WO2005021574A2 - Induction d'anticorps neutralisants antiviraux chez les hommes et les animaux - Google Patents

Induction d'anticorps neutralisants antiviraux chez les hommes et les animaux Download PDF

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WO2005021574A2
WO2005021574A2 PCT/DE2004/001897 DE2004001897W WO2005021574A2 WO 2005021574 A2 WO2005021574 A2 WO 2005021574A2 DE 2004001897 W DE2004001897 W DE 2004001897W WO 2005021574 A2 WO2005021574 A2 WO 2005021574A2
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Prior art keywords
seq
amino acid
virus
antibodies
hiv
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PCT/DE2004/001897
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German (de)
English (en)
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WO2005021574A3 (fr
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Uwe Fiebig
Mirco Schmolke
Joachim Denner
Reinhardt Kurth
Alexander Karlas
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Brd, Vertreten Durch Das Bundesministerium Für Gesundheit Und Soziale Sicherung, Letztvertreten Durch Den Präsidenten Des Robert-Koch-Institutes
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Priority claimed from DE10339966A external-priority patent/DE10339966A1/de
Application filed by Brd, Vertreten Durch Das Bundesministerium Für Gesundheit Und Soziale Sicherung, Letztvertreten Durch Den Präsidenten Des Robert-Koch-Institutes filed Critical Brd, Vertreten Durch Das Bundesministerium Für Gesundheit Und Soziale Sicherung, Letztvertreten Durch Den Präsidenten Des Robert-Koch-Institutes
Priority to CA002548483A priority Critical patent/CA2548483A1/fr
Priority to EP04786181A priority patent/EP1660523A2/fr
Publication of WO2005021574A2 publication Critical patent/WO2005021574A2/fr
Publication of WO2005021574A3 publication Critical patent/WO2005021574A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to immunogenic constructs and a pharmaceutical agent for inducing humoral neutralizing immune responses to virus infections and to kits for the detection of antibodies and virus antigens on the basis of these immunogenic constructs and the induced antibodies.
  • the invention further relates to a method for inducing an antibody response and a method for passive immunization of an organism using neutralizing antibodies which have been obtained with the aforementioned immunogenic constructs and a bioassay for the detection of infection with viruses.
  • HIV human immunodeficiency virus
  • AIDS acquired human deficiency syndrome
  • Artificial active immunization involves the application of defined antigens, which leads to actively acquired immunity through the formation of antibodies or cellular immunity. In passive immunization, the antibodies or immune cells are applied directly, which lead to passively acquired immunity.
  • Artificial active immunization of an organism against viruses can be carried out, for example, by inactivated viruses, viral proteins or peptides derived from viruses, which, for example, induce neutralizing antibodies. Neutralizing antibodies have been described for various viral diseases and can be induced in the organism by the administration of certain antigens, in particular virus proteins. Such an activation or induction of an immune response is called vaccination.
  • a human monoclonal antibody (2F5) described in 1993 shows that 20 can be induced in HIV-infected persons and that could be obtained from an HIV-positive patient via a cell culture.
  • This antibody has a virus-neutralizing spectrum that encompasses almost all subtypes of HIV; the antibodies bind to an epitope located at the N-terminal of the transmembrane passage of the transmembrane coat protein gp41. It could
  • gp41 In addition to the neutralizing antibodies that can be used for the therapy, a further therapy was developed which is directed against gp41.
  • T-20 One of these peptides, T-20, contains the ELDKWA sequence already described.
  • a major disadvantage of the vaccination strategies presented, especially against gp120, o is that the vaccines used are not able to prevent the emergence of new virus variants or modifications in the course of the virus disease (escape mutants).
  • the neutralizing antibodies formed against the original virus are unable to interact with the mutated virus in such a way that successful preventive protection or effective therapeutic treatment of the infected organism is possible.
  • the object of the invention was therefore to produce a vaccine which can be used in the prevention, diagnosis and therapy of viral diseases, in particular retroviral diseases.
  • an immunogenic construct comprising amino acid sequences selected from a viral transmembrane coat protein which is associated with the virus membrane via at least one membrane passage, comprises at least one fusion domain and at least two alpha-helical structures, the amino acid sequences being selected from ( i) a first region of the protein located between the membrane passage and a first alpha-helical structure as well
  • a construct is also suitable which comprises the DNA coding the respective amino acid sequence, the presence of both regions in turn being important in the coding sequence.
  • An immunogenic construct is thus claimed, which consists of different amino acid sequences.
  • the claimed amino acid sequences are to be selected from a viral transmembrane coat protein.
  • This coat protein has to fulfill the requirement that it is associated with the virus membrane via at least one membrane passage and that it has at least one fusion domain and at least two alpha-helical structures.
  • the skilled worker is aware that several coat proteins meet these requirements, such as. B.GP2, gp20, gp30, gp37, gp160, p15E, HA2, F2 and others
  • Each of these coat proteins is a viral transmembrane coat protein which has at least one membrane passage, a fusion domain and two alpha-helical structures.
  • the person skilled in the art has to select two regions of amino acid sequences from the viral transmembrane coat proteins mentioned.
  • the first region is a freely selectable segment of the coat protein between the membrane passage of this coat protein and the first alpha-helical structure which this coat protein has.
  • the second area to be selected from the above-mentioned coat protein is the Section that lies between the fusion domain of the transmembral coat protein and the second alpha-helical structure of this coat protein, wherein the alpha-helical structure, the membrane passage and / or the fusion domain can be part of the immunogenic construct. The combination of both areas leads to the induction of the neutralizing antibodies.
  • the immunogenic construct comprises two amino acid sequences, wherein these two amino acid sequences can be selected from very specific regions of viral transmembrane envelope proteins.
  • an immunogenic construct which comprises at least two amino acid sequences, can induce neutralizing antibodies in an organism and can thus optionally be used as vaccine together with auxiliaries known to the person skilled in the art.
  • an immunogenic construct is any agent which is suitable for inducing neutralizing antibodies.
  • Such an antibody response provides antibodies in the organism that have a neutralizing effect on the respective retrovirus or another virus.
  • the neutralization of a virus is understood to mean any mechanism which prevents viruses from infection in vivo or in vitro and prevents their multiplication in the preventive sense or inhibits the further multiplication of the viruses in the therapeutic sense or a Combination therapy can be more effective.
  • the construct according to the invention activates the immune system and induces the formation of neutralizing antibodies.
  • the neutralizing antibodies are advantageously directed, in particular, against the viral structures by which they were induced, cross-reacting antibodies not being excluded according to the invention.
  • transmembrane envelope proteins When infected with enveloped viruses, including retroviruses including HIV, there are interactions between the surface envelope proteins and cellular receptors, which then lead to changes in the conformation of the transmembrane envelope protein and to fusion with the cell membrane (Fig. 1, Fig. 2).
  • the transmembrane envelope proteins contain highly conserved domains that play an important role in the infection process and are therefore excellent targets for the induction of broadly neutralizing antibodies.
  • Envelope proteins have, among other things, a component that is anchored in the membrane and a component that at least partially and temporarily protrudes outwards from the membrane.
  • the selected amino acid sequences preferably originate from the part protruding from the membrane.
  • This transmembrane coat protein comprises an ectodomain, an anchor and a cytoplasmic part, the ectodomain having a fusion domain, a first alpha-helical structure, a cysteine ioop and a second alpha-helical structure. Following the second alpha-helical structure, the C-terminal is followed by an anchor domain that anchors the transmembrane coat protein gp41 in the virus membrane.
  • the selected amino acid sequences which can be, for example, domains, peptides or recombinant proteins or fragments thereof, can be obtained in various ways by the person skilled in the art, preferably by peptide synthesis or genetic engineering. Of course, it is also possible not to use the amino acid sequences, but rather the DNA encoding them. For example, the DNA can be packaged in a vector. The corresponding amino acid sequence is then encoded in the cells. Such methods are known to the person skilled in the art from gene therapy.
  • a region which can also be referred to as a peptide section, is selected between the fusion domain and the first alpha- helical structure.
  • a second peptide is selected from the area between the membrane and an alpha-helical structure facing it. In the sense of the invention means choose between two areas that the flanking areas such.
  • B. membrane passage, alpha-helical structure and / or fusion domain can be at least partially components of the selected section.
  • amino acid sequences selected according to the invention between the membrane passage and one alpha-helical structure and between the fusion domain and the other alpha-helical structure are only used in combination and regions of the membrane passage, the fusion domain and / or partially or completely include the alpha-helical structure or structures.
  • the alpha-helical structure facing the membrane has a smaller spatial distance from the membrane if the coat protein is represented or assumed as a linear, non-folded structure. Accordingly, the structure facing is spatially closer to the membrane passage of the linear protein. It is of course possible that the position of individual components of the coat protein is changed by natural or artificial folding processes.
  • amino acid sequences selected from the coat protein e.g. Peptides or protein domains that correspond to the regions in the vicinity of the alpha-helical structures can be removed from the total protein or, after their natural sequence sequence is known, can be obtained synthetically or by genetic engineering.
  • the peptides, recombinant or viral proteins thus obtained are used as an immunogenic construct by being applied to an organism.
  • the person skilled in the art can determine the size of the selected amino acid sequences by routine experiments, e.g. measure antibody production or response.
  • two amino acid sequences are selected from the area of an ectodomain of a transmembrane coat protein of a virus, the first amino acid sequence from the area between the fusion domain and a subsequent alpha-helical structure flanking or offset therefrom and the second amino acid sequence from the area between the Membrane of the virus and the next alpha-helical structure is selected.
  • Synthetic peptides, recombinant proteins or a DNA which codes these amino acid sequences are produced according to these amino acid sequences. The selection can be made in such a way that the amino acid sequences still encompass regions of the alpha-helical structure or the anchor structure that anchors the coat protein to the membrane.
  • enveloped viruses including retroviruses
  • retroviruses can also be used to obtain immunogenic constructs according to the invention, further preferred are: FeLV, MuLV, BIV, CAEV, EIAV1, FIV, OMVV, SIVmac, SIVcpz, VILV, RSV, ALV, JSRV, SMRV, SRV, GALV, BLV, HTLV-1, HTLV-2, Marburg Virus, Ebola, SARS virus, influenza
  • Virus, measles virus, mumps virus and / or HPV-1 All amino acid sequences, peptides or recombinant proteins or viral proteins obtained in this way can be used as an immunogenic construct in order to generate neutralizing antibodies against these viruses, although it cannot be ruled out according to the invention that cross-reactivities occur, so that, for example, an immunogenic construct which is used for Example from MuLV or complete artificially obtained, can also have an effect against, for example, HIV or FeLV.
  • the neutralizing antibodies induced by the immunogenic construct are directed against the viruses from which the respective peptides are obtained.
  • the immunogenic constructs can also be recombinant proteins which consist of parts of the transmembrane envelope protein of one virus and the two domains of another virus according to the invention, so-called hybrids.
  • DNA is used for the immunization, which corresponds to these peptides or recombinant proteins of these viruses.
  • a combination between DNA immunization and subsequent immunization with peptides or recombinant proteins or in a multiple sequence is also preferred.
  • the neutralizing effect of the antibodies on enveloped viruses, including the retroviruses, with regard to their prophylactic potential, is shown, for example, as an inhibition of virus infection, an inhibition of syncytium formation, an inhibition of the fusion between virus and target membrane, and a reduction or stabilization of the rate of virus multiplication in one Organism or otherwise.
  • the neutralizing effect with regard to its therapeutic effect can consist, for example, in that certain antiviral drugs work better, for example as a desired side effect, through the induction or application of the antibodies, or the number of side effects of these drugs is reduced by reducing the dose. This means that the effect of the antibodies in the sense of the invention is not limited to eliminating the viruses, but rather encompasses the entire spectrum of advantageous effects in therapy or prophylaxis.
  • the at least two peptides or recombinant proteins or their corresponding DNA can be used alone or in combination, if appropriate, with other antigens of interest.
  • they can be used with other antigens as physical mixtures or chemically bound to one another with or without a spacer molecule.
  • the two peptides or recombinant proteins according to the invention can be chemically or physically linked to one another.
  • carrier peptides, proteins or carrier substances can be, for example, albumins, the KLH, the MAP and other proteins which are known to the person skilled in the art for their immunogenic capacity; as
  • Carrier substances are furthermore preferably thyroglobulin or BSA. These proteins can be bound by non-peptide bonds, such as a disulfide bridge or calcium ion bonds, but it is also possible that they are linked by a peptide bond.
  • the peptides can of course be substitution, deletion and addition analogs of the peptides according to the invention.
  • the viruses can be all viruses which have a membrane with which envelope proteins or similar structures are associated, these structures having to have at least one ectodomain which contains a fusion domain and / or an alphachical structure.
  • the membrane of the virus can be any structure that includes lipids, as long as this enables a connection to proteins.
  • the introduction of the immunogenic construct into an organism can take place in any way which enables the immune system to be brought into contact with the construct in such a way that an antibody response is induced.
  • This can be done, for example, by ingesting the immunogenic construct orally or rectally, enterally or parenterally, or by injecting it directly into the body, for example into selected organs such as the spleen or into blood vessels Vaccination by injection.
  • Preferred injections are intradermal, subcutaneous, intramuscular or intravenous injection.
  • DE 198 51 282 A1 describes a method for applying antigens to mucous membranes, which is included in the disclosure of the present invention. Further applications are shown below in connection with the method according to the invention.
  • the application of the DNA used for the immunization happens e.g. with the help of so-called vaccination pistols, which insert the DNA into the skin using gold balls or with syringes, which can insert the DNA under the skin or into the muscle.
  • Amino acid sequences e.g. two peptides or recombinant proteins - which are parts of defined domains and are positioned between other defined domains - from a coat protein of a virus can be used as a simple and effective immunogen to be provided.
  • two peptides or recombinant proteins or the corresponding DNA Through the interaction of the two peptides or recombinant proteins or the corresponding DNA, at least one epitope is presented to the immune system so effectively that neutralizing antibodies against viruses are generated.
  • the construct according to the invention is artificial in the sense that it does not represent a complete coat protein which occurs naturally, ie the natural complete coat proteins are not encompassed by the teaching according to the invention.
  • the construct When used as a vaccine naturally occurring transmembrane coat proteins isolated from the virus, or corresponding soluble ectodomains of the transmembrane coat proteins (except HIV) are used.
  • the construct consists of two amino acid sequences that are not chemically or otherwise linked. However, it can be advantageous if the amino acid sequences are present directly or in association with one another via a linker or a support. It is further preferred that the selected amino acid sequences from one coat protein are introduced into another viral coat protein or fragments thereof by addition or substitution or other methods known to the person skilled in the art. Accordingly, the carrier of the selected amino acid sequences of one virus can be the transmembrane coat protein of another virus.
  • adjuvants can be added to the immunogenic construct or to all means which can be produced therefrom, in particular the vaccine, the vaccine become.
  • Known adjuvants for human vaccines are, for example, aluminum compounds such as aluminum hydroxide or aluminum phosphate.
  • Aluminum hydroxide is a component of numerous inactivated or subunit vaccines, inter alia in the case of hepatitis B virus vaccines, and is therefore well known to the person skilled in the art.
  • Other known adjuvants are, for example, MF 59 in the Fluad.
  • peptides or proteins enables a specific immune response against these, enhances or modifies an adjuvant in the sense of the invention.
  • an adjuvant in the sense of the invention.
  • the co-application of egg albumin in complete Freund's adjuvant can, under certain circumstances, cause an increased formation of cell-mediated immunity and thus support the effect of neutralizing antibodies.
  • Other adjuvants of interest are, for example, saponins, such as, for example, QS 21, muramyl dipeptide, muramyl tripeptide and compounds with a muramyl peptide nucleus, proteins such as, for example, gamma interferon and TNF or phosphate dibylcholine, squalene or the polyols known to those skilled in the art.
  • DNA which itself has an immunostimulatory property, or a protein can
  • Adjuvant effect including encoding cytokines, applied in parallel or in a construct.
  • immunogenic constructs which are present either as a recombinant protein or as a synthetic peptide with the above-mentioned modifications, can also be used in combination with others Vaccines, including those that are already commercially available, can be used regardless of whether the other vaccine is directed against the same virus or against other viruses (combination vaccination).
  • antigen refers to a compound containing one or more domains against which an immune response is desired.
  • the binding sites of the antibodies in these domains are called epitopes.
  • Complex mixtures of antigens are also included in this definition, such as killed cells, bacteria or viruses or fractions thereof, in each case in connection with the peptides according to the invention, recombinant proteins or the DNA used for immunization.
  • admixing denotes the addition of an excipient to the peptides according to the invention, recombinant proteins, complex mixtures and / or adjuvant of interest, for example by mixing dry reagents or mixing a dry reagent with a reagent in solution or suspension, or mixtures aqueous formulations of reagents.
  • excipient refers to a pharmaceutical
  • Non-therapeutic carrier added to the composition which is pharmaceutically acceptable, i.e. non-toxic to recipients at the dosages and concentrations used.
  • Suitable excipients and their formulations are known to the person skilled in the art, for example from Remington's pharmaceutical science, 16th edition, 1980.
  • Vaccine refers to a formulation of the peptides or recombinant proteins of the invention or the DNA useful for immunization, optionally in combination with another antigen or other DNA which is intended to provide a prophylactic, therapeutic or diagnostic response in or outside of one Provide host when the host is exposed to the antigens.
  • exemplary vaccines include
  • Vaccines against diseases such as HIV / AIDS, SARS, FeLV and others.
  • therapeutic amount refers to an amount that prevents or ameliorates symptoms of a disorder or responsive, pathologically physiological condition.
  • the amount of immunogenic construct to be used by a healthy person in the case of prophylaxis or to a patient in the case of therapy is formulated and the dose is determined in accordance with customary medical practice, the disorder to be treated, the condition of the individual patient, the administration site, the administration method and other factors known to treating physicians.
  • the dose of vaccines administered will depend on the properties of the antigen used, that of the immunogen, for example its binding activity and in vivo plasma half-life, as well as the concentration of the antigen in the formulation, route of administration, site and location Rate of dosage, the clinical tolerance of the individual (human and animal), the pathological affection of the patient and the like, as is known to doctors or other experts.
  • dosages of about 0.1 to 1000 mg per individual per administration are preferred.
  • Different doses can also be used during a sequence of successive vaccinations; the attending physician can administer a first vaccination and then boost (boost) with relatively low doses of adjuvants; here the options protein-protein, peptide-peptide, protein-peptide or vice versa, DNA-protein / peptide are preferred.
  • boost boost
  • injections are a preferred route for the therapeutic administration of the vaccines, such as the encapsulated or carrier-bound vaccines, although aerosol delivery via catheters or surgical tubing is also can be applied.
  • Alternative routes include suspensions, tablets, capsules and the like for oral administration, commercially available nebulizers for liquid formulations and inhalations of lyophilized or aerolyzed compounds and suppositories for rectal or vaginal administration.
  • Liquid formulations can be used, for example, from powder formulations. Proteins, peptides and DNA are injected for prophylactic immunization.
  • the suitability of the chosen vaccination parameters can be determined by taking serum aliquots from the patient - that is to say from humans or animals - and testing for antibody titers in the course of the immunization protocol.
  • the amount of T cells or other immune system cells can be determined in a conventional manner in order to obtain an overview of the patient's immunological constitution.
  • the clinical condition of the patient can be observed for the desired effect, for example the anti-infectious effect. Since, for example, HIV or other diseases can be associated with other infections, it is also possible to follow them up.
  • the patient can be boosted with further vaccinations and the vaccination parameters can be modified in a way that an improvement in the immune response can be expected, preferably an increase in the amount of peptide or antigen and / or adjuvant, complexation of the peptides with a carrier or its conjugation to an immunogenic protein or variation of the route of administration.
  • both an aqueous formulation and dry peptides or adjuvants can be mixed with an excipient in order to ensure a stabilizing effect before the treatment, for example with a solvent.
  • An aqueous solution of a peptide can be a peptide in suspension or a solution.
  • the recombinant protein according to the invention, the DNA construct and / or the peptide can be introduced in a solution with a preservative.
  • suitable preservatives of the suspension or solutions include phenol, benzyl alcohol, m-cresol,
  • the formulations of the peptides or the antigen constructs can contain components in amounts which are not detrimental to the production of stable forms and in amounts which are suitable for effective, safe pharmaceutical administrations.
  • other pharmaceutically acceptable excipients known to those skilled in the art may form part of the vaccines or formulations of the invention. These include, for example, salts, various fillers, additional buffering agents, chelating agents, antioxidants, cosolvents and the like.
  • the immunogenic construct is associated with a liposomal formulation. This can be done, for example, in such a way that the immunogenic construct is enclosed in a liposome or anchored on the liposome surface. It is known to the person skilled in the art that artificial or natural membranes of liposomes can have an immunostimulating effect, in particular if the antigenic components are coupled to the surface of the liposomes or inside of the liposomes are enclosed or simply mixed together with the liposomes. It is preferred that the immunostimulating effect is increased in that the liposomes are "spiked" with transmembrane or fusiogenic glycoproteins. Such formulations of liposomes can be applied parentrally.
  • a mucosal immune response that can be induced with the spray is preferably suitable for the treatment of SARS.
  • the antigenic component or the immunogenic construct must be applied to the mucous membrane in such a state that it is able to penetrate the mucous membrane or to be absorbed by it. Therefore, the vesicle must be biocompatible with the mucus and have a certain degree of hydrophilicity. Structures of this type are known to the person skilled in the art, for example from EP 0682528, the teaching of which is included in the disclosure content of the invention.
  • the liposomal composition can comprise one or more additional pharmaceutical carriers which are selected from surface-active substances and absorption promoters such as, for example, polyoxyethylene alcohol ether, bile salts and their derivatives, fusidic acid and their derivatives, oleic acid, lecithin, lysolecithins, Tween® 21 to 85, etc.
  • additional pharmaceutical carriers which are selected from surface-active substances and absorption promoters such as, for example, polyoxyethylene alcohol ether, bile salts and their derivatives, fusidic acid and their derivatives, oleic acid, lecithin, lysolecithins, Tween® 21 to 85, etc.
  • water absorbing polymers such as glycofurol, polyethylene glycol 200 to 7500, polyvinyl pyrrolidone, propylene glycol or polyacrylic acid, gelatin, cellulose and derivatives, etc .
  • Substances that inhibit enzymatic degradation, such as aprotinin, etc . organic solvents such as alcohols such as ethanol, glycerol, benzyl alcohol, etc .; or ethyl acetate, etc .
  • hydrophobic agents such as vegetable oil, soybean oil, peanut oil, coconut oil, corn oil, olive oil, sunflower oil, "miglyols" or mixtures thereof, etc .
  • pH regulators such as nitric acid, phosphoric acid, acetic acid, citrates, etc .
  • Preservatives and osmotic pressure regulators such as
  • Glycerol sodium chloride, methyl paraoxybenzoate, benzoic acid, etc .
  • Liposome and / or emulsion formulations such as lecithins, etc .
  • microencapsulated formulations Blowing agents such as butane.
  • the peptide segments are optionally associated with one another or enclosed in liposomes connected to a carrier, the inclusion in liposomes in the sense of the invention not necessarily meaning that the peptides are present inside the liposomes
  • inclusion in the sense of the invention can also mean that the peptides are associated with the membrane of the liposomes, for example in such a way that they are anchored on the outer membrane.
  • Such Representation of the peptides according to the invention in or on the liposomes is advantageous if the person skilled in the art selects the liposomes in such a way that they have an immunostimulating effect.
  • the lipids can be simple lipids such as esters and amides or complex lipids such as glycolipids such as cerebrosides or gangliosides, sphingolipids or phospholipids.
  • the first helical structure is a C-terminal helix and the second alpha-helical structure is an N-terminal helix of the viral coat protein.
  • the person skilled in the art can preferably choose a region between the fusion domain and the N-terminal helix and a region between the transmembrane passage and the C-terminal helical structure, the fusion domain, the region of the Transmembrane passage and / or the alpha-helical structure, if it is one, or the alpha-helical structures can be wholly or partly involved in the immunogenic construct.
  • the peptides according to the invention are obtained from HI viruses, they are preferably peptides or recombinant proteins or DNA which have the amino acids 519 to 564 (N-terminal sequence) and 650 to 683 (C-terminal sequence) of the HIV-1 reference Genome (NCBI database: K03455, HIV HXB2 CG) correspond, or fragments or subunits thereof, which are functionally analogous to the above-mentioned domains, that is to say which are able to induce neutralizing antibodies and, in the particularly preferred case, to give protection against infection , It is of course known to the person skilled in the art that, because of the variability in different subtypes of HIV-1, there are sequence variations within these sequences; Such sequence variations are also covered by the invention. This also applies to partial sequences of the consensus sequences 519 to 564 and 650 to 683, including chemical modifications of individual amino acids, the linking of the sequences by means of linkers and sequences with additional amino acids added for the purpose of multimerization of the sequences.
  • N-terminal sequences are:
  • Preferred C-terminal sequences are:
  • a peptide is selected in each case from the group of the N-terminal sequences and the C-terminal sequences, it being possible to provide the immunogenic construct according to the invention by combining at least two sequences. It can also be applied as a recombinant protein or DNA (vaccine, vaccine).
  • the vaccine is a fragment of coat proteins selected from the group comprising GP2, gp20, gp21, gp30, gp36, gp37, gp40, gp41, gp45, gp160, p15E, E2, HA2 and / or F2.
  • GP2 gp20, gp21, gp30, gp36, gp37, gp40, gp41, gp45, gp160, p15E, E2, HA2 and / or F2.
  • the at least two peptide sections, recombinant proteins or corresponding DNA are selected from the group (where N stands for N-terminal sequences and C for C-terminal sequences):
  • N AVGLAIFLLVLAIMAITSSLVAATTLVNQHTTAKV
  • C SLSDTQDTFGLETSIFDHLVQLFDWTSWKDWIK, preferred for BIV (transmembrane coat protein gp40);
  • N GVGLVIMLVIMAIVAAAGASLGVANAIQQSYTKAAVQTLAN C: AMTQLAEEQARRIPEVWESLKDVFDWSGWFSWLKYI, preferred for CAEV (transmembrane coat protein); N: FGISAIVAAIVAATAIARSATMSYVALTEVNKIMEVQNH C: LAQSMITFNTPDSIAQFGKDLWSHIGNWIPGLGASIIKY, preferred for EIAV1 (transmembrane coat protein gp45); N: SSSYSGTKMACPSNRGILRNWYNPVAGLRQSLEQYQVVKQPDYLLVPE C: MDIEQNNVQGKIGIQQLQKWEDWVRWIGNIPQYLK, preferred for FIV (transmembrane coat protein gp36); N: GIGLVIVLAIMAIIAAAGAGLGVANAVQQSSYTRTAVQSLANATAAQQN C: QVQIAQRDAQRIPDVWKALQEAFD
  • N LGALGFLGAAGSTMGAAAVTLTVQARQLLSGIVQQQNNLL
  • C EEAQSQQEKNERDLLELDQWASLWNWFDITKWLWYIK, preferred for SIVcpz (transmembrane protein gp41);
  • N FLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLLRAIEAQQ FLGAAGSTMGAASMTLTVQARQLLSGIVQQQQNNLLRAIEAQQHLL FLGAAGSTMGAASVTLTVQARLLLSGIVQQQNNLLRAIEAQQHML
  • C SQNQQEKNEQELLELDKWAGLWSWFSITNWLWY SQNQQEKNEQELLELDKWASLWNWFNITNWLWY SQTQQEKNEQELLELDKWASLWNWFDITNWLWY preferably HIV-1 (gp41 transmembrane envelope protein);
  • N GIGLVIVLAIMAIIAAAGAGLGVANAVQQSYTRTAVGSLANATAAQQE C: EAALQVHIAQRDARRIPDAWKAIQEAFNNWSSWFSWLKY, preferred for Visna virus (transmembrane coat protein gp41); N: LGFLGFLATAGSAMGARSLTLSAQSRTLLAGIVQQQQQLL C: EEAQIQEKNMYELQKLNSWDILGNWFDUSWVKYIQ, preferred for HIV-2 (transmembrane coat protein gp36); N: WGPTARIFASILAPGVAAAQALREIERLACWSVKQANLTTSLL C: KFQLMKKHVNKIGVDSDPIGSWLRGIFGGIGEWAVH, preferred for RSV (transmembrane coat protein gp37); N: SVSHLSSDCNDEVQLWSVTARIFASFFAOGVAAQALKEIERLA C: ALQAMKEHTEKIRVEDDOIGDWFTRTFGGLGGWL
  • N TAALITGPQQLEKGLSNLHRIVTEDLQALEKSVSNL
  • C DHSGAIRDSMSKLRERLERRRREREADQGWFEGWFNRS preferred for PERV (transmembrane coat protein p15E);
  • N TALIKGPIDLQQGLTSLQIAMDTDLRALQDSISKLED
  • C SMRRLKERLDKRQLEHQKNLSWYEGWFNRSPWLTT preferred at KoRV (transmembrane coat protein p15E);
  • N SPVAALTLGLALSVGLTGINVAVSALSHQRLTSLIHVLEQDQQ
  • C PLSQRVSTDWQWPWNWDLGLTAWVRET, preferred for BLV (transmembrane coat protein gp30);
  • N AVPVAWLVSALAMGAGVAGGITGSMSLASGKSLLHEV
  • C PILQERPPLENRVLTGWGLNWDLGLSQWAREALQ, preferred for HTLV-1 (transmembrane coat protein gp21);
  • N AVPIAVWSVSALAAGTGIAGGVTGSLSLASSKSLLLEVD
  • C SVLQERPPLEKRVITGWGLNWDLGLSQWAREALQ, preferred for HTLV-2 (transmembrane coat protein gp30);
  • N FPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGI
  • C KNISEQIDQIKKDEQKIGRGWGLGGKWWTSDWG, preferred for the Marburg virus (transmembrane glycoprotein gp36);
  • N LITGGRRTRREAIVNAQPKCNPNLHYWTQDEGAAIGLAWIPYFGPAA
  • C KNITDKIDQHHDFVDKTLPDQGDNDNWWTGWRQWI, preferred for Ebola (transmembrane protein GP2);
  • N LITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDF C: DRLNEVAKNLNESLIDLQELKYEQYEKWPWYVW, preferred for SARS virus (E2, transmembrane glycoprotein gp36);
  • C HDVYRDEALNNRFQ
  • N FAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAAAMKNSIQTNRA
  • C TELSKVNASLQNAVKQIKESNHQLQSVSVSSK, preferred for the mumps virus (fusion glycoprotein F2)
  • N FFGAVIGTIALGVATAAQ1TAGIALAEAREARKDIALIKDSIVKTH
  • C TNFLEESKTELMKARAIISVGGWHNTESTQ, preferred for HPV-1 (F2 glycoprotein)
  • BIV Bovine Immunodeficiency Virus
  • CAEV Caprines Arthritis Encephalitis Virus
  • EIAV1 Equine Infectious Anemia Virus
  • FIV Fine Immunodeficiency Virus
  • OMVV Outdoor Maedi-Visna Virus
  • SIVmac Simian Virus Makeficiency
  • SIVcpz Simian chimpanzee immunodeficiency virus
  • HIV-1 human immunodeficiency virus type 1
  • HIV-2 human immunodeficiency virus type 2
  • RSV rous sarcoma virus
  • ALV avian leukosis virus
  • JSRV Jaagsiezte sheep retrovirus
  • SMRV Simrrel Monkey Retrovirus
  • SRV Ses Retrovirus
  • GALV Gabbon monkey leukemia virus
  • MuLV MuLV
  • FeLV fine leukemia virus
  • KoRV kos Retrovirus
  • FLGFLGAAGSTMGAASITLTVQARQLLS FLGFLGAAGSTMGAASMTLTVQARQLLS, FLGFLGAAGSTMGAASLTLTVQARQLLS, LLGFLGAAGSTMGAASITLTVQARQLLS, FLGFLGAAGSTMGAASITLTVQVRQLLS, FLGVLSAAGSTMGAAATALTVQTHTLMK, FLGFLGAAGSTMGARSMTLTVQARQLLSG1VQQQNNLLRAIEAQQ FLGAAGSTMGAASMTLTVQARQLLSGIVQQQQNNLLRAIEAQQHLL FLGAAGSTMGAASVTLTVQARLLLSGIVQQQQNNLLRAIEAQQHML
  • AVGLAIFLLVLAIMAITSSLVAATTLVNQHTTAKV GVGLVIMLVIMAIVAAAGASLGVANAIQQSYTKAAVQTLAN, FGISAIVAAIVAATAIARSATMSYVALTEVNKIMEVQNH, SSSYSGTKMACPSNRGILRNWYNPVAGLRQSLEQYQVVKQPDYLLVPE, GIGLVIVLAIMAIIAAAGAGLGVANAVQQSSYTRTAVQSLANATAAQQN,
  • LGFLGFLATAGSAMGAASLVTAQSRTLLAVIVQQQQQLLDVV LGALGFLGAAGSTMGAAAVTLTVQARQLLSGIVQQQNNLL
  • GIGLVIVLAIMAIIAAAGAGLGVANAVQQSYTRTAVGSLANATAAQQE LGFLGFLATAGSAMGARSLTLSAQSRTLLAGIVQQQQLL
  • WGPTARIFASILAPGVAAAQALREIERLACWSVKQANLTTSLL SVSHLSSDCNDEVQLWSVTARIFASFFAOGVAAQALKEIERLA
  • GLSLIILGIVSLITLIATAVTACCSLAQSIQAAHTVDLSSQNVTKVMGT AVTLIPLLVGLGVSTAVATGTAGLGVAVQSYTKLSHQLINDVQALSSTI, AIQFIPLVIGLGITTAVSTGTAGLGVSLTWYTKLSHQLISDBQAISSTI, DPVSLTVALLLGGLTMGSLAAGIGTGTAALIETNQFKQLQ, DP
  • NEQDLLALDKWANLWNWFDISNWLWYIK NEQDLLALDKWANLWNWFDITNWLWYIR
  • NEQELLELDKWASLWNWFDITNWLWYIK NEKDLLALDSWQNLWNWFDITNWLWYIK
  • NEQELLELDKWASLWNWFSITQWLWYIK NEQELLALDKWASLWNWFDISNWLWYIK
  • NEQDLLALDKWDNLWSWFSITNWLWYIK NEQDLLALDKWASLWNWFDITKWLWYIK
  • NEQDLLALDKWASLWNWFSITNWLWYIK NEKKLLELDEWASIWNWLDITKWLWYIK
  • TNFLEESKTELMKARAIISVGGWHNTESTQ these are preferably C-terminal sequences.
  • amino acids have analogous physicochemical properties which advantageously result in these amino acids being able to be exchanged with one another.
  • amino acids include, for example, the group of amino acids (a) glycine, alanine, valine, leucine and / or isoleucine; or the amino acids (b) serine and threonine, the amino acids (c) asparagine and glutamine, the amino acids (d) aspartic acid and glutamic acid; the amino acids (e) lysine and arginine and the group of aromatic amino acids (f) phenylalanine, tyrosine and / or tryptophan.
  • Amino acids within one and the same group (a-f) can be interchanged. It is also possible for amino acids to be replaced by modified amino acids or specific enantiomers. Further modifications are possible according to the teaching according to WO 99/62933 or WO 02/38592.
  • the peptide comprises a linker and / or a spacer which is selected from the group comprising: ⁇ -aminocarboxylic acids and their homo- and heterooligomers, ⁇ , ⁇ -aminocarboxylic acids and their branched homo- or heterooligomers, other amino acids and the linear and branched homo- or heterooligomers (peptides); Amino-oligoalkoxy-alkylamines; Maleimidocarboxylic acid derivatives; Oligomers of alkyl amines; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkyl mercaptophenyl or 4-oligoalkyl mercaptophenoxy derivatives; 4-oligoalkylaminphenyl or 4-oligoalkylaminyphenoxy derivatives; (Oligoalkylbenzyl) phenyl or 4-oligoalkylbenzyl) phenoxy derivatives;
  • Synthetic peptides which correspond to the N-terminal and / or C-terminal sequences or are fragments thereof can preferably be multimerized by chemical crosslinkers or coupled to a carrier molecule such as BSA, dextran, KLH or others.
  • a carrier molecule such as BSA, dextran, KLH or others.
  • Preferred crosslinkers are homobifunctional crosslinkers, preferably: NHS esters, such as DSP, DTSSP, DSS, BS, DST, sulfo-DST, BSOCOES, sulfo-BSOCOES, EGS, sulfo-EGS, DSG or DSC, homobifunctional imidoesters, such as DMA, DMP , DMS or DTBP, homobifunctional sulfhydryl-reactive crosslinkers, such as DPDPB, BMH or BMOE, difuorobenzene derivatives, such as DFDNB or DFDNPS, homobifunctional photoreactive crosslinkers, such as BASED, homobifunctional aldehydes, such as formaldehyde or glutaraldehyde, 1-epoxydyl ether, such as 1-epoxides, such as 1-epoxides , homobifunctional hydrazides, such as adipic dihydrazi
  • Heterobifunctional crosslinkers in particular amine-reactive and sulfhydryl-reactive crosslinkers, such as SPDP, LC-SPDP, sulfo-LC-SPDP, SMPT, sulfo-LC-SMPT, SMCC, sulfo-SMCC, MBS, sulfo-MBS, SIAB, sulfo-SIAB, are also preferred , SMPB, sulfo-SMBP, GMBS, sulfo-GMBS, SIAX, SIAXX, SIAC, SIACX or NPIA, carbonyl-reactive and sulfhydryl-reactive crosslinkers such as MPBH, M 2 C 2 H or PDPH, amine-reactive and photoreactive crosslinkers such as NHS-ASA, sulfo -NHS-ASA, sulfo-NHS-LC-ASA, SASD, HSAB, sulfo-HSAB, SANPAH,
  • the peptides according to the invention and recombinantly produced structures are linked by peptide bridges with a length of 0 to 50 amino acids.
  • This also includes recombinant proteins consisting of two N-terminal and one C-terminal sequence or hexamers consisting of three N-terminal sequences and three C-terminal sequences, or multimers of the recombinant structures listed above, with the N- and the C-terminal sequences one each Peptide bridge from 0 to 50 amino acids can be present.
  • the peptides can be provided with specific fusion fractions either at the N- or at the C-terminus, such as, for example, CBP (calmodulin binding protein), His-Tag and / or others. Similar constructs can also be encoded by DNA used for immunization.
  • the peptide mixture and protein mixture is selected from the group comprising: a) at least two peptides or recombinant proteins comprising an amino acid sequence according to SEQ ID No 1 to 104, b) peptides or recombinant proteins comprising an amino acid sequence which a has sufficient homology to be functionally analogous to an amino acid sequence according to a), c) peptides or recombinant proteins according to an amino acid sequence a) or b) which are modified by deletions, additions, substitutions, translocations, inversions and / or insertions and functionally analogous to one Amino acid sequence according to a) or b).
  • amino acid sequence e.g. the peptide or recombinant protein, which has sufficient homology to be functionally analogous to one of the amino acid sequences according to SEQ ID No 1 to 104, at least 40% homologous to these.
  • these amino acid sequences are at least 60%, preferably 70%, preferably 80%, very particularly preferably 90%, homologous to one of the amino acid sequences according to SEQ ID No 1 to 104.
  • the peptide or recombinant protein essentially consists of the amino acid sequence
  • the peptide consists of modifications of these sequences which were obtained according to WO 99/62933 and WO 02/38592, these peptides naturally generating and binding antibodies in the sense of the invention.
  • the peptide sections of the immunogenic construct are associated with one another at other peptides or proteins or with a carrier.
  • the at least two peptides according to the invention of the immunogenic construct are linked to one another via peptide or non-peptide bonds.
  • the non-peptide bonds are known to the person skilled in the art and include, for example, imino, ester, azo, hydrazite, semicarbacite and other bonds.
  • the carriers in the sense of the invention can be proteins which stimulate the antibody response due to their immunogenic behavior, but also pharmaceutical auxiliaries known to the person skilled in the art, such as QS21, GPI-0100 or other saponins, water-oil emulsions such as, for example Montanide, polylysine, polyagenin compounds or others such as phosphate buffered saline, water, various types of detergents, sterile solutions and the like.
  • pharmaceutical auxiliaries known to the person skilled in the art, such as QS21, GPI-0100 or other saponins, water-oil emulsions such as, for example Montanide, polylysine, polyagenin compounds or others such as phosphate buffered saline, water, various types of detergents, sterile solutions and the like.
  • the invention also includes antibodies which are produced or induced by the peptides according to the invention or by the immunogenic construct according to the invention.
  • the peptides or proteins can be produced chemically or by recombinant DNA technology or otherwise.
  • at least one immunogenic construct according to the invention is brought into contact with an organism in such a way that it is able to produce antibodies against this construct which can be obtained and isolated by the person skilled in the art using the known methods.
  • the invention also relates to anti-idiotype antibodies.
  • Antibodies carry idiotypes, areas near their antigen recognition sites that are themselves antigenic and able to stimulate antibody production. Antibodies that are specific for the antigen binding sites are called paratope-specific anti-idiotype antibodies. These antibodies carry the same recognition site as the antigen that initially stimulated antibody production; Marx, "Making Antibodies without Antigens", 1986.
  • paratope-specific anti-idiotypic antibodies with partially the same structure as HIV, SARS or other viruses can be obtained by immunizing an animal with a monoclonal HIV-1, SARS- or FeLV antibodies to the viruses mentioned.
  • These paratope-specific anti-idiotypic antibodies which have a certain structure identical to that of the immunogenic parts of the viruses mentioned, are suitable for triggering an immune response and can accordingly also be used as a vaccine.
  • the invention also includes pharmaceutical compositions which comprise at least one of the immunogenic constructs according to the invention, optionally together with a pharmaceutically acceptable carrier.
  • a pharmaceutical agent in the sense of the invention is any agent in the field of medicine which can be used in the prophylaxis, diagnosis, therapy, follow-up or follow-up treatment of patients who have come into contact with enveloped viruses, including retroviruses, in such a way that at least temporarily could establish pathogenic modification of the overall condition or the condition of individual parts of the organism.
  • the pharmaceutical agent it is possible for the pharmaceutical agent to be a vaccine or an immunotherapeutic in the sense of the invention.
  • the pharmaceutical agent in the sense of the invention can comprise, for example, an acceptable salt or components thereof. These can be, for example, salts of inorganic acids, such as, for example, phosphoric acid or salts of organic acids.
  • the salts are free of carboxyl groups and have been derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides or from organic bases such as isopropylamine, trimethylamine, 2-ethyl aminoethanol, histidine and others.
  • inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides
  • organic bases such as isopropylamine, trimethylamine, 2-ethyl aminoethanol, histidine and others.
  • liquid carriers are sterile aqueous solutions which do not comprise any further materials or active ingredients and for example water or those which comprise a buffer such as sodium phosphate with a physiological pH or a physiological saline solution or both, such as phosphate-buffered sodium chloride solution.
  • Other liquid carriers may include more than just a buffer salt, such as sodium and potassium chloride, dextrose, propylene glycol, polyethylene glycol, or others.
  • Liquid compositions of the pharmaceutical compositions can additionally comprise a liquid phase, but with the exclusion of water.
  • additional liquid phases are glycerol, vegetable oils, organic esters or water-oil emulsions.
  • the pharmaceutical composition or the pharmaceutical composition typically contains at least 0.1% by weight of the peptides according to the invention, based on the total pharmaceutical composition.
  • the respective dose or the dose range for the administration of the pharmaceutical agent according to the invention is large enough to achieve the desired prophylactic or therapeutic effect of the formation of neutralizing antibodies.
  • the dose should not be chosen so that undesirable side effects dominate. In general, the dose will vary with the age, constitution, gender of the patient and, of course, the severity of the disease.
  • the individual dose can be set in relation to the primary illness as well as in relation to the occurrence of additional complications.
  • the exact dose can be determined by a person skilled in the art using known means and methods, for example by determining the antibody titer depending on the dose or depending on the vaccination schedule or the pharmaceutical carrier and the like.
  • the dose can be selected individually depending on the patient.
  • a dose of the pharmaceutical agent that is still tolerated by the patient can be one whose range in the plasma or in individual organs is locally in the range from 0.1 to 10000 ⁇ M, preferably between 1 and 100 ⁇ M.
  • the dose can also be calculated in relation to the patient's body weight.
  • a typical dose of the pharmaceutical agent would have to be set in a range between 0.1 ⁇ g to 100 ⁇ g per kg body weight, preferably between 1 and 50 ⁇ g / kg.
  • Organs to determine This would be the case, for example, if the pharmaceutical agent according to the invention is placed, for example in a biopolymer, introduced into the respective patient, near certain organs by means of an operation.
  • a biopolymer Several biopolymers are known to the person skilled in the art which can release peptides or recombinant proteins in a desired manner.
  • Such a gel can, for example, 1 to 1000 ⁇ g the amino acid sequences according to the invention, for example peptides or recombinant proteins or the pharmaceutical composition per ml of gel composition, preferably between 5 to 500 ⁇ g / ml and particularly preferably between 10 and 100 mg / ml.
  • the therapeutic agent is administered as a solid, gel-like or liquid composition.
  • the pharmaceutical agent is preferably used as a vaccine after infection or as a preventive vaccination.
  • the vaccination is advantageously carried out in such a way that active vaccination protection develops after application in the organism.
  • the vaccination it is also possible for the vaccination to be carried out immediately before or shortly after the infection or to be administered several times as therapy. It is known to the person skilled in the art that induction of a response of neutralizing antibodies can be advantageous at almost any time, even after infection, so that vaccination in the sense of the invention also results in the application of the pharmaceutical agent according to the invention weeks, months, years or decades later infection with the respective virus.
  • the pharmaceutical agent may be preferred
  • embodiment of the invention comprise further immunogenic components, in particular selected from the group consisting of Bordetella, Haemophilus, Borrelia, Pseudomonas, Corynebacteria, Mycobacteria, Streptococci, Salmonella, Pneumococci, Staphylococci and / or Clostridia. It is known that the sole administration of antigens often does not lead to an adequate formation of antibodies. Because epitopes under certain conditions
  • the immunogenic component can also be derived from another virus, preferably selected from the family of Hepadnaviridae, Herpesviridae, Poxviridae, Adenoviridae, Papovaviridae, Parvoviridae, Retroviridae, Togaviridae or Flaviviridae; the virus can be, for example, an HIV, herpes simplex virus, influenza virus, hepatitis virus.
  • the modifications already shown for the peptides can also be applied to the pharmaceutical agent.
  • the immunogenic component can be a protein of these viruses or a fragment - ie a peptide - of these.
  • the immunogenic component in the pharmaceutical agent with both peptides according to the invention or recombinant proteins, that is to say the construct according to the invention.
  • Such a combination in the sense of the invention is preferably carried out by a covalent bond.
  • the immunogenic component and the peptides according to the invention to be adsorbed together on a carrier, this carrier being for example an albumin, a KLH or another pharmaceutically acceptable carrier.
  • the invention accordingly also comprises a method for producing an immunogenic construct which consists of the (a) peptides according to the invention or recombinant proteins or (b) the pharmaceutical composition according to the invention and microorganisms used as (c) additional immunogens, such as bacteria or viruses ,
  • a preferred method comprises the steps of treating the peptides according to the invention and / or the immunogenic microbial structures with an activator suitable for the covalent binding, optionally removing excess activator, incubating the solution thus obtained and purifying it.
  • Homo-, hetero-, bifunctional cross-linkers such as, for example, N-hydroxysuccinimide esters, imido esters, maleimido derivatives, N-hydroxysuccinimides, pyridyl disulfides or compounds containing keto groups can preferably be used as activators for the covalent bond.
  • The. Purification can be done by centrifugation, filtration, precipitation, dialysis or a chromatographic method. Gel filtration, affinity-chromatographic purification or ion-exchange chromatography are preferred as the chromatographic method. If the peptides according to the invention in the pharmaceutical composition are applied together with an immunogenic microorganism component on an absorbable carrier material, these carriers can be
  • Gold or platinum or metals such as aluminum or iron are preferred for the metals; among the insoluble or colloidal metal compounds, adjuvants such as aluminum, zinc and / or iron hydroxide are preferred.
  • Both All resorbable or biodegradable materials can be used in polymer compounds. Of course, it is also possible to use non-degradable polymer compounds if they are physiologically accepted, such as latex.
  • nucleic acids can of course also be used to enhance an immune response to the peptides according to the invention.
  • the pharmaceutical compositions also comprise cytokines, in particular interleukin-2 and / or CSF.
  • cytokines in particular interleukin-2 and / or CSF.
  • Such substances can be used to increase the activity, preferably for immune stimulation.
  • the invention also relates to the amino acid sequences according to SEQ ID No. 1-104, in particular for use in medicine. These sequences represent a selection from known larger amino acid sequence regions, the selection leading to the surprising result of the induction of neutralizing antibodies, especially if at least two of the amino acid sequences are used together.
  • the amino acid sequences are particularly preferred in the context of a medical indication, ie. H. as therapeutically assigned amino acid sequences.
  • a medical indication ie. H. as therapeutically assigned amino acid sequences.
  • ELDKWA and other sequences disclosed in the references mentioned are not claimed.
  • the invention also relates to the neutralizing antibodies which are produced with the immunogenic construct according to the invention.
  • the invention also relates to a diagnostic kit which comprises a pharmaceutical agent according to the invention.
  • This kit can be used, for example, to diagnose and monitor the course and / or therapy of viral diseases, the following viruses being preferred:
  • BIV Bovine Immunodeficiency Virus
  • CAEV Caprines Arthritis Encephalitis Virus
  • EIAV1 Equine Infectious Anemia Virus
  • FIV Fine Immunodeficiency Virus
  • OMVV Green Maedi-Visna Virus
  • SIVmac Seimian Immunodeficiency Virus from Macaque
  • SIVc immunocytosis (SIV) virus HIV-1 Human Immunodeficiency Virus Type 1
  • HIV-2 Human Immunodeficiency Virus Type 2
  • RSV Rat Sarcoma Virus
  • JSRV Japanese leukosis virus
  • JSRV Jaagsiekte sheep retrovirus
  • GALV Gallbon monkey leukemia virus
  • BLV Bovine Leukemia Virus
  • HTLV-1 Human T-cell leukemia virus type 1
  • HTLV-2 Human T-cell leukemia virus type 2
  • SARS virus pathogen of severe acute respiratory syndrome
  • HPV-1 Human Parainfluenza Virus
  • the diagnostic kit is used for the detection of antiviral antibodies against epitopes on the immunogenic construct, for the detection of neutralizing antibodies against the immunogenic construct and / or for the detection of the virus.
  • the kit may include instructions or information on pharmaceutical delivery or therapeutic treatment procedures.
  • the information can be, for example, an instruction leaflet or another medium which gives the user information about the therapeutic method or vaccination schedule with which the substances mentioned, ie in particular the peptides according to the invention or the pharmaceutical agents according to the invention, in particular vaccines, are to be used .
  • the package insert contains in particular detailed and / or essential information about the healing process. Of course, it is not absolutely necessary for this information to be formulated on a package insert, it is also possible for this information to be communicated, for example, via the Internet.
  • the diagnostic kit relates on the one hand to an immunoassay for the detection of antibodies which are directed against the enveloped viruses, including the retroviruses, and on the other hand to detection of the virus load (virus antigens) in the organism.
  • Appropriate samples are taken in which antibodies or virus antigens are to be detected.
  • a sample In the sense of the invention, the term for a biological or chemical good or a part or a small amount of it, which is to be tested chemically, biologically, clinically or similarly, by sampling, in particular the presence of neutralizing antibodies.
  • Sampling takes place in such a way that the partial quantity taken corresponds to an average of the total quantity. Of course, it can also be preferred that the partial quantity removed should not correspond to the average of a larger quantity.
  • the features determined by examining the sample are used to assess the amount recorded by the sample, which allows conclusions to be drawn about the total amount, for example the blood in an organism or the lymph.
  • the samples can be pretreated by mixing, adding enzymes or markers or otherwise.
  • a sample can in particular be all biological materials, such as biological tissues and fluids, for example blood, lymph, urine, brain fluid and others.
  • a sample refers to any substance that contains or is believed to contain neutralizing antibodies and includes a sample of tissue or fluid that has been isolated from an individual or individuals, including, but not limited to, for example, skin, Plasma, serum, lymph, urine, tears, smears, tissue samples, organs, tumors and also on samples of components of cell cultures.
  • Such a sample can be examined, for example, for the presence of binding antibodies as follows: a) coating a solid phase with the immunogenic construct according to the invention, b) incubating the solid phase with the biological sample, c) incubating the solid phase with an anti human antibody which can detect the classes IgA, IgM, IgG, which is labeled with a detectable label and d) detection of the label to determine the presence of the binding antibodies against the viruses mentioned in the sample.
  • the sample can have different concentrations of urea but also no urea (avidity test). Since neutralizing antibodies can only be detected with difficulty and not quantitatively in this way, they are detected in an infection assay in which the inhibition of the infection of unified cells by the respective virus from the list above is detected by the neutralizing antibodies.
  • the proportion of antibodies against the epitopes on the immunogenic construct can be determined quantitatively from the amount of neutralizing antibodies. Since a correlation was found between the amount of antibodies found binding the immunogenic construct and the amount of neutralizing antibodies, a new rapid test can be carried out in which many sera for neutralizing antibodies can be tested for the first time in a short time. This test allows statements about the success of the immunization in the case of prophylactic application as well as about the progression of the infection. Monitoring the success of therapy in the case of therapeutic application is also possible.
  • kits for the detection of neutralizing antibodies against the immunogenic construct on the basis of a neutralization assay with corresponding competition analyzes and a novel rapid method for the detection of neutralizing antibodies on the basis of an ELISA with the complete immunogenic construct can also be provided.
  • the diagnostic kit also includes the detection of viral antigen for determining the viral load in the samples listed above as follows: a) coating a solid phase with antibodies against the sought-after viral antigens, which in an animal or a human after immunization with the invention were obtained according to the immunogenic construct, b) incubating the solid phase with the biological sample, c) incubating the solid phase with a second, different from the first, antibody against the sought-after viral antigens, which is also in an animal or a human after immunization with the immunogenic construct according to the invention were obtained, d) detection of the coupled second antibody so as to determine the amount of bound antigen.
  • the invention also relates to an amino acid sequence selected from the group comprising the sequences SEQ ID No 1 to 104 for use in the field of therapeutic treatment and diagnostic methods on the human or animal body.
  • the invention accordingly relates to peptides, peptide fragments, recombinant proteins or their fragments, the membrane-associated proteins of certain viruses for certain therapeutic or diagnostic applications. Preference is given to using a mixture of at least two peptides or recombinant proteins from the sequence sequence of an o viral coat protein in accordance with the selection described according to the invention.
  • the invention also relates to a method for inducing an antibody response, the immunogenic construct according to the invention and / or the pharmaceutical agent according to the invention being brought into contact5 with an organism, preferably a human or an animal.
  • This method preferably comprises the formulation of the neutralizing antibody with a pharmaceutically acceptable carrier.
  • the disclosure of the peptides according to the invention makes it possible for the person skilled in the art to use them as an antigen for inducing neutralizing antibodies. It is known to the person skilled in the art that he can apply the peptides according to the invention, which can of course also be used as the pharmaceutical agent 0 according to the invention, in different dosages to an organism, in particular a human patient. The application should be carried out in such a way that the largest possible amount of neutralizing antibodies is generated.
  • the concentration and the type of application can be determined by a person skilled in the art through routine tests.
  • the following applications are possible: orally in the form of powder, tablets, juice, drops, capsules and the like; rectally in the form of suppositories, solutions and the like; parentral in the form of injections, infusions of solutions, inhalations of vapors, aerosols, dusts and plasters as well as locally in the form of ointments, plasters, envelopes, rinses and the like.
  • the immunogenic construct and / or the pharmaceutical agent can preferably be brought into contact prophylactically or therapeutically.
  • the infection with the viruses mentioned should at least be prevented in such a way that, after individual viruses have penetrated, for example into a wound, further multiplication thereof is greatly reduced or that viruses which have penetrated are almost completely killed.
  • therapeutic induction of an immune response the patient is already infected and the induction of the neutralizing antibodies takes place in order to kill the viruses already in the body or to inhibit their replication.
  • the invention also relates to a method for generating an antibody, preferably a neutralizing antibody against a disease with enveloped viruses including retroviruses, an organism in contact with an immunogenic construct, optionally together with an immunogenic component and / or a pharmaceutical agent according to the invention is brought and thus a humoral immune response is induced by the formation of antibodies and subsequently the antibodies are obtained from the organism.
  • the neutralizing antibodies obtained in this way can preferably be used for passive immunization.
  • Monoclonal antibodies can also be obtained, which u. a. be used after appropriate humanization.
  • Antibody-producing cells can also be obtained from vaccinated or infected individuals, who produce neutralizing antibodies, which are directed against the immunogenic construct according to the invention, and are applied in the form of monoclonal antibodies during passive immunization.
  • passive immunization In the case of passive immunization, there is no own immune reaction in the patient's body or essentially no own immune reaction to certain viruses, but the antibodies are introduced into the patient, for example in the form of healing sera. In contrast to active immunization, passive immunization essentially has the task of curing an infection that has already occurred as quickly as possible or of immediately protecting it against infection with viruses.
  • Various immunization schemes for passive immunization are known to the person skilled in the art, for example, from passive immunization against hepatitis A, hepatitis B and the TBE.
  • Such vaccination schedules can be adapted to specific viruses such as HIV, feline leukemia virus and others through routine experimentation.
  • the antibodies used for passive immunization can preferably be monoclonal antibodies.
  • the immunogenic components according to the invention or the pharmaceutical agent according to the invention or the kit according to the invention are used for diagnosis, prophylaxis, therapy, follow-up and / or aftertreatment of retroviral diseases.
  • the invention is to be explained in more detail below with the aid of examples, without being restricted to these examples.
  • Hybrid I and II consisting of a PERV-p15E ectodomain backbone5 with HIV epitopes inserted, were generated using a two-stage mutagenesis PCR (27).
  • the coding area for HybridII is based on that of HybridI, it was only expanded on both sides by HIV sequences.
  • the coding regions for constructs which consist of the epitopes E1 and E2 of gp41 (hereinafter referred to as loop I) of HIV-1 and are connected by a short peptide bridge, were generated by hybridization of 80mer oligonucleotides (Sigma-ARK) and a filling reaction generated by PCR.
  • the PCR product was ligated into the multiple cloning site of the expression vector pCal-n (Stratagene) via a BamHI interface at the 5 ' end and an Xhol interface at the 3rd end of the coding strand.
  • E.coli BL21 DE3 (Stratagene) were transformed with these constructs.5 DNA from feline embryonic fibroblasts FEA that produce FeLV-A was isolated with a Qiagen DNA Isolation Kit. A sequence which corresponds to the ectodomain of p15E was amplified by means of PCR with the specific primers, then cloned in the pCal-n vector and expressed in E. coli BL21 DE3.
  • CBP calmodulin binding protein
  • rgp41 Xhol rev cac ccg ata ctc gag ata cca cag cca att tgt tat g
  • Hybrid I BamHI fw cgc cca tcc cta atc aca caa gcg aga cag ctg
  • Hybrid I Xhol rev cac ccg ata ctc gag tca gtt gaa cca gtt cca aag
  • Hybrid I E1 mut fw caa gcg aga cag ctg agt gat att gtt cag caa caa cga att gta acg gaa gat ctc caa gcc c
  • Hybrid I E1 mut rev ttg ttg ctg aac aat atc act cag cag ctg tct cgc ttg tgt gat tag gga tcc acg egg
  • Hybrid I E2mut fw gaa ctg gat aag tgg gcg tcg ctt tgg aac tgg ttc aac tga gaa ttc aga ctc cag ggg tcg act cga gc
  • Hybrid I E2mut rev gtt cca aag cca cgc cca ctt atc cag ttc ttc cet tcg acg cet ctc taa cet ttc tc
  • Hybrid II BamHI fw cg gga tcc gga gca tca gta acg ctg acgta cag egg aga caa ta ttg tgt gat ata 9
  • Hybrid II Xhol rev cg ctc gag cta ata cca cag cca att tct tat gtt aaa cca att cca caa act tgc cca tt 5 Loop I BamHI fw: ggg gat cec agc ca ttg gag atg tcg aac cagttc cac aa gaa gec cat ttg tcc agt tcc agc agt tcc tgt tcg tta gaa gac gga gaa gaa gaa gac a
  • Loop I Xhol rev ccg gat cec tgg gtg ctg ctg gtt cta cca tgg gtg ctg ctt ctg tta cec tga ccg ttc agg ctc l o gtc tgc tgc tgt ctt ctt ctc cgt cta acg a
  • the purification was carried out according to the Stratagene protocol for the CBP-fused proteins.
  • the purity of the recombinant proteins was checked by SDS-PAGE and Western blot analysis using the monoclonal gp41 -specific antibody 2F5 or a serum against p15E from FeLV.
  • E1 and E2 Synthetic peptides corresponding to the sequences of the epitopes E1 and E2 of HIV (E1 (LGAAGSTMGAASVTLTVQARLLLS) and E2 (NEQELLELDKWASLWNWFDIT NWL) were produced by the Jerini company and purified by HPLC. E1 and E2 were free antigen
  • dextran-peptide conjugates were carried out either via direct binding, the carboxyl groups contained in the activated dextran, to primary amino groups of the peptides (28) or via a heterobifunctional crosslinker (3- (2-pyridyldithio) propionyl hydrazide, PDPH, Pierce) , which enables targeted coupling via the cysteines attached to the C- or N-terminal end of the peptides (29), overlapping synthetic peptides that correspond to the entire Env (gp120 and gp41) of HIV have been developed by NIH, USA (HIV- Env peptide set derived from HIV-1 isolate MN, Cat # 6451) was obtained. Overlapping and immobilized peptides that correspond to FeLV's gp41 and p15E were produced by Jerine.
  • Wistar rats (BfR, female 54-58 days old), goats, Balb / c mice (female, 10-20 g) were treated with 500 ⁇ g-1mg protein, peptide or peptide conjugate in 800 ⁇ l PBS / Freund 's incomplete adjuvant mixture ( 1: 1) immunized, 200 ⁇ l of which were applied in each hind leg and 400 ⁇ l sc in the neck.
  • Three animals were immunized with the same antigen. The animals were boosted after 4 weeks with the same injection schedule and the same antigens.
  • the blood was drawn from the rats and mice by means of retrobulbar puncture, that of the goats from the jugular vein.
  • the blood was separated after 20 h storage at 4 ° C. by centrifugation into serum and cellular components.
  • the complement factors contained in the serum were inactivated by incubation at 56 ° C. for 30 min.
  • the serum was stored at -20 ° C until used.
  • the epitope mapping was carried out on a Pepspot membrane (Jerini, Berlin), on which peptides overlapping by 11 amino acids, which correspond to the sequence of gp41 and p15E from FeLV, were applied point by point, according to the manufacturer. 1.8 ELISA
  • mAb 2F5 50-100 ng peptides of the N-terminal region of the ectodomain of gp41 from an overlapping HIV-Env peptide set were applied in each case in combination with Dp 178 or peptide P6373 to Probind ELISA plates (NUNC ).
  • the mAb 2F5 was incubated in a dilution of 1: 25000 in the presence of 0-8M urea for 2 h at 37 ° C.
  • the detection was carried out using a polyclonal goat anti-human serum (1: 2000) conjugated to horseradish peroxidase (Sigma). It was measured with an OD 92 / 56o. All values were measured as triplicates.
  • the medium supernatant was removed and the cells were disrupted by freezing and thawing three times.
  • Interfering proteins were removed from the genomic DNA by adding Proteinase K (Invitrogen) in 100 ⁇ l IxPCR buffer (Röche) and 3 h incubation at 56 ° C. Proteinase K was inactivated by incubation at 95 ° C. for 30 min.
  • Real-time PCR was carried out on the basis of the cell lysate using HIV-env-specific primers and an env-specific, dabcyl-labeled probe (TIB MOLBIOL, Berlin) and PCR master mix (Stratagene).
  • the linear serum-specific inhibition was determined using the formula 2 ⁇ ct , where ⁇ ct is the difference between ct (immune serum) and ct (pre-immune serum). These linearized values were then converted into a percentage inhibition. All values were measured as triplicates.
  • a virus stock of the FeLV-A (Glasgow strain) was titrated on uninfected FEA cells, the titer was 10 4 - 76 TCIDso / ml.
  • 6000 FEA cells were seeded per well of a 96-well microtiter plate. Preimmunes and Immunsera were heat inactivated at 56 ° C for 30 min. 50 ul virus were added to 1: 2 dilutions of the serum or purified immunoglobulin, incubated at 37 ° C for 30 min and added to the cells. Alternatively, the serum concentration was kept constant at 1: 5 and the virus was diluted. After 3 The cells were broken open and lysed for days (20 mg / ml Proteinase K in PCR buffer, 50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.4). The cells were incubated at 56 ° C. for 3 hours, then at 95 ° C. for another 10 minutes in order to inhibit the activity of proteinase K. Provirus integration was quantified using PCR.
  • the human, monoclonal antibody (mAb) 2F5 has a broad neutralization spectrum against laboratory strains and various primary isolates.
  • Various research groups were able to show that the antibody binds to a linear sequence (ELDKWA) within the ectodomain of the transmembrane envelope protein gp41 of HIV-1. This sequence is located a few amino acids N-terminal of the transmembrane passage. With the help of an epitope mapping method based on a Pepspot membrane, this sequence could be confirmed as a binding site for 2F5 (Fig.3).
  • peptide P6373 (NEQELLEJJDKWASLW) with modified peptides (P6342 mut 1-5) was applied to ELISA plates and at a concentration of 3M urea incubated with 2F5.
  • Fig. 6 shows the evaluation of the ELISA after deducting the absorption of 2F5 on P6373 alone. It was found that amino acid exchanges in at least 4 positions of the peptide P6342 reduce the binding of the monoclonal antibody 2F50.
  • a stoichiometric ratio of 2: 1 shows a greater o inhibition of the neutralizing activity of 2F5 than a ratio of 1: 1, similar to that in the ELISA (Fig. ⁇ B).
  • the sequence of gp41 amplified on the basis of the NIH-HIV molecular clone pNL4-3 with the specific primers 1 and 2 in the PCR was ligated via the restriction sites of BamHI and Xhol into the vector pCal-n (Stratagene) opened via the same restriction enzymes ,
  • the closed vector (pCal-n rgp41) was transformed into BL21 Codon Plus (Stratagene) and there expressed IPTG dependent.
  • the amino acid sequence of the fusion protein produced is listed in section 2.1. This sequence was successfully expressed as a recombinant fusion protein. However, the fusion protein was not soluble and was therefore used as an aggregate after 8M urea precipitation to remove the soluble bacterial proteins.
  • sequences of gp41 from HIV-1 were inserted into the PERV-p15E sequence with specific primers and thereby the corresponding domains of p15E eliminated (substitution).
  • sequences of gp41 were inserted, at the N-terminal end and at the C-terminal end.
  • the respective substitutes were used in two different lengths (Hybrid I and Hybrid II).
  • the resulting PCR products were ligated via the restriction sites of BamHI and Xhol into the vector pCal-n (Stratagene) opened via the same restriction enzymes.
  • the closed vector (pCal-n rgp41) was transformed into BL21 Codon Plus (Stratagene) and there expressed IPTG dependent.
  • the amino acid sequence of the viral portions (p15E and gp41) of the fusion proteins Hybrid I and II produced are listed in section 2.1. These fusion proteins were also insoluble and were therefore used as an aggregate after 8M urea precipitation to remove the soluble bacterial proteins.
  • the coding sequences for the peptide E1 and E2 were connected through the coding region for a five amino acid long peptide bridge.
  • the resulting PCR product was ligated via the restriction sites of BamHI and Xhol into the vector pCal-n (Stratagene) opened via the same restriction enzymes.
  • the closed vector (pCal-n rgp41) was transformed into BL21 Codon Plus (Stratagene) and there expressed IPTG dependent.
  • the amino acid sequence of the loop 1 fusion protein produced is listed in section 2.1. These fusion proteins were also insoluble and were therefore used as an aggregate after 8M urea precipitation to remove the soluble bacterial proteins.
  • the immune sera were examined in a Western blot and in an ELISA for binding antibodies against gp41.
  • the sera were also examined for neutralizing antibodies in the neutralization assay.
  • Fig. 9 shows the percentage inhibition of the virus infection converted from the ⁇ ct of the inhibitory effect of
  • the recombinant gP41 which is actually not suitable as an antigen for immunization because it is insoluble, was unable to raise neutralizing antibodies (group N).
  • Fig. 1 Spatial structure of gp41 with the gp120 above it (Zwick et al. 2001 (5)).
  • Fig. 2 A Scheme of the ectodomain of gp41: Simplified representation of monomeric gp41. The fusion peptide at the N-terminus, the N-terminal helix region (NHR), the cysteine-cysteine loop, the C-terminal helix region (CHR) and the transmembrane passage are highlighted.
  • Hexamer gp41 The NHR and CHR are shown under supervision. Three N-helices are trimers consisting of three parallel ⁇ -helices with three external C-helices arranged on the outside. The interacting amino acids between the helices are entered.
  • Fig. 3 Epitope mapping for mAb 2F5 using a Pepspot membrane: 13 mer peptides were covalently bound to a nitrocellulose membrane using an acetyl linker. From spot to spot, these peptides overlap by 11 amino acids and cover the entire sequence of the gp41.
  • the mAb 2F5 was used in a concentration of 250 ng / ml.
  • the secondary antibody conjugate (anti-human POD) was used 1: 5000.
  • the epitope in the sequence of gp41 is underlined.
  • Fig. 4 SDS-PAGE (A) and Western blot (B) of Dp178 (lane 1) and recombinantly expressed CBP-rgp41 (lane 2): Although more Dpi 78, which corresponds to the C helix of the ectodomain of gp41, was applied , it is poorly detected in the Western blot than the recombinant CBP-rgp41, which contains the entire ectodomain of the gp41. 2F5 was used in the Western blot at a concentration of 500ng / ml. Fig.
  • the NIH peptide set consists of 15 mer peptides, each overlapping by 11 amino acids and which comprise the entire protein sequence of the coat protein complex. All peptides were tested in combination with Dpi 78, only the results for 10 peptides from the N-terminal region of the ectodomain of gp41 are shown here.
  • the peptide P6342 but not all others, increases the binding of 2F5 to Dpi 78.
  • the 2F5 binding is increased synergistically, since the mAb 2F5 does not recognize the P6342 alone, and can also be seen in 3M and 5M urea ,
  • the diagram shows the mean values from triplicates.
  • Fig. 7 ELISA with varying stoichiometric combinations P6342 / P6343 at four different concentrations of urea:
  • the ELISA shows that 2F5 binds particularly well to a 2: 1 ratio of P6342 / P6373.
  • a larger excess of P6342 means no increase in binding, while a 1: 1 ratio results in a significantly weaker binding of the mAb 2F ⁇ to the two peptides.
  • the diagram shows the mean values from triplicates.
  • the graphic shows the ct values when using cell lysate after treatment with different amounts of E1 (LGAAGSTMGAASVTLTVQARLLLS) and E2 (NEQELLELDKWASLWNWFDIT. NWL) during infection with HIV.
  • E1 LGAAGSTMGAASVTLTVQARLLLS
  • E2 NEQELLELDKWASLWNWFDIT. NWL
  • 2F5 was used in a concentration of 2.5 ⁇ g / ml.
  • Fig. 9 HIV neutralization by rat sera The columns show the percentage inhibition of HIV-1 IIIB provirus integration in C8166 in the presence of various rat sera or 2F5.
  • the rat sera were prediluted 1: 4. 2F5 was used in a concentration of 2.5 ⁇ g / ml. The conversion of the ct values into percentage inhibition is described under 2.9.
  • Fig. 10 Recombinant constructs derived from gp41 A): A recombinant protein was derived from gp41 of HIV-1, which contains only parts of the exodomain. (B) A recombinant protein was derived from the transmembrane coat protein p15E of the porcine endogenous retrovirus and in this protein the E1 and E2 domains of gp41 (gray) of HIV-1 were inserted at topographically identical positions (substitution of the corresponding epitopes of p15E). Different sized E1 and E2 domains were chosen.
  • Fig. 11 Recombinant loop protein derived from gp41 and free peptides as well as conjugates to a carrier molecule
  • the gray-colored E1 and E2 domains were produced (A) as a recombinant protein with an amino acid linker or (B) as free synthetic peptides or coupled to the carrier material dextran.
  • Goat 27 was immunized with the recombinant ectodomain, the antiserum inhibited the infection of FEA cells with the feline leukemia virus FeLV.
  • the infection was measured by PCR (detection of provirus infection).
  • A titration of the virus with preimmune serum
  • B titration of the virus with immune serum.
  • Immunizations with the recombinant ectodomain of p15 from FeLV were mapped as described in Fig. 3. Overlapping peptides that correspond to the ectodomain of p15 of FeLV, were used.
  • IgG Prot G-purified antibodies, pl ⁇ E - pl ⁇ E-purified antibodies. The four main epitope domains are boxed.
  • Fig. 14 Effect of joint immunization with the recombinant protein p15E from FeLV and the commercial anti-FeLV vaccine leucogen
  • Rats were immunized with the commercial vaccine leucogen (animals 56.1, 56.2, 56.3) or with the commercial vaccine leucogen and the recombinant protein p15E from FelV. Neutralization was measured using quantitative real time PCR and presented as a percentage of provirus integration. It is clear that group 54 sera neutralize FeLV (leucogen and p15E) than group 56 sera (leucogen) alone.

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Abstract

La présente invention concerne des constructions immunogènes qui renferment principalement deux zones différentes de la protéine transmembranaire de virus enveloppés, y compris les rétrovirus et le VIH, ainsi qu'un agent pharmaceutique conçu pour l'induction de réponses immunitaires neutralisantes humorales contre des infections virales et une trousse pour identifier des anticorps et des antigènes viraux. La présente invention concerne également un procédé pour induire une réponse d'anticorps et un procédé pour immuniser un organisme de manière passive, en utilisant des anticorps neutralisants qui sont obtenus avec lesdites constructions immunogènes, ainsi qu'un essai biologique pour identifier l'infection à virus et pour identifier la formation d'anticorps neutralisants.
PCT/DE2004/001897 2003-08-26 2004-08-26 Induction d'anticorps neutralisants antiviraux chez les hommes et les animaux WO2005021574A2 (fr)

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WO2007107597A2 (fr) * 2006-03-21 2007-09-27 Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Robert-Koch-Institut Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida
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US9717789B2 (en) 2005-04-12 2017-08-01 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US10076567B2 (en) 2013-09-27 2018-09-18 Duke University MPER-liposome conjugates and uses thereof

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US9717789B2 (en) 2005-04-12 2017-08-01 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US10588960B2 (en) 2005-04-12 2020-03-17 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
EP1754715A1 (fr) * 2005-08-19 2007-02-21 Bundesrepublik Deutschland vertreten durch das Bundesminsterium für Gesundheit, dieses vertr. durch das Robert-Koch-Institut Vaccin à base des anticorps neutralisant un virus
WO2007020243A2 (fr) * 2005-08-19 2007-02-22 Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Robert-Koch-Institut Vaccin a base d'anticorps de neutralisation de virus
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WO2007107597A2 (fr) * 2006-03-21 2007-09-27 Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Robert-Koch-Institut Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida
WO2007107597A3 (fr) * 2006-03-21 2007-12-21 Bundesrep Deutschland Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida
WO2010074656A1 (fr) * 2008-12-24 2010-07-01 Temasek Life Sciences Laboratory Limited Anticorps monoclonaux spécifiques du peptide de fusion de l'hémagglutine issu des virus influenza a et leurs applications
US8540995B2 (en) 2008-12-24 2013-09-24 Temasek Life Sciences Laboratory Limited Monoclonal antibodies specific to the fusion peptide from hemagglutinin from influenza A viruses and uses thereof
US10076567B2 (en) 2013-09-27 2018-09-18 Duke University MPER-liposome conjugates and uses thereof

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