EP2984171A2 - Peptides having immune suppresive domains for transfection - Google Patents

Peptides having immune suppresive domains for transfection

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Publication number
EP2984171A2
EP2984171A2 EP14722536.1A EP14722536A EP2984171A2 EP 2984171 A2 EP2984171 A2 EP 2984171A2 EP 14722536 A EP14722536 A EP 14722536A EP 2984171 A2 EP2984171 A2 EP 2984171A2
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EP
European Patent Office
Prior art keywords
peptide
kit
parts
virus
composition according
Prior art date
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EP14722536.1A
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German (de)
French (fr)
Inventor
Shervin Bahrami
Christian KANSTRUP HOLM
Søren RIIS PALUDAN
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Skau Aps
Aarhus Universitet
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Skau Aps
Aarhus Universitet
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Publication of EP2984171A2 publication Critical patent/EP2984171A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a use of a peptide, comprising an immune suppressive domain, for introducing entities, such as foreign entities, to cells.
  • the invention further relates to a use of a peptide, comprising an immune suppressive domain, for transfection.
  • the present invention relates to the use of the peptide for immune suppression in a transfection mix.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term is used notably for non-viral methods in eukaryotic cells.
  • Transfection is used for the introduction of DNA plasmids into live cells with the purpose of expressing either proteins or silencing / interfering RNA encoded in the plasmid.
  • Transfection is also used to introduce immune-stimulatory DNA or RNA into cells with the purpose of studying aspects of immune reactions to DNA or RNA. Further, transfection can be used to introduce non-DNA non-RNA molecules into cells, which directly interfere with cell function.
  • the International patent application WO 98/40502 discloses transfection compositions in which a peptide is covalently linked to a transfection agent. The application purports that enhanced transfection efficiency is obtained.
  • the International patent application WO 2009/79635 discloses a delivery system for delivering interfering RNA molecules into a cell and methods for using the delivery system.
  • Membrane fusion and STING pathway Membrane disturbance as induced by fusion induces type I interferon responses with expression of interferon-stimu!ated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toil- like receptor 7 (TLR7) and TLR9.
  • TLR7 Toil- like receptor 7
  • the fusion-dependent response is dependent on the stimulator of interferon genes STING.
  • Membrane disturbance can be induced by fusion of cellular membranes with viral envelopes, cellular membranes of other cells, and fusogenic liposomes.
  • STING stimulation of interferon genes
  • M ITA/MPYS/ERIS is also essential for cytosolic DNA-mediated type I IFNs induction.
  • STING contains multi-putative transmembrane regions in the amino terminal region, and is found to associate with membranes.
  • immune suppressive domains in the viral fusion proteins is expected to insert the immune suppressive activity partly through interference with this pathway either through direct or indirect interaction with STING.
  • an antagonist of this putative interaction will enhance the immune responses to proteins containing such immune suppressive domains and can be used as adjuvants.
  • fusion proteins typically undergo a conformational transition triggered by receptor recognition or low pH, leading to the insertion of a fusion peptide into the plasma membrane or the membrane of an endocytic vesicle.
  • fusion proteins typically include members of the paramyxovirus family, separate envelope proteins mediate attachment and fusion.
  • Membrane fusion can occur either at the plasma membrane or at an intracellular location following internalization of virus by receptor-mediated endocytosis. Fusion is mediated by viral
  • transmembrane proteins known as fusion proteins.
  • the fusion protein interacts with the target membrane through a hydrophobic fusion peptide and undergoes a conformational change that drives the membrane fusion reaction.
  • fusion triggers including various combinations of receptor binding, receptor/coreceptor binding, and exposure to the mildly acidic pH within the endocytic pathway. Fusion proteins from different viruses have different names in spite of the common functionality.
  • virus membrane fusion proteins are currently annotated to either the "class I" membrane fusion proteins exemplified by the influenza
  • hemagglutinin (HA) or HIV-1 gp41 or the "class II" proteins of the alphaviruses and flaviviruses.
  • the alphaviruses and flaviviruses are members of the Togaviridae and Flaviviridae families, respectively. These small enveloped positive-sense NAviruses are composed of a capsid protein that assembles with the RNA into the nucleocapsid, and a lipid bilayer containing the viral transmembrane (TM) proteins.
  • Class I fusion proteins are synthesized as single chain precursors, which then assemble into trimers.
  • the polypeptides are then cleaved by host proteases, which is an essential step in rendering the proteins fusion competent.
  • This proteolytic event occurs late in the biosynthetic process because the fusion proteins, once cleaved are metastable and readily activated. Once activated, the protein refolds into a highly stable conformation. The timing of this latter event is of crucial importance in the fusion process. Maintenance of the intact precursor polypeptide during folding and assembly of the oligomeric structure is essential if the free energy that is released during the refolding event is to be available to overcome the inherent barriers to membrane fusion.
  • the new amino-terminal region that is created by the cleavage event contains a hydrophobic sequence, which is known as the fusion peptide.
  • the authentic carboxy-terminal region of the precursor polypeptide contains the
  • transmembrane anchor In the carboxy-terminal polypeptide, there are sequences known as the heptad repeat that are predicted to have an alpha helical structure and to form a coiled coil structure. These sequences participate in the formation of highly stable structure that characterizes the post- fusion conformation of the fusion protein.
  • the class II fusion proteins are elongated finger-like molecules with three globular domains composed almost entirely of ⁇ -sheets. Domain I is a ⁇ -barrel that contains the N-terminus and two long insertions that connect adjacent ⁇ -strands and together form the elongated domain II.
  • the first of these insertions contains the highly conserved fusion peptide loop at its tip, connecting the c and d ⁇ -strands of domain II (termed the cd loop) and containing 4 conserved disulfide bonds including several that are located at the base of the fusion loop.
  • the second insertion contains the ij loop at its tip, adjacent to the fusion loop, and one conserved disulfide bond at its base.
  • a hinge region is located between domains I and II.
  • a short linker region connects domain I to domain III, a ⁇ -barrel with an immunoglobulin-like fold stabilized by three conserved disulfide bonds.
  • domain III is followed by a stem region that connects the protein to the virus TM anchor.
  • Fusion peptides are moderately hydrophobic segments of viral and non-viral membrane fusion proteins that enable these proteins to disrupt and connect two closely apposed biological membranes. This process, which results in membrane fusion occurs in a well-controlled manner with a surprisingly small amount of leakage of the contents of the encapsulated volumes to the outside world.
  • the sequences of fusion peptides are highly conserved within different groups of fusion proteins, for example within different virus families, but not between them. Most fusion peptides are located at the extreme N-termini of the transmembrane subunits of the fusion proteins.
  • Fusion proteins of a subset of enveloped Type I viruses have previously been shown to feature an immune suppressive activity. Inactivated retroviruses are able to inhibit proliferation of immune cells upon stimulation. Expression of these proteins is enough to enable allogenic cells to grow to a tumor in immune competent mice. In one study, introduction of ENV expressing construct into MCA205 murine tumor cells, which do not proliferate upon s.c.
  • immunosuppressive domains have been found in a variety of different viruses with type 1 fusion mechanism such as gamma-retroviruses like Mason pfeizer monkey virus (MPMV) and murine leukemia virus (MLV), lentiviruses such as HIV and in filoviruses such as Ebola and Marburg viruses.
  • MPMV Mason pfeizer monkey virus
  • MMV murine leukemia virus
  • lentiviruses such as HIV
  • filoviruses such as Ebola and Marburg viruses.
  • This immune suppressive activity was in all cases located to a very well-defined structure within the class I fusion proteins, more precisely at the bend in the heptad repeat just N-terminale of the transmembrane structure in the fusion protein.
  • the immunosuppressive effects range from significant inhibition of lymphocyte proliferation, cytokine skewing (up regulating IL-10; down regulating TNF-a, IL-12, IFN- ⁇ ) and inhibition of monocytic burst to cytotoxic T cell killing.
  • peptides spanning ISD in these assays must either be linked as dimers or coupled to a carrier (i.e. >monomeric) to be active.
  • Such peptides derived from immune-suppressive domains are able to reduce or abolish immune responses such as cytokine secretion or proliferation of T-cells upon stimulation.
  • the protection mediated by the immunosuppressive properties of the fusion protein from the immune system of the host is not limited to the fusion protein but covers all the viral envelope proteins displayed at viral or cellular membranes in particular also the protein mediating attachment of the virus to the cell.
  • immunosuppressive domains of viruses like but not limited to retro-, lenti-, Orthomyxo-, flavi- and filoviruses overlap structurally important parts of the fusion subunits of the surface
  • glycoproteins In several cases the primary structure (sequence) of the ISD can vary greatly from virus to virus, but the secondary structure, which is very well preserved among different virus families, is that of an alpha helix that bends in different ways during the fusion process This structure plays a crucial role during events that result in fusion of viral and cellular membranes. It is evident that the immunosuppressive domains of these (retroviral, lentiviral and filoviral) class I fusion proteins overlap with a very important protein structure needed for the fusion mechanistic function.
  • the energy needed for mediating the fusion of viral and cellular membranes is stored in the fusion proteins, which are thus found in a meta-stable conformation on the viral surface. Once the energy is released to drive the fusion event, the protein will find its most energetically stable conformation. In this regard fusion proteins can be compared with loaded springs that are ready to be sprung. This high energy conformation makes the viral fusion proteins very susceptible to modifications; Small changes in the primary structure of the protein often result in the protein to be folded in its stable post fusion conformation. The two conformations present very different tertiary structures of the same protein. It has been shown in the case of simple retroviruses that small structural changes in the envelope protein are sufficient to remove the immune suppressive effect without changing structure and hence the antigenic profile.
  • the mutated non-immune suppressive envelope proteins are much better antigens for vaccination.
  • the proteins can induce a 30-fold enhancement of anti-env antibody titers when used for vaccination and are much better at launching an effective CTL response.
  • viruses that contain the non-immunosuppressive form of the friend murine leukemia virus envelope protein although fully infectious in irradiated immunocompromised mice cannot establish an infection in
  • Immunosuppressive domains in the fusion proteins have been known since 1985 for retrovirus, since 1988 for lentivirus and since 1992 for filoviruses. These viruses, as mentioned above, all belong to enveloped RNA viruses with a type I fusion mechanism.
  • the immunosuppressive domains of lentivirus, retroviruses and filoviruses show large structural similarity. Furthermore the immunosuppressive domain of these viruses are all located at the same position in the structure of the fusion protein, more precisely in the linker between the two heptad repeat structures just N-terminal of the transmembrane domain in the fusion protein.
  • the immune suppressive domains can be located in relation to two well conserved cystein residues that are found in these structures. These cystein residues are between 4 and 6 amino acid residues from one another and in many cases are believed to form disulfide bridges that stabilize the fusion proteins.
  • the immune suppressive domains in all three cases include at least some of the first 22 amino acids that are located N-terminal to the first cysteine residue.
  • Immunosuppressive domains are found in type II fusion proteins. Immunosuppressive domains have been identified at different positions in different groups of viruses. For example an immune suppressive domain might co-localize with the fusion peptide exemplified by the identification of an common immunosuppressive domain in the fusion peptide of Flavirius (Dengue virus, west Nile virus etc), or with the hydrophobic alpha helix N-terminal of the transmembrane domain in the fusion protein exemplified by the finding of an immunosuppressive domain in said helixes of all flaviridae e.g. Hepatitis C virus, Dengue, west nile etc.
  • flaviridae e.g. Hepatitis C virus, Dengue, west nile etc.
  • the immune suppressive domains can also be located in the fusion peptide of the fusion protein among enveloped RNA viruses with type I fusion mechanism.
  • HIV or influenza A and B types have an immune suppressive domain that co-localized with their fusion peptide.
  • immunosuppressive domains are identified among enveloped RNA viruses with type II fusion mechanism at different positions in different groups of viruses: i. Co-localizing with the fusion peptide exemplified by the identification of an common
  • Immunosuppressive domains have been identified in the fusion protein among enveloped RNA viruses with type I fusion mechanism. This position co-localizes with the fusion peptide of said fusion protein as demonstrated by the identification of a common immunosuppressive domain in the fusion peptide of all Influenza A and B types as well as HIV.
  • Functional homolog refers to homologues of the molecules according to the present invention and is meant to comprise any molecule which is capable of mimicking the function of molecules as described herein. Thus, the terms refer to functional similarity or, interchangeably, functional identity, between two or more molecular entities.
  • functional homology is further used herein to describe that one molecular entity are able to mimic the function of one or more molecular entities.
  • Functional homologues according to the present invention may comprise any molecule that can function as an antagonist of the immune suppressive activity exerted by an immune suppressive domains.
  • a molecule when added to the composition containing said immune suppressive domains reduces the immune suppressive activity exerted by the latter in either an in vitro test system (e.g. CTLL-2 or PBMC proliferation assays) or in vivo seen as an enhanced T- and/or B-cell responses.
  • Functional homologues according to the present invention may comprise polypeptides with an amino acid sequence, which are sharing at least some homology with the predetermined polypeptide sequences as outlined herein.
  • polypeptides are at least about 40 percent, such as at least about 50 percent homologous, for example at least about 60 percent homologous, such as at least about 70 percent homologous, for example at least about 75 percent homologous, such as at least about 80 percent homologous, for example at least about 85 percent homologous, such as at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with the predetermined polypeptide sequences as outlined herein above.
  • the homology between amino acid sequences may be calculated using well known algorithms such as for example any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
  • Functional homologues may comprise an amino acid sequence that comprises at least one substitution of one amino acid for any other amino acid.
  • a substitution may be a conservative amino acid substitution or it may be a non-conservative substitution.
  • a conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within predetermined groups exhibit similar or substantially similar characteristics.
  • one amino acid may be substituted for another within groups of amino acids characterized by having i) hydrophilic (polar) side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ⁇ ) hydrophobic (non-polar) side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met) iii) aliphatic side chains (Gly, Ala Val, Leu, lie)
  • amino acids being monoamino-dicarboxylic acids or monoamino-monocarboxylic- monoamidocarboxylic acids (Asp, Glu, Asn, Gin).
  • Non-conservative substitutions are any other substitutions.
  • a non-conservative substitution leading to the formation of a functional homologue would for example i) differ substantially in hydrophobicity, for example a hydrophobic residue (Val, lie, Leu, Phe or Met) substituted for a hydrophilic residue such as Arg, Lys, Trp or Asn, or a hydrophilic residue such as Thr, Ser, His, Gin, Asn, Lys, Asp, Glu or Trp substituted for a hydrophobic residue; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having
  • Functional homologues according to the present invention may comprise more than one such substitution, such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution, such as from 75 to 100 amino acid substitutions, for example more than 100 amino acid substitutions.
  • substitutions such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution,
  • the addition or deletion of an amino acid may be an addition or deletion of from 2 to 5 amino acids, such as from 5 to 10 amino acids, for example from 10 to 20 amino acids, such as from 20 to 50 amino acids.
  • additions or deletions of more than 50 amino acids, such as additions from 50 to 200 amino acids are also comprised within the present invention.
  • polypeptides according to the present invention may in one embodiment comprise more than 5 amino acid residues, such as more than 10 amino acid residues, for example more than 20 amino acid residues, such as more than 25 amino acid residues, for example more than 50 amino acid residues, such as more than 75 amino acid residues, for example more than 100 amino acid residues, such as more than 150 amino acid residues, for example more than 200 amino acid residues.
  • the genetic code is the set of rules by which information encoded within genetic material (DNA or m NA sequences) is translated into proteins (amino acid sequences) by living cells. Biological decoding is accomplished by the ribosome, which links amino acids in an order specified by mRNA, using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time.
  • tRNA transfer RNA
  • the code defines how sequences of these nucleotide triplets, called codons, specify which amino acid will be added next during protein synthesis. With some exceptions a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded with exactly the same code (see the RNA codon table), this particular code is often referred to as the canonical or standard genetic code, or simply the genetic code, though in fact some variant codes have evolved. For example, protein synthesis in human mitochondria relies on a genetic code that differs from the standard genetic code. Not all genetic information is stored using the genetic code.
  • All organisms' DNA contains regulatory sequences, intergenic segments, chromosomal structural areas, and other non-coding DNA that can contribute greatly to phenotype. Those elements operate under sets of rules that are distinct from the codon-to-amino acid paradigm underlying the genetic code.
  • Codons Compressed Codons Compressed acid acid
  • GCU GCC, GCA, UUA, UUG, CUU,
  • ACU ACC, ACA,
  • L-amino acids represent all of the amino acids found in proteins during translation in the ribosome
  • D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria, and D-serine may act as a neurotransmitter in the brain.
  • L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of
  • glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotatory).
  • Fatty acids, or fatty acid residues when they form part of a lipid are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis. They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used as a building-block of more structurally complex lipids.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur.
  • a double bond exists, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration.
  • Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. This in turn plays an important role in the structure and function of cell membranes.
  • Most naturally occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and partially hydrogenated fats and oils.
  • biologically important fatty acids are the eicosanoids, derived primarily from arachidonic acid and eicosapentaenoic acid, that include prostaglandins, leukotrienes, and thromboxanes.
  • Docosahexaenoic acid is also important in biological systems, particularly with respect to sight.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines.
  • the fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide.
  • Glycerolipids are composed mainly of mono-, di-, and tri-substituted glycerols, the most well-known being the fatty acid triesters of glycerol, called triglycerides.
  • the word "triacylglycerol” is sometimes used synonymously with "triglyceride", though the latter lipid contains no hydroxyl group.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Because they function as an energy store, these lipids comprise the bulk of storage fat in animal tissues.
  • the hydrolysis of the ester bonds of triglycerides and the release of glycerol and fatty acids from adipose tissue are the initial steps in metabolising fat.
  • glycosylglycerols are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • structures in this category are the digalactosyldiacylglycerols found in plant membranes and seminolipid from mammalian sperm cells.
  • Glycerophospholipids usually referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and cell signaling.
  • Neural tissue including the brain contains relatively high amounts of glycerophospholipids, and alterations in their composition has been implicated in various neurological disorders.
  • Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or the sn-1 position in the case of archaebacteria.
  • Examples of glycerophospholipids found in biological membranes are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • glycerophospholipids in eukaryotic cells such as phosphatidylinositols and phosphatidic acids are either precursors of or, themselves, membrane- derived second messengers.
  • phosphatidylinositols and phosphatidic acids are either precursors of or, themselves, membrane- derived second messengers.
  • one or both of these hydroxyl groups are acylated with long- chain fatty acids, but there are also alkyl-linked and lZ-alkenyl-linked (plasmalogen)
  • glycerophospholipids as well as dialkylether variants in archaebacteria.
  • Sphingolipids are a complicated family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids and other compounds.
  • the major sphingoid base of mammals is commonly referred to as sphingosine.
  • Ceramides are a major subclass of sphingoid base derivatives with an amide-linked fatty acid.
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterol lipids such as cholesterol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • the steroids all derived from the same fused four-ring core structure, have different biological roles as hormones and signaling molecules.
  • the eighteen-carbon (C18) steroids include the estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone.
  • the C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids.
  • the secosteroids comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure.
  • sterols are the bile acids and their conjugates, which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver.
  • the plant equivalents are the phytosterols, such as ⁇ -sitosterol, stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth.
  • the predominant sterol in fungal cell membranes is ergosterol.
  • Prenol lipids are synthesized from the five-carbon-unit precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid (MVA) pathway.
  • the simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes.
  • Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A.
  • quinones and hydroquinones which contain an isoprenoid tail attached to a quinonoid core of non-isoprenoid origin.
  • Vitamin E and vitamin K as well as the ubiquinones, are examples of this class.
  • Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a saccharolipid In the saccharolipids, a
  • glycerophospholipids The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains.
  • the minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes.
  • anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, avermectins, and antitumor epothilones.
  • the glycerophospholipids are the main structural component of biological membranes, such as the cellular plasma membrane and the intracellular membranes of organelles; in animal cells the plasma membrane physically separates the intracellular components from the extracellular environment.
  • the glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage. While
  • glycerophospholipids are the major component of biological membranes, other non-glyceride lipid components such as sphingomyelin and sterols (mainly cholesterol in animal cell membranes) are also found in biological membranes.
  • sphingomyelin and sterols mainly cholesterol in animal cell membranes
  • galactosyldiacylglycerols, and sulfoquinovosyldiacylglycerol which lack a phosphate group, are important components of membranes of chloroplasts and related organelles and are the most abundant lipids in
  • Bilayers have been found to exhibit high levels of birefringence, which can be used to probe the degree of order (or disruption) within the bilayer using techniques such as dual polarization interferometry and Circular dichroism.
  • a biological membrane is a form of lipid bilayer.
  • the formation of lipid bilayers is an energetically preferred process when the glycerophospholipids described above are in an aqueous environment. This is known as the hydrophobic effect.
  • the hydrophobic tails minimize their contact with water and tend to cluster together, forming a vesicle; depending on the concentration of the lipid, this biophysical interaction may result in the formation of micelles, liposomes, or lipid bilayers.
  • Other aggregations are also observed and form part of the polymorphism of amphiphile (lipid) behavior.
  • Phase behavior is an area of study within biophysics and is the subject of current academic research.
  • Micelles and bilayers form in the polar medium by a process known as the hydrophobic effect.
  • the polar molecules i.e., water in an aqueous solution
  • the polar molecules become more ordered around the dissolved lipophilic substance, since the polar molecules cannot form hydrogen bonds to the lipophilic areas of the amphiphile. So in an aqueous environment, the water molecules form an ordered "clathrate" cage around the dissolved lipophilic molecule.
  • the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
  • a dimer or multimer may comprise more than one immune suppressive domain motif.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
  • the invention concerns a use of a molecule, which comprises at least two parts, each part comprising an immune suppressive domain, for transfection.
  • the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response.
  • the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
  • the invention concerns a cell obtainable with said method for transfecting a cell.
  • the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy.
  • the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term "for transfection” is meant to encompass that the peptide is used as part of the transfection process, for example together with at least one transfection agent, and/or for preparing a cell for additional steps of the transfection process.
  • immune suppressive activity means that it can inhibit proliferation of CTLL-2 or PBMCs in assays as described in the examples, i.e. by more than 20% or interfere with type I IFN production in response to lipid based formulations.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
  • peptide as used here, comprises shorter amino sequences as well as longer sequences such as proteins, and monomers as well as dimers and multimers.
  • the immune suppressive peptides do not need to be covalently bound to form a dimer or multimer. If they contain multimerization domains, the monomeric peptides may associate together through non-covalent interactions and form dimers or multimers that will also be functional.
  • the invention concerns the use of a peptide of the invention, wherein said peptide is a dimer.
  • the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
  • the word "or” is not necessarily an exclusive expression.
  • a dimer or multimer may comprise more than one immune suppressive domain motif, and still be within the scope of the present invention.
  • the invention concerns the use of the invention, wherein said at least two immune suppressive domain motifs are two identical or different motifs.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
  • the invention concerns the use of a peptide according to the invention, which is soluble in water.
  • the peptide is preferably sufficiently soluble to provide immune suppressive activity in an aqueous solution.
  • the invention concerns the use of a peptide according to the invention, wherein said immune suppressive domain is part of a virus.
  • the invention concerns the use of a peptide of the invention, wherein said virus is an influenza virus.
  • the invention concerns the use of a peptide according to the invention, wherein said peptide forms part of a protein.
  • the invention concerns a use of a molecule, which comprises at least two parts, each comprising an immune suppressive domain, for transfection.
  • the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns the kit-of-parts or composition of the invention, wherein said transfection agent is coupled covalentely to said peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based transfection agent.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said lipid based transfection agent is selected among an anionic and a cationic transfection agent.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based liposome or virosome or viral vector.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a nano particle.
  • a nanoparticle may e.g. be used for drug delivery, it may have an inherent therapeutic effect, or it may be fluorescent and used for tests.
  • the invention concerns the kit-of-parts or composition according to the invention, further comprising a hydrophobic vehicle for delivery of hydrophobic molecules.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a drug.
  • the immune suppressive domain may suppress an unwanted immune response induced by the drug, which e.g. may comprise a carrier oil, known to induce an immune response.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is an immune suppressive peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein of a pathogen.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said pathogen is a virus.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein on the surface of a pathogen.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a virus surface glycoprotein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide forms part of an enveloped virus surface glycoprotein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide has a length of at least 6, preferably 7, more preferred 8, preferably 9, more preferred 10, preferably 11, more preferred 12, preferably 13, more preferred 14, preferably 15, more preferred 16, preferably 17, more preferred 18, preferably 19, more preferred 20, preferably 21 more preferred 22, preferably 23, more preferred 24, preferably 25 amino acids.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has a length selected among 5 - 200, preferably 10 - 100, more preferred 20 - 50, preferably 30 - 40 amino acids.
  • the invention concerns the kit-of-parts or composition according to the invention, further comprising a fusion peptide from a fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from the fusion protein of an enveloped virus.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from a type I fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a fusion peptide from a type II fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide has 1, 2, 3 or 4 mutations, deletions or insertions with respect to the wild type.
  • mutation is used with a number about this number of point mutation(s), i.e. 3 mutations mean 3 point mutations.
  • deletion is used with a number about the deletion of this number of amino acid(s), i.e. 2 deletions means the deletion of 2 amino acids.
  • insertion is used with a number about insertion of this number of amino acid(s), i.e. 1 insertion means the insertion of 1 amino acid.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, binds either directly or indirectly to a cellular protein complex containing the protein STING encoded by the gene Tmeml73.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses induced by membrane fusion.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from selected among the group consisting of the lists of Table 1 and among the sequences with Seq. Id. 1 - 287.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from an influenza virus or a Flu peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has immune suppressive activity as dimer or mulitimer or when coupled to carrier proteins.
  • the coupling of peptides to larger proteins is one of the known methods for producing mulitimeric peptides or antigens.
  • Proteins such as Bovine serum albumin (BSA) or Keyhole limpet hemocyanin (KLH) can be used as carrier proteins.
  • BSA Bovine serum albumin
  • KLH Keyhole limpet hemocyanin
  • KLH is used extensively as a carrier protein in the production of antibodies for research
  • KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues used as coupling sites.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one non-genetically encoded amino acid residue.
  • the non-genetically encoded amino acid residues are amino acid residues, which are not genetically encoded.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid residue.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to any other molecule.
  • Two peptides may be joined via another molecule, and each of the two peptides may comprise an immune suppressive domain sequence.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to lipids.
  • Lipids are here defined as cationic, anionic or neutrally charged fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol).
  • Liposomes may be used as a delivery system, and vesicles may be at least partly covered by immune suppressive domains, thereby suppressing a potential immune system response to the delivery system.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a molecule through a peptide bond.
  • the molecule may e.g. be a ligand of a receptor, thereby targeting the peptide, or it may e.g. be a molecule providing different solubility characteristics of the combination of the peptide and the molecule as compared to the peptide alone, or the molecule may be a nanoparticle.
  • the peptide may further form part of a protein, which may provide advantages such as easy production, as the protein may be derived from natural sources.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is a circular peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to at least one biological membrane.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is modified in a way in which one of the peptide bonds is replaced by a non-peptide bond.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide interferes with an interferon response induced by the transfection agent upon addition to a cell.
  • the invention concerns the kit-of-parts or composition comprising a functional homologue of a peptide according to the invention.
  • a functional homologue may be replaced by an altered peptide, obtained from a peptide of the invention, by making 1, 2, 3, or 4 mutations, deletions or insertions of the immune suppressive domain.
  • An alternated peptide may have a %identity vis-a-vis the unaltered form of at least 60%, preferably at least 70%, more preferred at least 80%, preferably at least 90%, more preferred at least 95%, preferably at least 98%, more preferred at least 99%.
  • the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for introducing a molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a DNA or NA molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a tissue.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a gene-therapeutic pharmaceutical molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for initiating expression of proteins via transfected plasmids. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for targeting active genes by micro NA silencing.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for examining the immune response to DNA or RNA. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for gene therapy.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for delivery of any molecule using liposomes.
  • the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
  • the invention concerns a cell obtainable with said method for transfecting a cell.
  • the cells obtainable by this process produce less or no cytokines, in particular interferons, compared to cells obtained without the use of the peptides of the invention. I.e. when compared to untreated cells, cells treated with the INF-F#2 peptide produce less or no cytokines, in particular interferons, but appear to retain the ability to react to other and stronger stimuli.
  • the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. Such a cell may be used e.g.
  • the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the peptide may e.g. be bound to the surface of the cell, internally or externally, or may be present inside of the cell.
  • the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy.
  • the invention concerns the use according to the invention, for cell therapy and/or gene therapy.
  • Gene therapy and cell therapy are overlapping fields of biomedical research with similar therapeutic goals.
  • Gene therapy can be defined as the use of genetic material (usually deoxyribonucleic acid - DNA) to manipulate a patient's cells for the treatment of an inherited or acquired disease.
  • Cell Therapy can be defined as the infusion or transplantation of whole cells into a patient for the treatment of an inherited or acquired disease.
  • the invention concerns the use according to the invention, for stem cell transplantation.
  • Table 1 is provided below. Table 1 likewise provides a number of immunosuppressive domains and motifs.
  • Flavivirus sp Flavivirus sp .
  • Hepa Hepatitis C Hepatitis C virus genotype 1 a seqid3
  • Border disease virus 2 SYFQQYMLKGQYQYWFDLE
  • Rubella virus strain TO-336 MSVFALASYVQHPHKTVRVKFHT vaccine .
  • Phlebovirus JS24 Phlebovirus JS24
  • Phlebovir sp Phlebovir sp .
  • Bovine parainfluenza virus 3 Porcine paramyxovirus strain Frost Porcine paramyxovirus strain Texas Human parainfluenza virus 1 Human parainfluenza virus 3 Simian Agent 10
  • Tuhoko virus 3 unclassified Atlantic salmon paramyxovirus Paramyxovirinae Beilong virus
  • the I N F-F#2 peptide has the sequence GLFGAIAGFI ENGWEGCGGEKEKEK [Seq id 287] and is dimerized through a disulfide bridge between the cystein residues.
  • Fig. 1 shows type I IFN production in cells either untreated (UT) or treated with cationic liposomes.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • treatment with cationic liposomes induce the production of type I I FN and that this production was inhibited when cells were pre-treated with IN F-F#2.
  • the cells used were bonemarrow derived dendritic cells (BM DCs) from mice of the laboratory strain C57BL.
  • the peptide IN F-F#2 was used at a concentration of ⁇ and initiated 15minutes before treatment with cationic liposomes.
  • Cells were then left to incubate 16 hours at 5% C0 2 and 37.5 degrees Celcius. After 16 hours of incubation the supernatants were harvested. Type I I FN activity was then measured using a type I I FN bioassay.
  • Cationic liposomes were prepared using a mix of the lipids DOTAP/DOPE/L (Lissamine Rhodamine)- DOPE in a w/w/w of 1/1/0.1 dissolved in chloroform.
  • the mix was purchased from the company Avanti Polar Lipids, inc. Chloroform was evaporated using rotation evaporation using a dry nitrogen stream. The lipids film was then dried in vaccum for 4 hours. To created liposomes, the lipid film was hydrated in phosphate buffered saline solution at pH 7.4 at ambient temperature. Liposomes were then size restricted using a 0.2 ⁇ filter and an Avanti Mini-extruder purchased form Avanti Polar Lipids. Fig.
  • FIG. 2 shows type I I FN production in cells either untreated (UT) or treated with the transfection reagent Lipofectamine2000 (Lipofect) according to the manufactures instructions.
  • Lipofectamine2000 was used alone and not with the purpose of transfecting DNA or RNA into the cells.
  • the type I IFN produces therefore represent the cellular response to the transfection reagent Lipofectamine2000 itself.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • treatment with Lipofectamine2000 induced the production of type I IFN and that this production was inhibited when cells were pre-treated with IN F-F#2.
  • Cells used were BM DCs as described in the Figure 1 text.
  • Fig. 3 shows type I IFN production in cells either untreated (UT) or transfected with double stranded DNA (dsDNA) using the reagent Lipofectamine2000 according to the manufactures instructions.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • transfection with dsDNA induces the production of type I IFN and that this production was NOT inhibited when cells were pre-treated with IN F-F#2. Therefore, pretreatment does not interfere with type I I FN levels induced by the transfected dsDNA but only with the type I I FN induced by the transfection reagent itself.
  • Cells used were BM DCs as described in the Figure 1 text.
  • Beads were then collected by centrifugation at 2000xg for 2min. Beads were then washed x3 in PBS at pH 7.4. Beads were then headed to 95 deg. Celcius in reducing denaturating loading buffer for 5 min. yielding the pull-down lysate. This was then loaded and run on a SDS-PAGE for 45 min at 120V. Proteins were then transferred to protein PVDF membrane and blotted for STING and Giantin. The protein Giantin is a golgi-resident transmembrane protein unrelated to STING function. As depicted in figure 4, the INF-F#2 but not beads alone or the peptide DI6 was able to precipitate STING. Further, INF-F#2 was unable to precipitate other transmembrane proteins such as Giantin.
  • the peptide INF-F#2 therefore blocks STING dependent type I IFN production in response to membrane disturbance as introduced by liposome based transfection agents. This is possibly through direct or indirect interaction with STING itself as INF-F#2 binds either directly to STING or to a complex which contains STING. This occurs without significant interference with type I IFN production with response to transfected immuno-stimulatory DNA itself (Fig. 3)
  • Fig. 5 shows the effect of INF-F#2 (INF ISD) on STING dimerization in response to cationic liposomes.
  • Cells of the human moncytic cell line THP-1 were either culture in the presence or absence of INF-F#2 (INF ISD) as depicted. Cells were then either treated with or left untreated with cationic liposomes (as in Figure 1) for 3.5 hours. Cells were then lysed using IPA lysis buffer with
  • Pretreatment of cells with INF-F#2 resultet in an inhibition of the formation of STING dimers.
  • Light panel Lysates from cells treated with cationic liposomes either treated with the reducing agent DTT or left untreated for 5min.
  • INF-F#2 binds either directly to STING or to a STING containing complex ( Figure 4), and interferes with STING dimerization in response to cellular treatment with cationic liposomes.
  • Figure 6 shows inflammation-related enzyme and transcription factor gene expression kinetics of THP-1 monocytes stimulated with ⁇ g/ml LPS. Gene expression was expressed as relative gene expression towards RPL13a-expression and non-stimulated cells at time zero (AACt). Data shown are means + standard deviation from two independent biological replications.
  • FIG. 7 shows effects of INF-F#2 peptide on expression of NF-kappaB mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 8 shows effects of INF-F#2 peptide on expression of SP-1 mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG 9 shows effects of INF-F#2 peptide on protein secretion of IL-8 in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the median ⁇ standard deviation from three independent experiments performed in duplicates.
  • Figure 10 shows effects of INF-F#2 peptide on protein secretion of IL-10 in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS.
  • Figure 12 shows expression kinetics of IFN gamma expression in response to PMA/ionomycin treatment. Gene expression was expressed as relative gene expression towards RPL13a expression and non-stimulated cells at time zero ( ⁇ Ct). Data shown are the medians ⁇ standard deviation from three independent technical replicates.
  • Figure 13 shows effect of INF-F#2 peptide on secretion of protein of IFN-gamma in PMA/ionomycin stimulated PBMCs.
  • PBMCs were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ or 60 ⁇ control peptide, and stimulated with 50ng/ml PMA and ⁇ g/ml ionomycin.
  • Data shown are the medians ⁇ standard deviation from three independent experiments performed in duplicates.
  • Figure 14 shows effects of SARS ([Seq id 285] AEVQIDRLITGRLQSLQTYVCGGEKEKEK) or Filo ISD ([Seq id 286] GAAIGLAWIPYFGPAAECGGEKEKEK) on expression of TNF-alpha mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 15 shows effects of SARS or Filo ISD on expression of IL-1 ⁇ mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 16 shows effects of SARS or Filo ISD on expression of IL-1 ⁇ mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • Figure 17 shows interactions between INF-F#2 peptide and STING depends on distinct STING domains.
  • STING was either in a wt form or with deletions. Lysates from tansfected cells were used for pulldown using biotinylated INF-F#2 peptide and streptavidin coated beads. The bead eluate was then immunoblotted using antibodies against HA-tag.
  • FIG. 18 Bonemarrow derived dendritic cells (BM DCs) were pretreated with indicated peptides at 5 ⁇ . After 30min cells were then treated with cationic liposomes (Lipo) or iwht lmw(low molecular weight) ds NA (Poly l:C). After 18 hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells.
  • VSV vesicular stomatitis virus
  • INF wt is the INF-F#2 peptide.
  • INF D4-6 and INF DI6 are deletion mutants of the INF ISD (negative controls) INF mono is the monomeric form of the peptide that does not have any effect.
  • the data show that the INF-F#2 peptide inhibits interferon production induced by liposom fusion with the cells.
  • Figure 19 a+b) BMDCs were pretreated with indicated peptides at 5 ⁇ . After 30min cells were then treated with dsDNA ⁇ g/mL) by transfection using lipofectamine 2000. After 18hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells, c-e) Human monocyte derived macrophages were treated with virus like particles (Vlp) or cationic liposomes. Cells were either not pretreated or pretreated with INF-F#2 peptide (pFlu). After 4 hours cells were fixed, stained for STING (green) and with DAPI (blue).
  • VSV vesicular stomatitis virus
  • Vlp virus like particles
  • pFlu cationic liposomes
  • Influenza peptide or pFlu are identical to INF ISD peptide which is identical to INF-F#2 peptide.
  • Figure 20 BMDCs were treated with Iipofectamine2000 according to manufactures directions for usage of Iipofectamine2000 for transfection with DNA (but in this instance without DNA). After 18 hours supernatants were harvested and analyzed for type I IFN by bioassay. Before Iipofectamin2000 treatment cells received either no pretreatment or pretreatment for 30 minutes with INF-F#2 peptide (product). The data shows that the INF-F#2 peptide prevents production of IFN caused by lipofectamine transfection.
  • INF-F#2 a peptide from Influenza HA2, blocks type I IFN production induced by membrane fusion.
  • transfection reagents such as Lipofectamine2000 from Invitrogen
  • Lipofectamine2000 is based on cationic liposomes and function because such liposomes can transport charged molecules such as DNA into living cells.
  • the reagent itself is harmful to the treated cells and induce these to produce cytokines such as type I IFN.
  • INF-F#2 could also inhibit the response to Lipofectamine2000 we pretreated BMDCs with INF-F#2 for 30min and then treated the cells with Lipofectamine2000 according to instructions by Invitrogen. As seen in Figure 2, INF-F#2 completely blocked the type I IFN response to Lipofectamine.
  • INF-F#2 blocks STING signalling in response to cationic liposomes

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Abstract

The present invention relates to a use of a peptide, comprising an immune suppressive domain, for transfection. In particular, the present invention relates to the use of the peptide for immune suppression in a transfection mix, wherein the peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.

Description

PEPTIDES HAVING IM MUNE SUPPRESIVE DOMAINS FOR TRANSFECTION
The present invention relates to a use of a peptide, comprising an immune suppressive domain, for introducing entities, such as foreign entities, to cells. The invention further relates to a use of a peptide, comprising an immune suppressive domain, for transfection. In particular, the present invention relates to the use of the peptide for immune suppression in a transfection mix.
Technical Background
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells. Transfection is used for the introduction of DNA plasmids into live cells with the purpose of expressing either proteins or silencing / interfering RNA encoded in the plasmid. Transfection is also used to introduce immune-stimulatory DNA or RNA into cells with the purpose of studying aspects of immune reactions to DNA or RNA. Further, transfection can be used to introduce non-DNA non-RNA molecules into cells, which directly interfere with cell function.
The International patent application WO 98/40502 discloses transfection compositions in which a peptide is covalently linked to a transfection agent. The application purports that enhanced transfection efficiency is obtained.
The International patent application WO 2009/79635 discloses a delivery system for delivering interfering RNA molecules into a cell and methods for using the delivery system.
Membrane fusion and STING pathway Membrane disturbance as induced by fusion induces type I interferon responses with expression of interferon-stimu!ated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toil- like receptor 7 (TLR7) and TLR9. The fusion-dependent response is dependent on the stimulator of interferon genes STING. Membrane disturbance can be induced by fusion of cellular membranes with viral envelopes, cellular membranes of other cells, and fusogenic liposomes. The molecule referred to as STING (stimulator of interferon genes) also known as M ITA/MPYS/ERIS is also essential for cytosolic DNA-mediated type I IFNs induction. STING contains multi-putative transmembrane regions in the amino terminal region, and is found to associate with membranes.
The existence of immune suppressive domains in the viral fusion proteins is expected to insert the immune suppressive activity partly through interference with this pathway either through direct or indirect interaction with STING. Hence an antagonist of this putative interaction will enhance the immune responses to proteins containing such immune suppressive domains and can be used as adjuvants.
Increased cellular production of type I IFNs and other cytokines in response to treatment with liposome based transfection reagent is a considerable problem. In the cases of introducing plasmids into live cells, the reaction to the transfection reagent itself (the increased production of type ί IFN and cytokines) can result in undesirable responds to the introduced plasmid. Further, when studying immune responses to DNA or RNA, production of type I IFN and other cytokines are important measures for such responses. Therefore, for the transfection reagent itself to induce of type I IFN and other cytokines is undesirable. In some cases, the response to the transfection reagent itself overwhelms the response to the delivered DNA or RNA. Typically, in viruses one or mores transmembrane glycoproteins, fusion proteins, undergo a conformational transition triggered by receptor recognition or low pH, leading to the insertion of a fusion peptide into the plasma membrane or the membrane of an endocytic vesicle. For some viruses, for example members of the paramyxovirus family, separate envelope proteins mediate attachment and fusion.
Membrane fusion can occur either at the plasma membrane or at an intracellular location following internalization of virus by receptor-mediated endocytosis. Fusion is mediated by viral
transmembrane proteins known as fusion proteins. Upon appropriate triggering, the fusion protein interacts with the target membrane through a hydrophobic fusion peptide and undergoes a conformational change that drives the membrane fusion reaction. There are a variety of fusion triggers, including various combinations of receptor binding, receptor/coreceptor binding, and exposure to the mildly acidic pH within the endocytic pathway. Fusion proteins from different viruses have different names in spite of the common functionality.
Based on important structural features, many virus membrane fusion proteins are currently annotated to either the "class I" membrane fusion proteins exemplified by the influenza
hemagglutinin (HA) or HIV-1 gp41, or the "class II" proteins of the alphaviruses and flaviviruses. The alphaviruses and flaviviruses are members of the Togaviridae and Flaviviridae families, respectively. These small enveloped positive-sense NAviruses are composed of a capsid protein that assembles with the RNA into the nucleocapsid, and a lipid bilayer containing the viral transmembrane (TM) proteins.
Class I fusion proteins are synthesized as single chain precursors, which then assemble into trimers. The polypeptides are then cleaved by host proteases, which is an essential step in rendering the proteins fusion competent. This proteolytic event occurs late in the biosynthetic process because the fusion proteins, once cleaved are metastable and readily activated. Once activated, the protein refolds into a highly stable conformation. The timing of this latter event is of crucial importance in the fusion process. Maintenance of the intact precursor polypeptide during folding and assembly of the oligomeric structure is essential if the free energy that is released during the refolding event is to be available to overcome the inherent barriers to membrane fusion. The new amino-terminal region that is created by the cleavage event contains a hydrophobic sequence, which is known as the fusion peptide. The authentic carboxy-terminal region of the precursor polypeptide contains the
transmembrane anchor. In the carboxy-terminal polypeptide, there are sequences known as the heptad repeat that are predicted to have an alpha helical structure and to form a coiled coil structure. These sequences participate in the formation of highly stable structure that characterizes the post- fusion conformation of the fusion protein. The class II fusion proteins are elongated finger-like molecules with three globular domains composed almost entirely of β-sheets. Domain I is a β-barrel that contains the N-terminus and two long insertions that connect adjacent β-strands and together form the elongated domain II. The first of these insertions contains the highly conserved fusion peptide loop at its tip, connecting the c and d β-strands of domain II (termed the cd loop) and containing 4 conserved disulfide bonds including several that are located at the base of the fusion loop. The second insertion contains the ij loop at its tip, adjacent to the fusion loop, and one conserved disulfide bond at its base. A hinge region is located between domains I and II. A short linker region connects domain I to domain III, a β-barrel with an immunoglobulin-like fold stabilized by three conserved disulfide bonds. In the full-length molecule, domain III is followed by a stem region that connects the protein to the virus TM anchor. Fitting of the structure of alphavirus El to cryo-electron microscopy reconstructions of the virus particle reveals that El is located almost parallel to the virus membrane, and that El-El interactions form the an icosahedral lattice. Fusion peptides
Fusion peptides are moderately hydrophobic segments of viral and non-viral membrane fusion proteins that enable these proteins to disrupt and connect two closely apposed biological membranes. This process, which results in membrane fusion occurs in a well-controlled manner with a surprisingly small amount of leakage of the contents of the encapsulated volumes to the outside world. The sequences of fusion peptides are highly conserved within different groups of fusion proteins, for example within different virus families, but not between them. Most fusion peptides are located at the extreme N-termini of the transmembrane subunits of the fusion proteins. However, in a few cases such as the sperm protein fertilin-a, vesicular stomatitis virus G, baculovirus gp64, and Rous sarcoma virus gp37, internal fusion peptides have been found. Deletion of the fusion peptide and, in many cases, even relatively conservative single amino acid changes in the fusion peptide completely abolish the ability of fusion proteins to fuse membranes, while other structural and functional properties of these proteins may remain intact. Conversely, single amino acid changes in many other regions of these proteins are less deleterious to their function. Such mutagenesis experiments clearly point to a central role of the fusion peptides in membrane fusion. It has further been shown in a number of cases that even isolated fusion peptides alone can support membrane fusion in model systems. (Tamm and Han, Bioscience Reports, Vol. 20, No. 6, 2000).
Immune suppressive domains - Immunosuppressive properties of enveloped viruses
Fusion proteins of a subset of enveloped Type I viruses (retrovirus, lentivirus and filoviruses) have previously been shown to feature an immune suppressive activity. Inactivated retroviruses are able to inhibit proliferation of immune cells upon stimulation. Expression of these proteins is enough to enable allogenic cells to grow to a tumor in immune competent mice. In one study, introduction of ENV expressing construct into MCA205 murine tumor cells, which do not proliferate upon s.c.
injection into an allogeneic host, or into CL8.1 murine tumor cells (which overexpress class I antigens and are rejected in a syngeneic host) resulted in tumor growth in both cases. Such
immunosuppressive domains have been found in a variety of different viruses with type 1 fusion mechanism such as gamma-retroviruses like Mason pfeizer monkey virus (MPMV) and murine leukemia virus (MLV), lentiviruses such as HIV and in filoviruses such as Ebola and Marburg viruses.
This immune suppressive activity was in all cases located to a very well-defined structure within the class I fusion proteins, more precisely at the bend in the heptad repeat just N-terminale of the transmembrane structure in the fusion protein. The immunosuppressive effects range from significant inhibition of lymphocyte proliferation, cytokine skewing (up regulating IL-10; down regulating TNF-a, IL-12, IFN-γ) and inhibition of monocytic burst to cytotoxic T cell killing. Importantly, peptides spanning ISD in these assays must either be linked as dimers or coupled to a carrier (i.e. >monomeric) to be active. Such peptides derived from immune-suppressive domains are able to reduce or abolish immune responses such as cytokine secretion or proliferation of T-cells upon stimulation. The protection mediated by the immunosuppressive properties of the fusion protein from the immune system of the host is not limited to the fusion protein but covers all the viral envelope proteins displayed at viral or cellular membranes in particular also the protein mediating attachment of the virus to the cell.
Co-location of the immunosuppression domain and the fusion domain
The immunosuppressive domains of viruses like but not limited to retro-, lenti-, Orthomyxo-, flavi- and filoviruses overlap structurally important parts of the fusion subunits of the surface
glycoproteins. In several cases the primary structure (sequence) of the ISD can vary greatly from virus to virus, but the secondary structure, which is very well preserved among different virus families, is that of an alpha helix that bends in different ways during the fusion process This structure plays a crucial role during events that result in fusion of viral and cellular membranes. It is evident that the immunosuppressive domains of these (retroviral, lentiviral and filoviral) class I fusion proteins overlap with a very important protein structure needed for the fusion mechanistic function.
The energy needed for mediating the fusion of viral and cellular membranes is stored in the fusion proteins, which are thus found in a meta-stable conformation on the viral surface. Once the energy is released to drive the fusion event, the protein will find its most energetically stable conformation. In this regard fusion proteins can be compared with loaded springs that are ready to be sprung. This high energy conformation makes the viral fusion proteins very susceptible to modifications; Small changes in the primary structure of the protein often result in the protein to be folded in its stable post fusion conformation. The two conformations present very different tertiary structures of the same protein. It has been shown in the case of simple retroviruses that small structural changes in the envelope protein are sufficient to remove the immune suppressive effect without changing structure and hence the antigenic profile.
The mutated non-immune suppressive envelope proteins are much better antigens for vaccination. The proteins can induce a 30-fold enhancement of anti-env antibody titers when used for vaccination and are much better at launching an effective CTL response. Furthermore, viruses that contain the non-immunosuppressive form of the friend murine leukemia virus envelope protein, although fully infectious in irradiated immunocompromised mice cannot establish an infection in
immunocompetent animals. Interestingly in the latter group the non-immunosuppressive viruses induce both a higher cellular and humeral immune response, which fully protect the animals from subsequent challenge by wild type viruses.
Immunosuppressive domains in the fusion proteins (viral envelope proteins) from Retroviruses, lentiviruses and Filoviruses have been known since 1985 for retrovirus, since 1988 for lentivirus and since 1992 for filoviruses. These viruses, as mentioned above, all belong to enveloped RNA viruses with a type I fusion mechanism. The immunosuppressive domains of lentivirus, retroviruses and filoviruses show large structural similarity. Furthermore the immunosuppressive domain of these viruses are all located at the same position in the structure of the fusion protein, more precisely in the linker between the two heptad repeat structures just N-terminal of the transmembrane domain in the fusion protein. These heptad repeat regions constitute two alpha helices that play a critical role in the active mechanism of membrane fusion by these proteins. The immune suppressive domains can be located in relation to two well conserved cystein residues that are found in these structures. These cystein residues are between 4 and 6 amino acid residues from one another and in many cases are believed to form disulfide bridges that stabilize the fusion proteins. The immune suppressive domains in all three cases include at least some of the first 22 amino acids that are located N-terminal to the first cysteine residue. Recently the immunosuppressive domains in the fusion protein of these viruses have been successfully altered in such a way that the fusogenic properties of the fusion protein have been preserved. Such mutated fusion proteins with decreased immunosuppressive properties have been shown to be superior antigens for vaccination purposes.
Other immunosuppressive domains are found in type II fusion proteins. Immunosuppressive domains have been identified at different positions in different groups of viruses. For example an immune suppressive domain might co-localize with the fusion peptide exemplified by the identification of an common immunosuppressive domain in the fusion peptide of Flavirius (Dengue virus, west Nile virus etc), or with the hydrophobic alpha helix N-terminal of the transmembrane domain in the fusion protein exemplified by the finding of an immunosuppressive domain in said helixes of all flaviridae e.g. Hepatitis C virus, Dengue, west nile etc.
The immune suppressive domains can also be located in the fusion peptide of the fusion protein among enveloped RNA viruses with type I fusion mechanism. For example HIV or influenza A and B types have an immune suppressive domain that co-localized with their fusion peptide. immunosuppressive domains are identified among enveloped RNA viruses with type II fusion mechanism at different positions in different groups of viruses: i. Co-localizing with the fusion peptide exemplified by the identification of an common
immunosuppressive domain in the fusion peptide of Flavirius (Dengue virus, west Nile virus etc), and
ii. In the hydrophobic alpha helix N-terminal of the transmembrane domain in the fusion
protein exemplified by the finding of an immunosuppressive domain in said helixes of all flaviridae e.g. Hepatitis C virus, Dengue, west nile etc. 2: Immunosuppressive domains have been identified in the fusion protein among enveloped RNA viruses with type I fusion mechanism. This position co-localizes with the fusion peptide of said fusion protein as demonstrated by the identification of a common immunosuppressive domain in the fusion peptide of all Influenza A and B types as well as HIV.
Functional homolog The term "functional homologue" or "functional equivalent" refers to homologues of the molecules according to the present invention and is meant to comprise any molecule which is capable of mimicking the function of molecules as described herein. Thus, the terms refer to functional similarity or, interchangeably, functional identity, between two or more molecular entities. The term "functional homology" is further used herein to describe that one molecular entity are able to mimic the function of one or more molecular entities.
Functional homologues according to the present invention may comprise any molecule that can function as an antagonist of the immune suppressive activity exerted by an immune suppressive domains. Such a molecule when added to the composition containing said immune suppressive domains reduces the immune suppressive activity exerted by the latter in either an in vitro test system (e.g. CTLL-2 or PBMC proliferation assays) or in vivo seen as an enhanced T- and/or B-cell responses.
Functional homologues according to the present invention may comprise polypeptides with an amino acid sequence, which are sharing at least some homology with the predetermined polypeptide sequences as outlined herein. For example such polypeptides are at least about 40 percent, such as at least about 50 percent homologous, for example at least about 60 percent homologous, such as at least about 70 percent homologous, for example at least about 75 percent homologous, such as at least about 80 percent homologous, for example at least about 85 percent homologous, such as at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with the predetermined polypeptide sequences as outlined herein above. The homology between amino acid sequences may be calculated using well known algorithms such as for example any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
Functional homologues may comprise an amino acid sequence that comprises at least one substitution of one amino acid for any other amino acid. For example such a substitution may be a conservative amino acid substitution or it may be a non-conservative substitution. A conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within predetermined groups exhibit similar or substantially similar characteristics. Within the meaning of the term
"conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within groups of amino acids characterized by having i) hydrophilic (polar) side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ϋ) hydrophobic (non-polar) side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met) iii) aliphatic side chains (Gly, Ala Val, Leu, lie)
iv) cyclic side chains (Phe, Tyr, Trp, His, Pro)
v) aromatic side chains (Phe, Tyr, Trp)
vi) acidic side chains (Asp, Glu)
vii) basic side chains (Lys, Arg, His)
viii) amide side chains (Asn, Gin)
ix) hydroxy side chains (Ser, Thr)
x) sulphor-containing side chains (Cys, Met), and
xi) amino acids being monoamino-dicarboxylic acids or monoamino-monocarboxylic- monoamidocarboxylic acids (Asp, Glu, Asn, Gin).
Non-conservative substitutions are any other substitutions. A non-conservative substitution leading to the formation of a functional homologue would for example i) differ substantially in hydrophobicity, for example a hydrophobic residue (Val, lie, Leu, Phe or Met) substituted for a hydrophilic residue such as Arg, Lys, Trp or Asn, or a hydrophilic residue such as Thr, Ser, His, Gin, Asn, Lys, Asp, Glu or Trp substituted for a hydrophobic residue; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice versa).
Functional homologues according to the present invention may comprise more than one such substitution, such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution, such as from 75 to 100 amino acid substitutions, for example more than 100 amino acid substitutions. The addition or deletion of an amino acid may be an addition or deletion of from 2 to 5 amino acids, such as from 5 to 10 amino acids, for example from 10 to 20 amino acids, such as from 20 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 200 amino acids, are also comprised within the present invention. The polypeptides according to the present invention, including any variants and functional homologues thereof, may in one embodiment comprise more than 5 amino acid residues, such as more than 10 amino acid residues, for example more than 20 amino acid residues, such as more than 25 amino acid residues, for example more than 50 amino acid residues, such as more than 75 amino acid residues, for example more than 100 amino acid residues, such as more than 150 amino acid residues, for example more than 200 amino acid residues.
Genetic code
The genetic code is the set of rules by which information encoded within genetic material (DNA or m NA sequences) is translated into proteins (amino acid sequences) by living cells. Biological decoding is accomplished by the ribosome, which links amino acids in an order specified by mRNA, using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly similar among all organisms, and can be expressed in a simple table with 64 entries.
The code defines how sequences of these nucleotide triplets, called codons, specify which amino acid will be added next during protein synthesis. With some exceptions a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded with exactly the same code (see the RNA codon table), this particular code is often referred to as the canonical or standard genetic code, or simply the genetic code, though in fact some variant codes have evolved. For example, protein synthesis in human mitochondria relies on a genetic code that differs from the standard genetic code. Not all genetic information is stored using the genetic code. All organisms' DNA contains regulatory sequences, intergenic segments, chromosomal structural areas, and other non-coding DNA that can contribute greatly to phenotype. Those elements operate under sets of rules that are distinct from the codon-to-amino acid paradigm underlying the genetic code.
Genetically encoded amino acids are as described below. Any other amino acid except for the 20 described below is considered a non-genetically encoded amio acid.
Amino Amino
Codons Compressed Codons Compressed acid acid
GCU, GCC, GCA, UUA, UUG, CUU,
Ala/A GCN Leu L YUR, CUN GCG CUC, CUA, CUG
CGU, CGC, CGA,
Arg R CGN, MGR Lys K AAA, AAG AAR
CGG, AGA, AGG
Asn N AAU, AAC AAY Met M AUG
Asp D GAU GAC GAY Phe F UUU, UUC UUY
CCU, CCC, CCA,
Cys/C UGU, UGC UGY Pro P CCN
CCG
UCU, UCC, UCA,
Gln/Q CAA, CAG CAR Ser/S UCN, AGY
UCG, AGU, AGC
ACU, ACC, ACA,
Glu E GAA, GAG GAR Thr/T ACN
ACG
GGU, GGC, GGA,
Gly/G GGN Trp/W UGG
GGG
His H CAU, CAC CAY Tyr/Y UAU, UAC UAY
GUU, GUC, GUA,
Ile I AUU, AUC, AUA AUH Val/V GUN
GUG
D- and L-Amino acids
Of the standard a-amino acids, all but glycine can exist in either of two enantiomers, called L or D amino acids, which are mirror images of each other. While L-amino acids represent all of the amino acids found in proteins during translation in the ribosome, D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria, and D-serine may act as a neurotransmitter in the brain. The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of
glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotatory).
Lipids Fatty acids
Fatty acids, or fatty acid residues when they form part of a lipid, are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis. They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used as a building-block of more structurally complex lipids. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. Where a double bond exists, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. This in turn plays an important role in the structure and function of cell membranes. Most naturally occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and partially hydrogenated fats and oils.
Examples of biologically important fatty acids are the eicosanoids, derived primarily from arachidonic acid and eicosapentaenoic acid, that include prostaglandins, leukotrienes, and thromboxanes.
Docosahexaenoic acid is also important in biological systems, particularly with respect to sight. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines. The fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide. Glycerolipids
Glycerolipids are composed mainly of mono-, di-, and tri-substituted glycerols, the most well-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride", though the latter lipid contains no hydroxyl group. In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Because they function as an energy store, these lipids comprise the bulk of storage fat in animal tissues. The hydrolysis of the ester bonds of triglycerides and the release of glycerol and fatty acids from adipose tissue are the initial steps in metabolising fat.
Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage. Examples of structures in this category are the digalactosyldiacylglycerols found in plant membranes and seminolipid from mammalian sperm cells.
Glycerophospholipids
Glycerophospholipids, usually referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and cell signaling. Neural tissue (including the brain) contains relatively high amounts of glycerophospholipids, and alterations in their composition has been implicated in various neurological disorders.
Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or the sn-1 position in the case of archaebacteria. Examples of glycerophospholipids found in biological membranes are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer). In addition to serving as a primary component of cellular membranes and binding sites for intra- and intercellular proteins, some glycerophospholipids in eukaryotic cells, such as phosphatidylinositols and phosphatidic acids are either precursors of or, themselves, membrane- derived second messengers. Typically, one or both of these hydroxyl groups are acylated with long- chain fatty acids, but there are also alkyl-linked and lZ-alkenyl-linked (plasmalogen)
glycerophospholipids, as well as dialkylether variants in archaebacteria.
Sphingolipids Sphingolipids are a complicated family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids and other compounds. The major sphingoid base of mammals is commonly referred to as sphingosine.
Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
Sterol lipids
Sterol lipids, such as cholesterol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins. The steroids, all derived from the same fused four-ring core structure, have different biological roles as hormones and signaling molecules. The eighteen-carbon (C18) steroids include the estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone. The C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids. The secosteroids, comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure. Other examples of sterols are the bile acids and their conjugates, which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver. The plant equivalents are the phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth. The predominant sterol in fungal cell membranes is ergosterol. Prenol lipids
Prenol lipids are synthesized from the five-carbon-unit precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid (MVA) pathway. The simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A. Another biologically important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of non-isoprenoid origin. Vitamin E and vitamin K, as well as the ubiquinones, are examples of this class. Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced.
Saccharolipids
Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a
monosaccharide substitutes for the glycerol backbone present in glycerolipids and
glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
Polyketides
Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Many commonly used anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, avermectins, and antitumor epothilones.
Biological functions in membranes
Eukaryotic cells are compartmentalized into membrane-bound organelles that carry out different biological functions. The glycerophospholipids are the main structural component of biological membranes, such as the cellular plasma membrane and the intracellular membranes of organelles; in animal cells the plasma membrane physically separates the intracellular components from the extracellular environment. The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. While
glycerophospholipids are the major component of biological membranes, other non-glyceride lipid components such as sphingomyelin and sterols (mainly cholesterol in animal cell membranes) are also found in biological membranes. In plants and algae, the galactosyldiacylglycerols, and sulfoquinovosyldiacylglycerol, which lack a phosphate group, are important components of membranes of chloroplasts and related organelles and are the most abundant lipids in
photosynthetic tissues, including those of higher plants, algae and certain bacteria. Bilayers have been found to exhibit high levels of birefringence, which can be used to probe the degree of order (or disruption) within the bilayer using techniques such as dual polarization interferometry and Circular dichroism.
A biological membrane is a form of lipid bilayer. The formation of lipid bilayers is an energetically preferred process when the glycerophospholipids described above are in an aqueous environment. This is known as the hydrophobic effect. In an aqueous system, the polar heads of lipids align towards the polar, aqueous environment, while the hydrophobic tails minimize their contact with water and tend to cluster together, forming a vesicle; depending on the concentration of the lipid, this biophysical interaction may result in the formation of micelles, liposomes, or lipid bilayers. Other aggregations are also observed and form part of the polymorphism of amphiphile (lipid) behavior. Phase behavior is an area of study within biophysics and is the subject of current academic research. Micelles and bilayers form in the polar medium by a process known as the hydrophobic effect. When dissolving a lipophilic or amphiphilic substance in a polar environment, the polar molecules (i.e., water in an aqueous solution) become more ordered around the dissolved lipophilic substance, since the polar molecules cannot form hydrogen bonds to the lipophilic areas of the amphiphile. So in an aqueous environment, the water molecules form an ordered "clathrate" cage around the dissolved lipophilic molecule.
Summary of the invention
According to an aspect, the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection.
According to another aspect, the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences. According to an aspect, the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
It will be clear for the person skilled in the art that the word "or" is not necessarily an exclusive expression. Thus, a dimer or multimer may comprise more than one immune suppressive domain motif.
According to an aspect, the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
According to an aspect, the invention concerns a use of a molecule, which comprises at least two parts, each part comprising an immune suppressive domain, for transfection. According to an aspect, the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response.
According to an aspect, the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
According to an aspect, the invention concerns a cell obtainable with said method for transfecting a cell.
According to an aspect, the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. According to an aspect, the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an aspect, the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy.
Detailed Disclosure
According to an embodiment, the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection. Transfection is the process of deliberately introducing nucleic acids into cells. The term "for transfection" is meant to encompass that the peptide is used as part of the transfection process, for example together with at least one transfection agent, and/or for preparing a cell for additional steps of the transfection process.
An immune suppressive peptide exhibits immune suppressive activity. The term "immune suppressive activity" means that it can inhibit proliferation of CTLL-2 or PBMCs in assays as described in the examples, i.e. by more than 20% or interfere with type I IFN production in response to lipid based formulations.
According to an embodiment, the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
The term "peptide" as used here, comprises shorter amino sequences as well as longer sequences such as proteins, and monomers as well as dimers and multimers.
The immune suppressive peptides do not need to be covalently bound to form a dimer or multimer. If they contain multimerization domains, the monomeric peptides may associate together through non-covalent interactions and form dimers or multimers that will also be functional.
According to an embodiment, the invention concerns the use of a peptide of the invention, wherein said peptide is a dimer.
According to an embodiment, the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
It will be clear for the person skilled in the art that the word "or" is not necessarily an exclusive expression. Thus, a dimer or multimer may comprise more than one immune suppressive domain motif, and still be within the scope of the present invention. According to an embodiment, the invention concerns the use of the invention, wherein said at least two immune suppressive domain motifs are two identical or different motifs. According to an embodiment, the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
According to an embodiment, the invention concerns the use of a peptide according to the invention, which is soluble in water. The peptide is preferably sufficiently soluble to provide immune suppressive activity in an aqueous solution.
According to an embodiment, the invention concerns the use of a peptide according to the invention, wherein said immune suppressive domain is part of a virus.
According to an embodiment, the invention concerns the use of a peptide of the invention, wherein said virus is an influenza virus.
According to an embodiment, the invention concerns the use of a peptide according to the invention, wherein said peptide forms part of a protein.
According to an embodiment, the invention concerns a use of a molecule, which comprises at least two parts, each comprising an immune suppressive domain, for transfection. According to an embodiment, the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. According to an embodiment, the invention concerns the kit-of-parts or composition of the invention, wherein said transfection agent is coupled covalentely to said peptide.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based transfection agent.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said lipid based transfection agent is selected among an anionic and a cationic transfection agent.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based liposome or virosome or viral vector.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, comprising a nano particle.
A nanoparticle may e.g. be used for drug delivery, it may have an inherent therapeutic effect, or it may be fluorescent and used for tests.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, further comprising a hydrophobic vehicle for delivery of hydrophobic molecules. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, comprising a drug.
The immune suppressive domain may suppress an unwanted immune response induced by the drug, which e.g. may comprise a carrier oil, known to induce an immune response. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is an immune suppressive peptide.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein of a pathogen.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said pathogen is a virus.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein on the surface of a pathogen.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a virus surface glycoprotein. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide forms part of an enveloped virus surface glycoprotein.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide has a length of at least 6, preferably 7, more preferred 8, preferably 9, more preferred 10, preferably 11, more preferred 12, preferably 13, more preferred 14, preferably 15, more preferred 16, preferably 17, more preferred 18, preferably 19, more preferred 20, preferably 21 more preferred 22, preferably 23, more preferred 24, preferably 25 amino acids.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has a length selected among 5 - 200, preferably 10 - 100, more preferred 20 - 50, preferably 30 - 40 amino acids.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, further comprising a fusion peptide from a fusion protein.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from the fusion protein of an enveloped virus.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from a type I fusion protein.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, comprising a fusion peptide from a type II fusion protein. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide has 1, 2, 3 or 4 mutations, deletions or insertions with respect to the wild type.
The term "mutation" is used with a number about this number of point mutation(s), i.e. 3 mutations mean 3 point mutations. The term "deletion" is used with a number about the deletion of this number of amino acid(s), i.e. 2 deletions means the deletion of 2 amino acids. The term "insertion" is used with a number about insertion of this number of amino acid(s), i.e. 1 insertion means the insertion of 1 amino acid.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, binds either directly or indirectly to a cellular protein complex containing the protein STING encoded by the gene Tmeml73.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses induced by membrane fusion.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from selected among the group consisting of the lists of Table 1 and among the sequences with Seq. Id. 1 - 287.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from an influenza virus or a Flu peptide.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has immune suppressive activity as dimer or mulitimer or when coupled to carrier proteins.
The coupling of peptides to larger proteins is one of the known methods for producing mulitimeric peptides or antigens. Proteins such as Bovine serum albumin (BSA) or Keyhole limpet hemocyanin (KLH) can be used as carrier proteins.
KLH is used extensively as a carrier protein in the production of antibodies for research,
biotechnology and therapeutic applications. KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues used as coupling sites.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one non-genetically encoded amino acid residue. The non-genetically encoded amino acid residues are amino acid residues, which are not genetically encoded.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid residue.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to any other molecule. Two peptides may be joined via another molecule, and each of the two peptides may comprise an immune suppressive domain sequence.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to lipids.
Lipids are here defined as cationic, anionic or neutrally charged fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol).
Liposomes may be used as a delivery system, and vesicles may be at least partly covered by immune suppressive domains, thereby suppressing a potential immune system response to the delivery system. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a molecule through a peptide bond.
The molecule may e.g. be a ligand of a receptor, thereby targeting the peptide, or it may e.g. be a molecule providing different solubility characteristics of the combination of the peptide and the molecule as compared to the peptide alone, or the molecule may be a nanoparticle. The peptide may further form part of a protein, which may provide advantages such as easy production, as the protein may be derived from natural sources.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a protein.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is a circular peptide.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to at least one biological membrane.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is modified in a way in which one of the peptide bonds is replaced by a non-peptide bond.
According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide interferes with an interferon response induced by the transfection agent upon addition to a cell.
According to an embodiment, the invention concerns the kit-of-parts or composition comprising a functional homologue of a peptide according to the invention. A functional homologue may be replaced by an altered peptide, obtained from a peptide of the invention, by making 1, 2, 3, or 4 mutations, deletions or insertions of the immune suppressive domain. An alternated peptide may have a %identity vis-a-vis the unaltered form of at least 60%, preferably at least 70%, more preferred at least 80%, preferably at least 90%, more preferred at least 95%, preferably at least 98%, more preferred at least 99%.
According to an embodiment, the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. According to an embodiment, the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an embodiment, the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an embodiment, the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
According to an embodiment, the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for introducing a molecule into a cell.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a DNA or NA molecule into a cell.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a cell.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a tissue.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a gene-therapeutic pharmaceutical molecule into a cell.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for initiating expression of proteins via transfected plasmids. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for targeting active genes by micro NA silencing.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for examining the immune response to DNA or RNA. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for gene therapy.
According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for delivery of any molecule using liposomes.
According to an embodiment, the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
According to an embodiment, the invention concerns a cell obtainable with said method for transfecting a cell.
The cells obtainable by this process produce less or no cytokines, in particular interferons, compared to cells obtained without the use of the peptides of the invention. I.e. when compared to untreated cells, cells treated with the INF-F#2 peptide produce less or no cytokines, in particular interferons, but appear to retain the ability to react to other and stronger stimuli. According to an embodiment, the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. Such a cell may be used e.g. as an intermediate product for producing a transfected cell with suppressed immune activity. According to an embodiment, the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. The peptide may e.g. be bound to the surface of the cell, internally or externally, or may be present inside of the cell. According to an embodiment, the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy. According to an embodiment, the invention concerns the use according to the invention, for cell therapy and/or gene therapy.
Gene therapy and cell therapy are overlapping fields of biomedical research with similar therapeutic goals. Gene therapy can be defined as the use of genetic material (usually deoxyribonucleic acid - DNA) to manipulate a patient's cells for the treatment of an inherited or acquired disease. Cell Therapy can be defined as the infusion or transplantation of whole cells into a patient for the treatment of an inherited or acquired disease.
According to an embodiment, the invention concerns the use according to the invention, for stem cell transplantation.
The co-pending patent application PCT/DK2012/050381 provides a number of immunosuppressive domains and motifs.
Table 1 is provided below. Table 1 likewise provides a number of immunosuppressive domains and motifs.
Group 1 Type II Fusion mechanism IU group and fusion type
6800£0/Η0Ώΐα/13<Ι 00S99l/M0Z OAV *******************
Dengue 4 seqidlO seqid2
GETAWDFGSVGGLLTSLGK DRGWGNGCGLFGKG
seqidl73
KGS SI GKMFEATARGARRMAILG
Japanese encephalitis virus seqidll seqid2
LGDTAWDFGSIGGVFNSIG DRGWGNGCGLFGKG
*** ***************
Koutango virus seqidl2 seqid2
LGDTAWDFGSVGGIFTSLG DRGWGNGCGLFGKG
Murray Valley encephalitis virus seqidl3 seqid2
LGDTAWDFGSVGGVFNSIG DRGWGNGCGLFGKG
Japanese
encephalitis
St. Louis encephalitis virus seqidll seqid2
virus group
LGDTAWDFGSIGGVFNSIG DRGWGNGCGLFGKG
Usutu virus seqidl4 seqid2
LGDTAWDFGSVGGI FNSVG DRGWGNGCGLFGKG
West Nile virus seqidl5 seqid2
LGDTAWDFGSVGGVFTSVG DRGWGNGCGLFGKG
********** ********
Kokobera Kokobera virus unclassified seqidl6 seqid2
group Kokobera virus group IGDDAWDFGSVGGILNSVG DRGWGNGCGLFGKG
Modoc virus Modoc virus seqidl7
group VGSAFWNSDQRFSAINLMD
seqidl8
DRGWGNGCALFGKG
Cowbone Ridge virus
Jutiapa virus
Sal Vieja virus
San Perlita virus
mosquito-borne Ilheus virus seqid84 seqid2
viruses LGDTAWDFGSVGGIFNSIG DRGWGNGCGLFGKG
Sepik virus seqidl9 seqid2
TGEHSWDFGSTGGFFASVG DRGWGNGCGLFGKG
Ntaya virus Bagaza virus seqid20 seqid2
group LGDTAWDFGSVGGFFTSLG DRGWGNGCGLFGKG
Tembusu virus seqid83 seqid2
LGDTAWDFGSVGGVLTSIG DRGWGNGCGLFGKG
Yokose virus seqid21 seqid2
IGDDAWDFGSTGGIFNTIG DRGWGNGCGLFGKG
Rio Bravo virus Apoi virus seqid22 seqid2 group SSAFWNSDEPFHFSNLISII DRGWGNGCGLFGKG
Entebbe bat virus seqid23 seqid2
GDDAWDFGSTGGIFNTIGKA DRGWGNGCGLFGKG
Rio Bravo virus seqid24 seqid2
SSAYWSSSEPFTSAGIMRIL DRGWGNGCGLFGKG
Saboya virus seqidl8
DRGWGNGCALFGKG
seqid25
GSSSWDFSSAGGFFGSIGKA
Seaborne tick- Meaban virus seqid26
borne virus GDAAWDFGSVGGFMTSIGRA
group seqid27
DRGWGNHCGLFGKG
Saumarez Reef virus seqid28
GETAWDFGSAGGFFTSVGRG
seqid27
DRGWGNHCGLFGKG
Tyuleniy virus seqid29
GEAAWDFGSAGGFFQSVGRG
seqid27
DRGWGNHCGLFGKG
Spondweni virus Zika virus seqid30 seqid2 group LGDTAWDFGSVGGVFNSLGK DRGWGNGCGLFGKG
Kyasanur forest disease virus seqid31
VGEHAWDFGSVGGMLSSVG
seqid27
DRGWGNHCGLFGKG
Langat virus seqid32
VLGEHAWDFGSVGGVMTSIG
seqid27
DRGWGNHCGLFGKG
Louping ill virus seqid33
I GEHAWDFGSAGGFFS SI G
seqid27
DRGWGNHCGLFGKG
Omsk hemorrhagic fever virus seqid34
LGEHAWDFGSTGGFLSSIG
seqid27
DRGWGNHCGLFGKG
Powassan virus seqid35
VGEHAWDFGSVGGI LS SVG
Jugra virus
Kadam virus
Kamiti River virus
Kedougou virus
Montana myotis leukoencephalitis
virus
Mosquito flavivirus
Ngoye virus
Nounane virus
Phlebotomus flavivirus Alg F19
Phlebotomus flavivirus Alg F8
Quang Binh virus
Russian Spring-Summer encephalitis
virus
Sokoluk virus
Spanish sheep encephalitis virus
T'Ho virus
Tax forest virus B31
Tamana bat virus
Tick-borne flavivirus
Wang Thong virus
Flavivirus sp .
Aedes flavivirus seqid45
NRGWGTGCFEWGLG
seqid46
HVAGRYSKHGMAGIGSVWEDLVR
Culex flavivirus seqid44
NRGWGTGCFKWGIG
seqid47
VDKYRRFGTAGVGG
Hepa Hepatitis C Hepatitis C virus genotype 1 a seqid3
civi virus GLIHLHQNIVDVQYLYG ω rus seqidl75 ω
PALSTGLIHLHQNIVDVQ
Hepatitis C virus genotype lb seqid48
GLIHLHRNIVDVQYLYG
seqidl76
PALSTGLIHLHRNIVDVQ
Hepatitis C virus genotype 2 seqid49
GLIHLHQNIVDVQYMYG
seqidl75
PALSTGLIHLHQNIVDVQ
Hepatitis C virus genotype 3 seqidl75 seqid3
PALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG
Hepatitis C virus genotype 4 seqidl75 seqid3
PALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG
Hepatitis C virus genotype 5 seqid50
GLIHLHQNIVDTQYLYG
seqidl77
PALSTGLIHLHQNIVDTQ
Hepatitis C virus genotype 6 seqidl75 seqid3
PALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG
All Hepatitis C virus seqid3
GLIHLHQNIVDVQYLYG
Border disease Border disease virus - seqid51
virus Border disease virus - X818 NTTLLNGSAFQLICPYGWVGRVEC ω
Border disease virus 1 seqid52
Border disease virus 2 SYFQQYMLKGQYQYWFDLE
Border disease virus 3
Border disease virus isolates
Bovine viral Bovin viral diarrhea virus 1-CP7 seqid53
diarrhea virus Bovin viral diarrhea virus 1-NADL NTTLLNGPAFQMVCPLGWTGTVSC
1 Bovin viral diarrhea virus 1- seqid54
Oslos SYFQQYMLKGEYQYWFDLE
Bovin viral diarrhea virus 1-SDl
Bovin viral diarrhea virus
isolates and strains
Bovine viral diarrhea virus type
la
Bovine viral diarrhea virus type
lb
Pestivirus isolate 97-360
Pestivirus isolate Hay 87/2210
Pestivirus strain mousedeer
Pestivirus type 1 isolates
Bovine viral Bovine viral diarrhea virus 2 seqid55
diarrhea virus Pestivirus sp . strain 178003 SLLNGPAFQMVCPQGWTGTIEC
2 Pestivirus sp . strain 5250Giessen- seqid56
(BVDV-2) 3 DRYFQQYMLKGKWQYWFDLD
Bovin viral diarrhea virus
isola SCP
Classical swine Classical swine fever virus seqid57
fever virus Hog cholera virus strain Zoelen TLLNGSAFYLVCPIGWTGVIEC
seqid58
SYFQQYMLKGEYQYWFDLD
unclassified Bovine viral diarrhea virus 3 seqid59
Pestivirus TLLNGPAFQLVCPYGWTGTIEC
seqid60
DNYFQQYMLKGKYQYWFDLEATD
Rubella virus Rubella virus ( strain BRD1) seqid72
Rubella virus ( strain BRDII) AC FWAVNAYSSGGYAQLASYFNPGGSYYK
Rubella virus ( strain Cendehill
seqid73
Rubella virus ( strain M33)
QYHPTACEVEPAFGHSDAACWGFPTDT
■9 Rubella virus ( strain RN-UK86)
Rubella virus ( strain THERIEN) seqid74
Rubella virus ( strain TO-336 MSVFALASYVQHPHKTVRVKFHT vaccine )
Rubella virus ( strain TO-336) seqidl59
Rubella virus (vaccine strain ETRTVWQLSVAGVSC
RA27/3)
seqid76
NVTTEHPFCNMPHGQLEVQVPP
seqid77
DPGDLVEYIMNYTGNQQSRW
seqid78
GSPNCHGPDWASPVCQRHSPDCS
seqid79
RLVGATPERPRLRLV
seqid80
DADDPLLRTAPGP
*oo**********
seqid81
GEVWVTPVIGSQARKCGL
seqid86
HIRAGPYGHATVEM
seqid87
PEWIHAHTTSDPWHP
seqid88
PGPLGLKFKTVRPVALPR
seqid89
ALAPPRNVRVTGCYQCGTPAL
seqid90
EGLAPGGGNCHLTVNGEDVG
seqid207
LLNTPPPYQVSCGG
Amur virus seqid94
Bayou virus NPPDCPGVGTGCTACGVYLD Black Creek
Canal virus seqid95
Cano Delgadito RKVCIQLGTEQTCKTIDSNDC virus *oo*o*o*o*oo**oo*o***
Calabazo virus seqid96
Catacamas virus DTLLFLGPLEEGGMI KQWCTTTCQFGDP Choclo virus GDIM
Dobrava- seqid97
Belgrade virus GSFRKKCSFATLPSCQYDGNTVSG El Moro Canyon
virus seqid98
Hantaan virus ATKDSFQSFNITEPH
Isla Vista
virus seqid99
Khabarovsk GSGVGFNLVCSVSLTEC virus
Laguna Negra seqidlOO
virus KACDSAMCYGS STANLVRGQNT
Limestone
Canyon virus seqidlOl
Monongahela GKGGHSGSKFMCCHDKKCSATGLVAAAPH virus L
Muleshoe virus
Muju virus
New York virus seqidl02
Oran virus DDGAPQCGVHCWFKKSGEW Playa de Oro
virus
Prospect Hill
virus
Puumala virus
Anopheles A seqidl03 virus KHDELCTGPCPVNINHQTGWLT
Anopheles B * o*o* * * o* *oooooooo* o* o virus seqidl04
Bakau virus WGCEEFGCLAVSDGCVFGSCQD Batama virus **o*oo**o*ooo**oo***** Bwamba virus seqidl05
o Caraparu virus GNGVPRFDYLCHLASRKEVIVRKC Kaeng Khoi *o*ooo*ooo*oooo*ooooo*o*
I O virus seqidl06
Kairi virus SCAGCINCFQNIHC
Madrid virus * o* *ooooooooo*
Main Drain
virus
Marxtuba virus
Nyando virus
Oriboca virus
Oropouche virus
Sathuperx virus
Shamonda virus
Shuni virus
Simbu virus
Tacaxuma virus
Tete virus
Turlock virus
unclassified
Orthobunyavxrus
Akabane virus Sabo virus
Tinaroo virus
Yaba-7 virus
Caraparu virus Apeu virus
Bruconha virus Ossa virus
Vinces virus
Manzanxlla Buttonwxllow virus virus Ingwavuma virus
Mermet virus
Marituba virus Gumbo Limbo virus
Murutucu virus Nepuyo virus Restan virus
Wyeomyia virus Anhembi virus
BeAr328208 virus Macaua virus Sororoca virus Taiassui virus
Bujaru virus
Candxruvxrus
> Chilibre virus
ο Frijoles virus
Punta
TorDSalehabad
virus
Sandflyfever
Naples virus
Uukunxemx
viruso virus
Anhanga virus
Arumowot virus
Chagres virus
Corfou virus
Gabek Forest virus
Itaporanga virus
Phlebovirus Adria/ALBl/2005
Phlebovirus Adria/ALB5/2005
Phlebovirus AH12
Phlebovirus AH12/China/2010
Phlebovirus AH15/China/2010
Phlebovirus B105-05
Phlebovirus B151-04 o Phlebovirus B43-02
Phlebovirus B68-03
<D Phlebovirus B79-02
■H Phlebovirus Chios-A
Phlebovirus Cyprus o
ϋ Phlebovirus HB29/China/2010
Phlebovirus HN13/China/2010
Phlebovirus HN6/China/2010
Phlebovirus Hu/Xinyangl/ China/ 2010
> Phlebovirus Hu/Xinyang2/ China/ 2010 o Phlebovirus IB13-04
<D Phlebovirus JS2007-01
Phlebovirus JS24
TS Phlebovirus JS26
Phlebovirus JS3/China/2010
Phlebovirus JS4/China/2010
Phlebovirus JS6
Phlebovirus JSD1
Phlebovirus LN2/China/2010
Phlebovirus LN3/China/2010
Phlebovir
sandflies /Gr29/Spain/2004
Phlebovirus
sandflies /Gr36/Spain/2004
Phlebovirus
sandflies /Gr44/Spain/2004
Phlebovirus
sandflies /Gr49/Spain/2004
Phlebovirus
sandflies /Gr52/Spain/2004
Phlebovirus
sandflies /Gr65/Spain/2004
Phlebovirus
sandflies /Gr98/Spain/2004
Phlebovirus SD24/China/2010
Phlebovirus SD4/China/2010
Phlebovirus tick/XCQ-2011
Phlebovirus XLL/China/2009
Rio Grande virus
Salobo virus
Sandfly fever Sicilian virus
Sandfly Sicilian Turkey virus
Utique vi rus
Phlebovir sp .
Phlebovir sp . Be An 24262
Phlebovir sp . Be An 356637
Phlebovir sp . Be An 416992
Phlebovir sp . Be An 578142
Phlebovir sp . Be Ar 371637
Phlebovir sp . Co Ar 170255
Phlebovir sp . Co Ar 171616
Phlebovir sp . GML 902878
Phlebovir sp . Pa Ar 2381
Phlebovir sp . PAN 479603
Phlebovir sp . PAN 483391
Phlebovir sp . VP-161A
Phlebovir sp . VP-334K
Phlebovir sp . VP-366G
<D Influenza A INFA HI seqidll9
■H
> > virus GLFGAIAGFIEGGWTG 1
0 ■H seqidl78
X
a WTYNAELLVLLENERTLD
0 3 seqidl79 <
NAELLVLLENERTLDYHD
O INFA H2 seqidl20
Canine distemper virus
Cetacean morbillivirus_Dolphin morbillivirus Pilot whale morbillivirus Porpoise
morbillivirus
Measles virus
I-a Peste-des-petits-ruminants virus
Phocine distemper virus
Phocine distemper virus 1
Phocine distemper virus-2
Rinderpest virus
Bovine parainfluenza virus 3 Porcine paramyxovirus strain Frost Porcine paramyxovirus strain Texas Human parainfluenza virus 1 Human parainfluenza virus 3 Simian Agent 10
Sendai virus
unclassified Respirovirus
Atlantic salmon respirovirus Guinea pig parainfluenza virus TS- 9
Pacific salmon paramyxovirus Trask River 1983 Swine
parainfluenza virus 3
Tursiops truncatus parainfluenza virus 1
Human paraxnfluenza virus
Human parainfluenza virus
( strain Greer)
Human parainfluenza virus
(strain Toshiba ) Human parainfluenza virus
Human parainfluenza virus
Human parainfluenza virus
Mapuera viru
Mumps virus
Parainfluenza vxrus Porcine rubulavirus Simian virus 41
unclassified Rubulavirus
Porcine parainfluenza virus Tuhoko virus 1
Tuhoko virus 2
Tuhoko virus 3 unclassified Atlantic salmon paramyxovirus Paramyxovirinae Beilong virus
Canine parainfluenza virus
Chimeric human parainfluenza virus rPIV3-2
Fer-de-lance virus J-virus
Menangle virus
Mossman virus
Murayama virus
Ovine parainfluenza virus 3 Pacific salmon paramyxovirus Paramyxovirus GonoGER85
Recombinant PIV3/PIV1 virus Reptilian paramyxovirus
Salem virus
Salmo salar paramyxovirus
Snake ATCC-VR-1408 paramyxovirus
Snake ATCC-VR-1409 paramyxovirus
Tioman virus
Tupaxa paramyxovirus
unclassified Alpaca coronavirus CA08-1/2008
coronavi ruses Bat coronavirus seqid209
Bird droppings coronavirus EVFAQVKQMYKTPTLKYFGGFNFSQIL
Bovine respiratory coronavirus seqid210
Chicken enteric coronavirus EVFAQVKQMYKTPAIKDFGGFNFSQIL
Coronavirus Anas Segid211
Coronavirus S FI EDLLFNKVTLADAGF
oystercatcher/pl7/2006/GBR Segid212
Coronavirus red knot/p60/2006/GBR SAIEDLLFNKVRLSDVGF
Ferret enteric coronavirus 1202 Seqid213
Ferret systemic coronavirus MSU-S SLLEDLLFNKVKLSDVGF
Ferret systemic coronavirus WADL Seqid214
Guangxi coronaviridae SAIEDLLFSKVKLADVGF
Human coronavirus NO Seqid215
Human enteric coronavirus strain SAIEDLLFDKVKLSDVGF
4408
Kenya bat coronavirus
Mink coronavirus strain WD1133
Parrot coronavirus AV71/99
Quail coronavirus Italy/Elvia/2005
Tax Forest coronavirus
unidentified coronavirus
unidentified human coronavirus
LCMV-Lassa Ippy virus seqidl49 U —- virus (Old Lassa virus NALINDQLIMKNHLRDIMGI PYC Ά World) complex Lu o virus o*o*
Lymphocytic choriomeningitis virus seqidl50
Ά
Mobala virus FTWTLSDSEGKDTPGGYCLT
Mopeia virus oo* ooo* oo*ooo* * *o* * o
I < seqidl51
< KCFGNTAIAKCNQKHDEEFCDMLRLFDFN
seqidl52
MLQKEYMERQGKTPLGLVDLFVFS
Aravan virus
Australian bat
lyssavirus
Duvenhage virus
European bat
I J lyssavirus 1
European bat
lyssavirus 2
Irkut virus
Khu and virus
Lagos bat virus
Mokola virus
West Caucasian
bat virus
Rabies virus Rabies virus AB21 seqid5
Rabies virus AB22 GFTCTGWTEAETYTNFVGYVT
Rabies virus AV01
Rabies virus BNG4 seqid6
Rabies virus BNG5 SLHNPYPDYRWLRTVKTT
Rabies virus China/DRV * ooooooooooo* * *o*
Rabies virus China/MRV Seqidl38
© Rabies virus CVS-11 ESLVIISPSVADLDPYDRSLHS
Rabies virus ERA * ooo* * * oooo*o* * ooo
Rabies virus Eth2003 Seqid91
Rabies virus HEP-FLURY CKLKLCGVLGLRLMDGT
Rabies virus India * ooo* * * *oooo*ooo*
Rabies virus Nishigahara RCEH Seqid206
Rabies virus Ontario fox ILGPDGNVLIPEMQSS
Rabies virus Ontario skunk Ό*οοο
Rabies virus PM seqid82
Rabies virus red fox/08RS- QHMELLESSVIPLVHPL
1981/Udine/2 008
Rabies virus SAD B19
Rabies virus silver-haired bat- associated SHBRV
Rabies virus strain Pasteur va
Rabies virus strain Street
Rabies virus vnukovo-32
Thailand genotype 1 dog lyssavirus
Seqid264
GTIGAMFLGFLGAAGSTMGAASMTLTVQA
RLLL
Seqid265
IGALFLGFLGAAGSTMGAASVTLTVQARL LLSG
Bovine lentivirus group Seqid266
AVGMVIFLLVLAIMAMTASVTAA
Seqid267
Equine lentivirus group FGI SAIVAAIVAATAIAASA
Seqid268
Feline lentivirus group TLALVTATTAGLIGTTTGTSA
Seqid269
HVMLALATVLSMAGAGTGATA
Seqid270
Ovine/caprine lentivirus group GIGLVIMLVTMAIVAAAGAS
Human immunodeficiency virus 2 Seqid271
GVMVLGFLGFLAMAGSAMGA
Simian immunodeficiency virus ooo* * * o* * oooo* oooooo
Seqid272
GVFVLGFLGFLATAGSAMGA
Simian immunodeficiency virus oooo* * oo* o* oo* * ooooo others Seqid273
GAIVLGLLGFLGLAGSAMG
Ovine lentivirus * ooooooo* o* ooo* * ooo
Seqid274
GIGLVIVLAIMAI lAAAGAGLGVANAVQ
Porcint PRRS Type I seqid275
Reproductions SRKLGRSLIPHSCFWWLFLLC og Respirations seqid276
Syndrome (PRRS) GNGNSSTYQYIYNLTIC
seqid277
GTAWLSTHFSWAVETFVLYHILSL
seqid278
GFLTTSHFFDTLGLGAVSITGFC
seqid279
RYAHTRFTNFIVDDRGRIHRW
PRRS Type II seqid280
SNNNSSHIQLIYNLTLC
seqid281
GTDWLAQKFDWAVETFVIFPVLTH
seqid282
GALTTSHFLDTVGLATVSTAGYY
seqid283
IYAVCALAALICFVIRLAKNC
seqid284
VSTAGYYHGRYVLSSIYAVCALAALICFV IRL
Figures
In all the figure texts, the I N F-F#2 peptide has the sequence GLFGAIAGFI ENGWEGCGGEKEKEK [Seq id 287] and is dimerized through a disulfide bridge between the cystein residues.
Fig. 1 shows type I IFN production in cells either untreated (UT) or treated with cationic liposomes. Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance. In figure 1 it can be seen that treatment with cationic liposomes induce the production of type I I FN and that this production was inhibited when cells were pre-treated with IN F-F#2. The cells used were bonemarrow derived dendritic cells (BM DCs) from mice of the laboratory strain C57BL. The peptide IN F-F#2 was used at a concentration of ΙΟμΜ and initiated 15minutes before treatment with cationic liposomes. Cells were then left to incubate 16 hours at 5% C02 and 37.5 degrees Celcius. After 16 hours of incubation the supernatants were harvested. Type I I FN activity was then measured using a type I I FN bioassay.
Cationic liposomes were prepared using a mix of the lipids DOTAP/DOPE/L (Lissamine Rhodamine)- DOPE in a w/w/w of 1/1/0.1 dissolved in chloroform. The mix was purchased from the company Avanti Polar Lipids, inc. Chloroform was evaporated using rotation evaporation using a dry nitrogen stream. The lipids film was then dried in vaccum for 4 hours. To created liposomes, the lipid film was hydrated in phosphate buffered saline solution at pH 7.4 at ambient temperature. Liposomes were then size restricted using a 0.2μιη filter and an Avanti Mini-extruder purchased form Avanti Polar Lipids. Fig. 2 shows type I I FN production in cells either untreated (UT) or treated with the transfection reagent Lipofectamine2000 (Lipofect) according to the manufactures instructions. In this case Lipofectamine2000 was used alone and not with the purpose of transfecting DNA or RNA into the cells. The type I IFN produces therefore represent the cellular response to the transfection reagent Lipofectamine2000 itself. Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance. In figure 2 it can be seen that treatment with Lipofectamine2000 induced the production of type I IFN and that this production was inhibited when cells were pre-treated with IN F-F#2. Cells used were BM DCs as described in the Figure 1 text.
Fig. 3 shows type I IFN production in cells either untreated (UT) or transfected with double stranded DNA (dsDNA) using the reagent Lipofectamine2000 according to the manufactures instructions. Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance. In figure 3 it can be seen that transfection with dsDNA induces the production of type I IFN and that this production was NOT inhibited when cells were pre-treated with IN F-F#2. Therefore, pretreatment does not interfere with type I I FN levels induced by the transfected dsDNA but only with the type I I FN induced by the transfection reagent itself. Cells used were BM DCs as described in the Figure 1 text. Fig. 4 shows co-precipitation of endogenous STING protein with biotinylated IN F-F#2 but not the with the IN F-F#2 mutant DI6. In this experiment, the human monocytic cell line TH P-1 was lysed using a native lysis buffer. Cell lysates were cleared by centrifugation at 300xg for 5 min. Supernatants were then incubated with a biotinylated form of IN F-F#2 (B), with a biotinylated form of the peptide mutant DI6 (C) or with no peptide (A) for 30min on ice. Cells lysates were then mixed with biotin binding streptavidin beads and mixed for 30 min on ice. Beads were then collected by centrifugation at 2000xg for 2min. Beads were then washed x3 in PBS at pH 7.4. Beads were then headed to 95 deg. Celcius in reducing denaturating loading buffer for 5 min. yielding the pull-down lysate. This was then loaded and run on a SDS-PAGE for 45 min at 120V. Proteins were then transferred to protein PVDF membrane and blotted for STING and Giantin. The protein Giantin is a golgi-resident transmembrane protein unrelated to STING function. As depicted in figure 4, the INF-F#2 but not beads alone or the peptide DI6 was able to precipitate STING. Further, INF-F#2 was unable to precipitate other transmembrane proteins such as Giantin.
The peptide INF-F#2 therefore blocks STING dependent type I IFN production in response to membrane disturbance as introduced by liposome based transfection agents. This is possibly through direct or indirect interaction with STING itself as INF-F#2 binds either directly to STING or to a complex which contains STING. This occurs without significant interference with type I IFN production with response to transfected immuno-stimulatory DNA itself (Fig. 3)
Fig. 5, (left panel): shows the effect of INF-F#2 (INF ISD) on STING dimerization in response to cationic liposomes. Cells of the human moncytic cell line THP-1 were either culture in the presence or absence of INF-F#2 (INF ISD) as depicted. Cells were then either treated with or left untreated with cationic liposomes (as in Figure 1) for 3.5 hours. Cells were then lysed using IPA lysis buffer with
0.1% SDS. Lysates were then run on a SDS-PAGE at 120V for 45min, transferred to a PVDF membrane, and blottet using anti-STING antibodies. As seen in figure 5, cationic liposomes were able to induce STING dimerization, which is a necessary step in STING dependent induction of type I IFN.
Pretreatment of cells with INF-F#2 resultet in an inhibition of the formation of STING dimers. (Right panel): Lysates from cells treated with cationic liposomes either treated with the reducing agent DTT or left untreated for 5min.
Therefore, INF-F#2 binds either directly to STING or to a STING containing complex (Figure 4), and interferes with STING dimerization in response to cellular treatment with cationic liposomes.
Figure 6 shows inflammation-related enzyme and transcription factor gene expression kinetics of THP-1 monocytes stimulated with ^g/ml LPS. Gene expression was expressed as relative gene expression towards RPL13a-expression and non-stimulated cells at time zero (AACt). Data shown are means + standard deviation from two independent biological replications.
Figure 7 shows effects of INF-F#2 peptide on expression of NF-kappaB mRNA in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ, 60μΜ INF-F#2 peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the medians ± standard deviation from two independent biological replications.
Figure 8 shows effects of INF-F#2 peptide on expression of SP-1 mRNA in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ, 60μΜ INF-F#2 peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the medians ± standard deviation from two independent biological replications.
Figure 9 shows effects of INF-F#2 peptide on protein secretion of IL-8 in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ or 60μΜ INF-F#2 peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the median ± standard deviation from three independent experiments performed in duplicates. Figure 10 shows effects of INF-F#2 peptide on protein secretion of IL-10 in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ or 60μΜ INF-F#2 peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the median ± standard deviation from three independent experiments performed in duplicates. Figure 11 shows effect of different stimulus on the secretion of IFN-gamma in PBMCs. PBMCs were incubated either with ^g/ml or 50ng/ml PMA and ^g/ml ionomycin or lOng/ml SEB for indicated time periods. Data shown are the medians ± standard deviation from three independent technical replicates.
Figure 12 shows expression kinetics of IFN gamma expression in response to PMA/ionomycin treatment. Gene expression was expressed as relative gene expression towards RPL13a expression and non-stimulated cells at time zero (ΔΔ Ct). Data shown are the medians ± standard deviation from three independent technical replicates.
Figure 13 shows effect of INF-F#2 peptide on secretion of protein of IFN-gamma in PMA/ionomycin stimulated PBMCs. PBMCs were incubated with either medium alone, 30μΜ or 60μΜ INF-F#2 peptide or 30μΜ or 60μΜ control peptide, and stimulated with 50ng/ml PMA and ^g/ml ionomycin. Data shown are the medians ± standard deviation from three independent experiments performed in duplicates.
Figure 14 shows effects of SARS ([Seq id 285] AEVQIDRLITGRLQSLQTYVCGGEKEKEK) or Filo ISD ([Seq id 286] GAAIGLAWIPYFGPAAECGGEKEKEK) on expression of TNF-alpha mRNA in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ, 60μΜ SARS or Filo ISD peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the medians ± standard deviation from two independent biological replications.
Figure 15 shows effects of SARS or Filo ISD on expression of IL-1 β mRNA in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ, 60μΜ SARS or Filo ISD peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the medians ± standard deviation from two independent biological replications.
Figure 16 shows effects of SARS or Filo ISD on expression of IL-1 β mRNA in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30μΜ, 60μΜ SARS or Filo ISD peptide or 30μΜ, 60μΜ control peptide, and stimulated with ^g/ml LPS. Data shown are the medians ± standard deviation from two independent biological replications.
Figure 17 shows interactions between INF-F#2 peptide and STING depends on distinct STING domains. To investigate further the interaction between STING and INF-F#2 peptide the C-terminal domian of STING was expressed with a HA-tag in HEK293 cells. STING was either in a wt form or with deletions. Lysates from tansfected cells were used for pulldown using biotinylated INF-F#2 peptide and streptavidin coated beads. The bead eluate was then immunoblotted using antibodies against HA-tag. As seen in the figure wt STING and the deletion mutant DN5 (162-N) was readily pulled down using INF-F#2 peptide whereas the deletion mutants DN6 (172-N) was not. These data indicate that amino acids 162-172 are necessary for interactions between INF-F#2 peptide and STING.
Figure 18: Bonemarrow derived dendritic cells (BM DCs) were pretreated with indicated peptides at 5μΜ. After 30min cells were then treated with cationic liposomes (Lipo) or iwht lmw(low molecular weight) ds NA (Poly l:C). After 18 hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells.
INF wt is the INF-F#2 peptide.
INF D4-6 and INF DI6 are deletion mutants of the INF ISD (negative controls) INF mono is the monomeric form of the peptide that does not have any effect.
The data show that the INF-F#2 peptide inhibits interferon production induced by liposom fusion with the cells.
Figure 19: a+b) BMDCs were pretreated with indicated peptides at 5μΜ. After 30min cells were then treated with dsDNA ^g/mL) by transfection using lipofectamine 2000. After 18hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells, c-e) Human monocyte derived macrophages were treated with virus like particles (Vlp) or cationic liposomes. Cells were either not pretreated or pretreated with INF-F#2 peptide (pFlu). After 4 hours cells were fixed, stained for STING (green) and with DAPI (blue). The quantity of large STING foci were then quantified by confocal microscopy (d+e). f) Left panel: Cells of the human monocytic cell line THP-1 were stimulated with cationic liposomes. Cells were either not pretreated or pretreated with INF-F#2 peptide (pFlu). STING protein was then assessed using immunoblotting after non-reducing SDS-PAGE. Right panel: Lysate from liposome treated cells were either analyzed under non-reducing conditions or under reducing conditions (DTT).
Influenza peptide or pFlu are identical to INF ISD peptide which is identical to INF-F#2 peptide. Figure 20: BMDCs were treated with Iipofectamine2000 according to manufactures directions for usage of Iipofectamine2000 for transfection with DNA (but in this instance without DNA). After 18 hours supernatants were harvested and analyzed for type I IFN by bioassay. Before Iipofectamin2000 treatment cells received either no pretreatment or pretreatment for 30 minutes with INF-F#2 peptide (product). The data shows that the INF-F#2 peptide prevents production of IFN caused by lipofectamine transfection.
All cited references are incorporated by reference.
The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments and claims of the present invention may be combined.
Examples
INF-F#2, a peptide from Influenza HA2, blocks type I IFN production induced by membrane fusion.
We wanted to investigate the ability of a conserved peptide (INF-F#2) from Influenza HA2 to interfere with type I IFN induced by membrane fusion. To do this we used bone marrow -derived dendritic cells (BM DCs), which were either left untreated or pretreated with a soluble form of IN F- F#2. The cells were then stimulated with highly fusogenic cationic liposomes (Holm et al., 2012) (Figure 1) and incubated overnight. Supernatants were then analyzed for type I IFN. Interestingly, INF-F#2 pretreatment completely blocked type I IFN production. Many commercially available transfection reagents (such as Lipofectamine2000 from Invitrogen) are based on cationic liposomes and function because such liposomes can transport charged molecules such as DNA into living cells. In many cases the reagent itself is harmful to the treated cells and induce these to produce cytokines such as type I IFN. To test whether INF-F#2 could also inhibit the response to Lipofectamine2000 we pretreated BMDCs with INF-F#2 for 30min and then treated the cells with Lipofectamine2000 according to instructions by Invitrogen. As seen in Figure 2, INF-F#2 completely blocked the type I IFN response to Lipofectamine. We have previously shown that type I IFN production in response to membrane fusion depends on STING (Holm et al., 2012). Therefore, we were interested in clarifying whether INF-F#2 also inhibited type I IFN production in response to intracellular dsDNA, which also depends on STING. To do so, we again pretreated BMDCs with INF-F#2 and then treated the cells with dsDNA delivered using Lipofectamine2000 according to Invitrogens instructions. As seen in Figure 3, pre-treatment with INF-F#2 did not interfere with dsDNA induced type I IFN (Figure 3). These data show that INF-F#2 treatment specifically inhibits STING dependent type I IFN induction in response to membrane fusion as induced in BMDCs by lipid based transfection reagents such as Lipofectamin2000. pFlu co-precipitates with STING
To investigate further how INF-F#2 inhibits signalling in response to membrane fusion fusion, we used biotinylated INF-F#2 and DI6 peptides. To find proteins interacting with pFlu, non-denatured, non-reduced lysates from THP-1 cells were mixed with either of the biotinylated peptides and precipitated with streptavidin immobilized on resin beads. Precipitated proteins were separated using reducing SDS-PAGE with subsequent probing with anti-STING antibodies. Interestingly, STING was detectable in the bio-pFlu preparations but not in the bio-DI6 or no-peptide preparations (Figure 4). This did not seem to be due to an unspecific pull-down of membrane fragments caused by membrane associating properties of pFlu since the abundant transmembrane golgi resident protein giantin was not pulled down by pFlu. Instead, these data suggest that STING specifically co- precipitates with pFlu. The interaction between STING and pFlu was mapped further by using expression of C-terminal domain of CTD of STING in HEK293 cells. Either wt STING or deletion mutants with stepwise deletions of STING amino acids were used. By this approach it was possible to identify aminoacids 162-172 as being necessary for pFLu and STING interactions (Figure 17).
INF-F#2 blocks STING signalling in response to cationic liposomes
It has recently been shown that STING forms redox-sensitive homodimers in response to stimulation with cyclic di-nucleotides. We wanted to examine whether this was also the case when STING was liposome induced membrane fusion, and further whether homodimer formation was affected by IFN- F#2 pre-treatment. Here we found that membrane fusion indeed did induce homodimer formation and that this was abrogated by pre-treatment with INF-F#2 (Figure 5).

Claims

Claims
1. A use of a peptide comprising an immune suppressive domain for transfection.
The use of a peptide according to claim 1, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
The use of a peptide according to claim 1 or 2, wherein said peptide is a dimer.
A use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
5. The use according to any of the preceding claims, wherein said at least two immune
suppressive domain motifs are two identical or different motifs.
6. A use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
7. The use of a peptide according to any of the preceding claims, which is soluble in water.
8. The use of a peptide according to any of the preceding claims, wherein said immune
suppressive domain is part of a virus.
9. The use of a peptide according to claim 8, wherein said virus is an influenza virus.
10. The use of a peptide according to any of the preceding claims, wherein said peptide forms part of a protein.
11. A use of a molecule, which comprises at least two parts, each part comprising an immune suppressive domain, for transfection.
12. A kit-of-parts or composition comprising:
a. a transfection agent; and
b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
13. The kit-of-parts or composition according to claim 12, wherein said transfection agent is coupled covalently to said peptide.
14. The kit-of-parts or composition according to any of the claims 12 - 13, wherein said
transfection agent is a lipid based transfection agent.
15. The kit-of-parts or composition according to claim 14, wherein said lipid based transfection agent is selected among an anionic and a cationic transfection agent.
16. The kit-of-parts or composition according to any of the claims 12 - 15, wherein said
transfection agent is a lipid based liposome or virosome or viral vector.
17. The kit-of-parts or composition according to any of the claims 12 - 16, comprising a nano particle.
18. The kit-of-parts or composition according to any of the claims 12 - 17, further comprising a hydrophobic vehicle for delivery of hydrophobic molecules.
19. The kit-of-parts or composition according to any of the claims 12 - 18, comprising a drug.
20. The kit-of-parts or composition according to any of the claims 12 - 19, wherein said peptide an immune suppressive peptide.
21. The kit-of-parts or composition according to any of the claims 12 - 20, wherein said peptide forms part of a protein of a pathogen.
22. The kit-of-parts or composition according to claim 21, wherein said pathogen is a virus.
23. The kit-of-parts or composition according to claim 22, wherein said peptide forms part of a protein on the surface of a pathogen.
24. The kit-of-parts or composition according to claim 23, wherein said peptide forms part of a virus surface glycoprotein.
25. The kit-of-parts or composition according to claim 24, wherein said immune suppressive peptide forms part of an enveloped virus surface glycoprotein.
26. The kit-of-parts or composition according to any of the claims 12 - 25, wherein said immune suppressive peptide has a length of at least 6, preferably 7, more preferred 8, preferably 9, more preferred 10, preferably 11, more preferred 12, preferably 13, more preferred 14, preferably 15, more preferred 16, preferably 17, more preferred 18, preferably 19, more preferred 20, preferably 21 more preferred 22, preferably 23, more preferred 24, preferably 25 amino acids.
27. The kit-of-parts or composition according to any of the claims 12 - 26, wherein said peptide has a length selected among 5 - 200, preferably 10 - 100, more preferred 20 - 50, preferably 30 - 40 amino acids.
28. The kit-of-parts or composition according to any of the claims 12 - 27, further comprising a fusion peptide from a fusion protein.
29. The kit-of-parts or composition according to claim 28, wherein said fusion peptide is from the fusion protein of an enveloped virus.
30. The kit-of-parts or composition according to claim 29, wherein said fusion peptide is from a type I fusion protein.
31. The kit-of-parts or composition according to claim 29, comprising a fusion peptide from a type II fusion protein.
32. The kit-of-parts or composition according to any of the claims 28 - 31, wherein said fusion peptide has 1, 2, 3 or 4 mutations, deletions or insertions with respect to the wild type.
33. The kit-of-parts or composition according to any of the claims 12 - 32, wherein said peptide, or a functional homologue thereof, binds either directly or indirectly to a cellular protein complex containing the protein STING encoded by the gene Tmeml73.
34. The kit-of-parts or composition according to any of the claims 12 - 33, wherein said peptide, or a functional homologue thereof, affects type I interferon responses.
35. The kit-of-parts or composition according to any of the claims 12 - 34, wherein said peptide, or a functional homologue thereof, affects type I interferon responses induced by membrane fusion.
36. The kit-of-parts or composition according to any of the claims 12 - 35, comprising a peptide selected among the group consisting of the lists of Table 1 and the sequences with Seq. Id. 1 - 287, preferably Seq. Id. 287.
37. The kit-of-parts or composition according to any of the claims 12 - 36, comprising a peptide from an influenza virus or a Flu peptide.
38. The kit-of-parts or composition according to any of the claims 12 - 37, wherein said peptide has immune suppressive activity as dimer or mulitimer or when coupled to carrier proteins.
39. The kit-of-parts or composition according to any of the claims 12 - 38, wherein said peptide comprises at least one non-genetically encoded amino acid residue.
40. The kit-of-parts or composition according to any of the claims 12 - 39, wherein said peptide comprises at least one D-amino acid.
41. The kit-of-parts or composition according to any of the claims 12 - 40, wherein said peptide comprises at least one D-amino acid residue.
42. The kit-of-parts or composition according to any of the claims 12 - 41, wherein said peptide is coupled to any other molecule.
43. The kit-of-parts or composition according to any of the claims 12 - 42, wherein said peptide is attached to lipids.
44. The kit-of-parts or composition according to any of the claims 12 - 43, wherein said peptide is coupled to a molecule through a peptide bond.
45. The kit-of-parts or composition according to any of the claims 12 - 44, wherein said peptide is coupled to a protein.
46. The kit-of-parts or composition according to any of the claims 12 - 45, wherein said peptide is a circular peptide.
47. The kit-of-parts or composition according to any of the claims 12 - 46, wherein said peptide is attached to at least one biological membrane.
48. The kit-of-parts or composition according to any of the claims 12 - 47, wherein said peptide is modified in a way in which one of the peptide bonds is replaced by a non-peptide bond.
49. The kit-of-parts or composition according to any of the claims 12 - 48, wherein said peptide interferes with an interferon response induced by the transfection agent upon addition to a cell.
50. The kit-of-parts or composition comprising a functional homologue of a peptide according to the any of the claims 12 - 49.
51. A transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
52. A nano particle coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
53. A hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
A drug coupled covalently to a peptide comprising an immune suppressive domain, wherei said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
55. A use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a
hydrophobic vehicle or a drug according to any of the preceding claims for inhibiting immune response.
56. The use of a peptide, composition, or a kit of parts according to any preceding claims for introducing a molecule into a cell.
The use of a peptide, composition, or a kit of parts according to claim 56, for introducing a DNA or NA molecule into a cell.
The use of a peptide, composition, or a kit of parts according to claim 56, for introducing a pharmaceutical molecule into a cell.
The use of a peptide, composition, or a kit of parts according to claim 56, for introducing a pharmaceutical molecule into a tissue.
60. The use of a peptide, composition, or a kit of parts according to claim 59, for introducing a gene-therapeutic pharmaceutical molecule into a cell.
61. The use of a peptide, composition, or a kit of parts according to any preceding claims for initiating expression of proteins via transfected plasmids.
62. The use of a peptide, composition, or a kit of parts according to any preceding claims for targeting active genes by micro NA silencing.
63. The use of a peptide, composition, or a kit of parts according to any preceding claims for examining the immune response to DNA or RNA.
64. The use of a peptide, composition, or a kit of parts according to any preceding claims for gene therapy.
65. The use of a peptide, composition, or a kit of parts according to any preceding claims for delivery of any molecule using liposomes.
66. A method for transfecting a cell, said method comprising:
a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences;
b. Incubating the cell with said peptide;
c. Providing a transfection agent;
d. Providing a material to be transfected into the cell; and
e. Further incubating the cell with said transfection agent and said material.
67. A cell obtainable with the method according to claim 66.
68. A cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
69. A cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
70. A use of a cell according to any of the claims 67 to 69, for diagnostics, prophylaxis or therapy.
The use according to claim 70, for cell therapy and/or gene therapy.
The use according to claim 70 or 71, for stem cell transplantation.
EP14722536.1A 2013-04-10 2014-04-10 Peptides having immune suppresive domains for transfection Withdrawn EP2984171A2 (en)

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