other embodiments, the pharmaceutical compositions comprise
  • the pharmaceutical composition is a vector mentioned herein, which encodes and can effect expression of at least 2 nucleic acid fragments of the invention.
  • RNA as the active principle, i .e. at least one mRNA encoding a polypeptide of the invention.
  • compositions of the invention at least 2 (such as 2, 3, 4, 5,6, 7, 8, 9, or 10) distinct polypeptides of the invention described above.
  • composition of the invention comprises at least 2 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) distinct nucleic acid molecules (such as DNA and RNA) each encoding a polypeptide of the invention.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants") . Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H . pylori, etc. pathogen, cf. the description of immunogenic carriers supra.
  • the pharmaceutical compositions of the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following :
  • Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulphate, etc. ; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
  • aluminium salts alum
  • aluminium hydroxide aluminium hydroxide
  • aluminium phosphate aluminium phosphate
  • aluminium sulphate aluminium phosphate
  • bacterial cell wall components such as for example
  • MF59 WO 90/14837
  • Span 85 containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton,
  • Ribi adjuvant system Ribi Immunochem, Hamilton
  • MT containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM) ; (3) saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4)
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • cytokines such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg.
  • Alum and MF59TM adjuvants are preferred together with CFA and IFA.
  • muramyl peptides include, but are not limited to, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • thr-MDP N-acetyl-muramyl-L- threonyl-D-isoglutamine
  • nor- MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
  • MTP-PE N-acetylmuramyl-L-alanyl-D-
  • polypeptide vaccine formulation Another possibility for a polypeptide vaccine formulation is to include the vaccine
  • polypeptide(s) of the present invention in a virus-like particle, i.e. a non-infectious self assembling structure composed of envelope or capsid proteins, where the protein(s) of the invention are incorporated.
  • the effect is multiple presentations of the polypeptides of the invention on the surface of the VLP, which in turn provides for improved immune recognition of the polypeptides.
  • VLPs exert immunological adjuvant effects, too.
  • the immunogenic compositions typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg . non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors.
  • the amount of immunogen will fall in a relatively broad range that can be determined through routine trials.
  • the amount administered per immunization is typically in the range between 0.5 pg and 500 mg (however, often not higher than 5,000 pg) .
  • the amount of polypeptide of the invention can therefore be between 1 and 400 pg, between 2 and 350 pg, between 4 and 300 pg, between 5 and 250 pg, and between 10 and 200 pg .
  • the composition will typically contain between 0.1-500 pg of protein of the invention per g of vaccine composition.
  • the immunogenic compositions are conventionally administered parenterally, eg, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg . W0 98/20734) .
  • Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
  • nucleic acid vaccination also the intravenous or intraarterial routes may be applicable.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunction with other immunoregulatory agents.
  • DNA vaccination also termed nucleic acid vaccination or gene vaccination
  • Donnelly et al. (1997) Annu Rev Immunol 15 : 617-648).
  • a further aspect of the invention is as mentioned above the recognition that combination vaccines can be provided, wherein 2 or more polypeptide antigens disclosed herein and in the above-referenced filings by the present assignee are combined to enhance the immune response by the vaccinated individual, including to optimize initial immune response and duration of immunity.
  • polypeptide antigens disclosed herein and in the above-referenced filings by the present assignee are combined to enhance the immune response by the vaccinated individual, including to optimize initial immune response and duration of immunity.
  • multiple antigenic fragments derived from the same, longer protein can also be used, such as the use of a combination of different lengths of polypeptide sequence fragments from one protein.
  • compositions relate to a composition (or the use as a vaccine thereof) comprising at least 2 distinct (i.e. non-identical) proteinaceous immunogens disclosed herein.
  • the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO
  • the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or with any of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536.
  • the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO
  • the present invention also relates to immunogenic compositions that include a polypeptide of the present invention derived from SEQ ID NO: 1 (i.e. being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1 in combination with any one of the chimeric polypeptides disclosed in PCT/EP2017/068694, in particular those chimeric polypeptides disclosed on pages 10-46 and defined in the claims in PCT/EP2017/068694.
  • the present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-19 in WO 2012/136653 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 9-18 in WO 2012/136653.
  • a nucleic acid is typically an expression vector, either DNA-based or RNA-based.
  • the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein.
  • the "combination of nucleic acids” can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the
  • the present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-16 and 49 in WO 2015/082536 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-25 in WO 2015/082536.
  • a nucleic acid is typically an expression vector, either DNA-based or RNA-based.
  • the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein.
  • the "combination of nucleic acids” can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the
  • the present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the polypeptides disclosed as SEQ ID NOs: 1-2 in WO 2017/144523 or 2) any one of the fragments and variants of these polypeptides disclosed on pages 10-19 in WO 2017/144523.
  • a nucleic acid is typically an expression vector, either DNA-based or RNA-based.
  • the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein.
  • the "combination of nucleic acids” can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the
  • the present invention also relates to immunogenic compositions that include a nucleic acid that encodes and is capable of effecting expression of a (poly)peptide of the present invention derived from SEQ ID NO: 1 (i.e. the (poly)peptide being one of the fragments or sequence variants disclosed herein) or being identical to SEQ ID NO: 1, in combination with a nucleic acid encoding and being capable of effecting expression of 1) any one of the chimeric polypeptides disclosed in the claims of PCT/EP2017/068694 as well as on pages 10-42 in PCT/EP2017/068694.
  • a nucleic acid is typically an expression vector, either DNA-based or RNA-based. It is also within the ambit of the present invention that the 2 nucleic acids may be fused and part of one single nucleic acid that is capable of expressing two different immunogens or one single immunogen which constitutes a fusion protein.
  • “combination of nucleic acids” can be in the form of separate nucleic acids that each encode a (poly)peptide or it can be in the form of a single nucleic acid that includes coding sequences from both of the (poly)peptides.
  • compositions may in the above cases comprise a cocktail of several proteins or nucleic acids disclosed in the patent applications discussed above.
  • PCT/EP2017/068694 are for these reasons incorporated in their entirety by reference herein.
  • the method of this aspect of the invention generally relates to induction of immunity and as such also entails methods that are prophylactic as well as therapeutic.
  • immunization methods entail that a polypeptide of the invention or a composition comprising such a polypeptide is administered the animal (e.g. the human) typically receives between 0.5 and 5,000 pg of the polypeptide of the invention per administration, cf. the above indications concerning dosages.
  • the immunization scheme includes that the a primary
  • Preferred embodiments comprise that the administration is for the purpose of inducing protective immunity against S. aureus.
  • the protective immunity is effective in reducing the risk of attracting infection with S. aureus.
  • the some vaccines of the invention induce humoral immunity, so it is preferred that the administration is for the purpose of inducing antibodies specific for S aureus.
  • the immunization method may also be useful in antibody production, so in other embodiments the administration is for the purpose of inducing antibodies specific for S. aureus wherein B-lymphocytes producing said antibodies are subsequently recovered from the animal and used for preparation of monoclonal antibodies.
  • compositions for immunization can as mentioned above comprise polypeptides, nucleic acids, or vectors of the invention.
  • the pharmaceutical compositions will comprise a therapeutically effective amount thereof.
  • terapéuticaally effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable preventative effect in a group of mammals such as humans The effect can be detected by, for example, chemical markers or antigen levels. Reference is made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.
  • an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the animal to which it is administered.
  • a pharmaceutical composition can also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.
  • the term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions may contain liquids such as water, saline, glycerol and ethanol . Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared . Liposomes are included within the definition of a
  • mice Female NMRI mice were immunized with recombinant polypeptide in combination with the adjuvant aluminium hydroxide (Alhydrogel®, Brenntag, Cat. no. 21645-51-2) and Freund's incomplete adjuvant.
  • the adjuvant aluminium hydroxide Alhydrogel®, Brenntag, Cat. no. 21645-51-2
  • Freund's incomplete adjuvant 20 mice were administered the vaccine, in experiment 2, only 8 mice received the vaccinations.
  • the adjuvant alone was administered .
  • Each mouse was immunized subcutaneously three times at approximately two week intervals. At each immunization the mice were immunized with a formulation of 100
  • Alhydrogel adjuvant mixed with 25 pg polypeptide followed by addition of an equal volume of Freund's incomplete adjuvant. Only aluminium hydroxide was used as adjuvant in the following immunizations (100 pi mixed with 25 pg polypeptide) .
  • the bacteria used in the animal model of peritonitis were prepared in advance and frozen at -80°C in aliquots; bacterial matter was streaked out on a blood agar plate and incubated at 37°C overnight. The following day, a single colony of S. aureus was used for the inoculation of 30 mL tryptic soy broth (TSB) media. The culture was incubated overnight at 37°C, with continuous shaking. The following day 1 L of TSB media was inoculated with 10 mL of the overnight culture and incubated at 37°C under continuous shaking for 6 hours. The bacterial suspension was centrifuged at 3000xG for 10 minutes and the pellet washed twice in 400 mL sterile PBS.
  • TTB tryptic soy broth
  • the bacterial suspension was centrifuged at 3000xG for 10 minutes. The bacterial pellet was resuspended in 10-15 mL PBS and glycerol added to a final concentration of 16%. The suspension was thoroughly mixed, aliquoted in 1 mL aliquots and stored at -80°C. The number of colony forming units (CFU) per mL was determined for the frozen stock, as aliquots were thawed on ice and serially diluted in sterile saline. The dilutions were plated on TSB agar plates and incubated overnight at 37°C. The number of CFU per mL was established the following day. The procedure was repeated with an additional aliquot to confirm homogeny among the aliquots. Immediately prior to challenge, aliquots were thawed and diluted in sterile saline to the desired number of CFU.
  • CFU colony forming units
  • mice were housed at the at the animal facility of the Panum Institute, University of Copenhagen.
  • mice were kept in an environment characterized by a 12-hours light-dark cycle and temperature and humidity control.
  • the mice had access to food and water ad libitum.
  • the experimental procedures were carried out in accordance with the guidelines of the Danish National Animal Ethics Committee.
  • mice were challenged intraperitoneally with 6.5 x 10 9 CFU S. aureus strain MRSA252. The seven days following the challenge, the mice were assessed daily to register symptoms and development of disease. To ensure a consistent evaluation of all animals, each animal was scored individually following the criteria for clinical symptoms set forth here: Score
  • mice were individually assessed on their physical appearance and behaviour, noting the presence or absence of the given characteristics.
  • mice were euthanized if either of the following humane endpoints were reached : a body temperature below 34°C or a weight loss above 20% of the initial body weight. Additionally, mice scored 3 over three successive days, without signs of improvements such as weight gain, or 4 once were euthanized .

Abstract

L'invention concerne des protéines et des acides nucléiques ainsi que des compositions vaccinales où les immunogènes sont dérivés de/liés à un Staphylococcus aureus comprenant SEQ ID NO : 1 ou les séquences de codage SEQ ID NO : 2 et 3. L'invention concerne également des acides nucléiques, des vecteurs, des organismes hôtes transformés, ainsi que des utilisations et des procédés prophylactiques et thérapeutiques. Enfin, l'invention concerne également des procédés de préparation faisant partie de l'invention.
PCT/EP2019/051710 2018-01-24 2019-01-24 Vaccins pour la prophylaxie d'infections par s. aureus WO2019145399A1 (fr)

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Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4684611A (en) 1982-02-11 1987-08-04 Rijksuniversiteit Leiden Process for the in-vitro transformation of plant protoplasts with plasmid DNA
US4879236A (en) 1984-05-16 1989-11-07 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US4952500A (en) 1988-02-01 1990-08-28 University Of Georgia Research Foundation, Inc. Cloning systems for Rhodococcus and related bacteria
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
WO1994009699A1 (fr) 1992-10-30 1994-05-11 British Technology Group Limited Methode d'examen corporel
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5384253A (en) 1990-12-28 1995-01-24 Dekalb Genetics Corporation Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes
WO1995006128A2 (fr) 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production
US5538877A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5591616A (en) 1992-07-07 1997-01-07 Japan Tobacco, Inc. Method for transforming monocotyledons
US5610042A (en) 1991-10-07 1997-03-11 Ciba-Geigy Corporation Methods for stable transformation of wheat
US5656610A (en) 1994-06-21 1997-08-12 University Of Southern California Producing a protein in a mammal by injection of a DNA-sequence into the tongue
US5702932A (en) 1992-07-20 1997-12-30 University Of Florida Microinjection methods to transform arthropods with exogenous DNA
US5736524A (en) 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
WO1998020734A1 (fr) 1996-11-14 1998-05-22 The Government Of The United States Of America, As Represented By The Secretary Of The Army Adjuvant pour immunisation transcutanee
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
US5789215A (en) 1991-08-20 1998-08-04 Genpharm International Gene targeting in animal cells using isogenic DNA constructs
US5871986A (en) 1994-09-23 1999-02-16 The General Hospital Corporation Use of a baculovirus to express and exogenous gene in a mammalian cell
US5925565A (en) 1994-07-05 1999-07-20 Institut National De La Sante Et De La Recherche Medicale Internal ribosome entry site, vector containing it and therapeutic use
US5935819A (en) 1992-08-27 1999-08-10 Eichner; Wolfram Process for producing a pharmaceutical preparation of PDGF-AB
US5945100A (en) 1996-07-31 1999-08-31 Fbp Corporation Tumor delivery vehicles
US5981274A (en) 1996-09-18 1999-11-09 Tyrrell; D. Lorne J. Recombinant hepatitis virus vectors
US5994624A (en) 1997-10-20 1999-11-30 Cotton Incorporated In planta method for the production of transgenic plants
EP1769068A2 (fr) 2004-02-20 2007-04-04 Fraunhofer USA, Inc. Systemes et methodes d'expression clonale dans des plantes
EP2192172A1 (fr) 2003-02-03 2010-06-02 Fraunhofer USA, Inc. Système d'expression de gènes chez les plantes
WO2012136653A1 (fr) 2011-04-08 2012-10-11 Novvac Aps Protéines et acides nucléiques utiles dans des vaccins ciblant le staphylococcus aureus
US20120282247A1 (en) * 2011-05-06 2012-11-08 Olaf Schneewind Methods and compositions involving protective staphylococcal antigens, such as ebh polypeptides
EP2853599A1 (fr) 2002-11-12 2015-04-01 iBio, Inc. Production De Proteines Pharmaceutiquement Actives Dans Des Semis Germes
WO2015082536A1 (fr) 2013-12-03 2015-06-11 Evaxion Biotech Aps Protéines et acides nucléiques utiles dans des vaccins ciblant staphylococcus aureus
WO2017144523A1 (fr) 2016-02-22 2017-08-31 Evaxion Biotech Aps Protéines et acides nucléiques utiles dans des vaccins ciblant staphylococcus aureus
WO2018015575A1 (fr) * 2016-07-22 2018-01-25 Evaxion Biotech Aps Protéines chimériques pour induire une immunité vis-à-vis d'une infection à s. aureus

Patent Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4684611A (en) 1982-02-11 1987-08-04 Rijksuniversiteit Leiden Process for the in-vitro transformation of plant protoplasts with plasmid DNA
US4879236A (en) 1984-05-16 1989-11-07 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US4952500A (en) 1988-02-01 1990-08-28 University Of Georgia Research Foundation, Inc. Cloning systems for Rhodococcus and related bacteria
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
US5464765A (en) 1989-06-21 1995-11-07 Zeneca Limited Transformation of plant cells
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5538877A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5538880A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5384253A (en) 1990-12-28 1995-01-24 Dekalb Genetics Corporation Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes
US5789215A (en) 1991-08-20 1998-08-04 Genpharm International Gene targeting in animal cells using isogenic DNA constructs
US5610042A (en) 1991-10-07 1997-03-11 Ciba-Geigy Corporation Methods for stable transformation of wheat
US5591616A (en) 1992-07-07 1997-01-07 Japan Tobacco, Inc. Method for transforming monocotyledons
US5702932A (en) 1992-07-20 1997-12-30 University Of Florida Microinjection methods to transform arthropods with exogenous DNA
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5935819A (en) 1992-08-27 1999-08-10 Eichner; Wolfram Process for producing a pharmaceutical preparation of PDGF-AB
WO1994009699A1 (fr) 1992-10-30 1994-05-11 British Technology Group Limited Methode d'examen corporel
WO1995006128A2 (fr) 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production
US5656610A (en) 1994-06-21 1997-08-12 University Of Southern California Producing a protein in a mammal by injection of a DNA-sequence into the tongue
US5925565A (en) 1994-07-05 1999-07-20 Institut National De La Sante Et De La Recherche Medicale Internal ribosome entry site, vector containing it and therapeutic use
US5871986A (en) 1994-09-23 1999-02-16 The General Hospital Corporation Use of a baculovirus to express and exogenous gene in a mammalian cell
US5736524A (en) 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
US5945100A (en) 1996-07-31 1999-08-31 Fbp Corporation Tumor delivery vehicles
US5981274A (en) 1996-09-18 1999-11-09 Tyrrell; D. Lorne J. Recombinant hepatitis virus vectors
WO1998020734A1 (fr) 1996-11-14 1998-05-22 The Government Of The United States Of America, As Represented By The Secretary Of The Army Adjuvant pour immunisation transcutanee
US5994624A (en) 1997-10-20 1999-11-30 Cotton Incorporated In planta method for the production of transgenic plants
EP2853599A1 (fr) 2002-11-12 2015-04-01 iBio, Inc. Production De Proteines Pharmaceutiquement Actives Dans Des Semis Germes
EP2192172A1 (fr) 2003-02-03 2010-06-02 Fraunhofer USA, Inc. Système d'expression de gènes chez les plantes
EP1769068A2 (fr) 2004-02-20 2007-04-04 Fraunhofer USA, Inc. Systemes et methodes d'expression clonale dans des plantes
WO2012136653A1 (fr) 2011-04-08 2012-10-11 Novvac Aps Protéines et acides nucléiques utiles dans des vaccins ciblant le staphylococcus aureus
US20120282247A1 (en) * 2011-05-06 2012-11-08 Olaf Schneewind Methods and compositions involving protective staphylococcal antigens, such as ebh polypeptides
WO2015082536A1 (fr) 2013-12-03 2015-06-11 Evaxion Biotech Aps Protéines et acides nucléiques utiles dans des vaccins ciblant staphylococcus aureus
WO2017144523A1 (fr) 2016-02-22 2017-08-31 Evaxion Biotech Aps Protéines et acides nucléiques utiles dans des vaccins ciblant staphylococcus aureus
WO2018015575A1 (fr) * 2016-07-22 2018-01-25 Evaxion Biotech Aps Protéines chimériques pour induire une immunité vis-à-vis d'une infection à s. aureus

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", JOHN WILEY
"Vaccine design: the subunit and adjuvant approach", 1995, PLENUM PRESS
CHEN X ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 65, no. 10, 2013, pages 1357 - 1369
DATABASE Geneseq [online] 3 January 2013 (2013-01-03), "Staphylococcus aureus sta094 antigen, SEQ ID 127.", XP002789679, retrieved from EBI accession no. GSP:BAG33452 Database accession no. BAG33452 *
DATABASE Geneseq [online] 8 March 2018 (2018-03-08), "Staphylococcus aureus derived protein, SEQ ID 5.", XP002789689, retrieved from EBI accession no. GSP:BEY30287 Database accession no. BEY30287 *
DATABASE Geneseq [online] 8 March 2018 (2018-03-08), "Therapeutic chimeric protein USA300HOU_1728-88-452, SEQ ID 96.", XP002789688, retrieved from EBI accession no. GSP:BEY30378 Database accession no. BEY30378 *
DATABASE UniProt [online] 27 May 2015 (2015-05-27), "SubName: Full=Maebl {ECO:0000313|EMBL:AGY89852.1};", XP002789680, retrieved from EBI accession no. UNIPROT:A0A0E1AHA2 Database accession no. A0A0E1AHA2 *
DEERING R.P. ET AL., EXPERT OPIN DRUG DELIV, vol. ll, no. 6, June 2014 (2014-06-01), pages 885 - 99
DONNELLY ET AL., ANNU REV IMMUNOL, vol. 15, 1997, pages 617 - 648
GREEN MR; SAMBROOK J ET AL.: "Molecular Cloning, A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
LARSEN J E P ET AL., IMMUNOME RESEARCH, vol. 2, April 2006 (2006-04-01), pages 2
PETERSEN B ET AL., BMC STRUCTURAL BIOLOGY, vol. 9, July 2009 (2009-07-01), pages 51
PETERSEN B ET AL., PLOS ONE, vol. 5, no. 11, November 2010 (2010-11-01), pages el5079
REMINGTON: "Pharmaceutical Sciences", 1991, MACK PUB. CO.
ROBINSON; TORRES, SEMINARS IN IMMUNOL, vol. 9, 1997, pages 271 - 283

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