EP0728203A1 - Zu innerem bild eines virenprotein ahnliche peptide - Google Patents

Zu innerem bild eines virenprotein ahnliche peptide

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Publication number
EP0728203A1
EP0728203A1 EP93905412A EP93905412A EP0728203A1 EP 0728203 A1 EP0728203 A1 EP 0728203A1 EP 93905412 A EP93905412 A EP 93905412A EP 93905412 A EP93905412 A EP 93905412A EP 0728203 A1 EP0728203 A1 EP 0728203A1
Authority
EP
European Patent Office
Prior art keywords
peptides
antibodies
peptide
sequence
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93905412A
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English (en)
French (fr)
Inventor
Vélibor KRSMANOVIC
Irena Cosic
Jean-Michel Biquard
Milton T.W. Hearn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monash University
Centre National de la Recherche Scientifique CNRS
Original Assignee
Monash University
Centre National de la Recherche Scientifique CNRS
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Application filed by Monash University, Centre National de la Recherche Scientifique CNRS filed Critical Monash University
Publication of EP0728203A1 publication Critical patent/EP0728203A1/de
Withdrawn legal-status Critical Current

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the subject of the invention is peptides immunologically related to the proteins of a viral agent, that is to say constituting, with regard to their immunological properties, analogues of the proteins involved in a viral pathology or of the antibodies formed against these proteins. .
  • infectious agent as used in the description and the claims, is meant viral pathogens, this term covering retroviral agents, or even cells infected with these.
  • This immunological approach is based on the use of antibodies capable of specifically recognizing the infectious antigen, or on the induction of the body's immune responses, also including autoantibodies.
  • the production of antibodies is done by immunization of animals against the infectious agent previously inactivated or against proteins purified from this agent.
  • the antibodies must exert a neutralizing effect in vivo with respect to the infectious agent.
  • HIV denotes the retroviruses of the HIV-1 and HIV-2 type isolated to date in humans and the retroviruses of the SIV type isolated in the monkey, which are different from HIV-1 and HIV-2 as well than their many variants.
  • the effectiveness of such vaccines is far from established and the development of an AIDS vaccine raises many problems.
  • the major problem resides in the fact that in seropositive subjects, as well as in patients, most of the circulating antibodies are not capable of neutralizing the virus and / or of eliminating the cells carrying it in the body of patients. Although some circulating antibodies are neutralizing in vitro, their protective effect in the body has been shown to be ineffective.
  • Another immunological approach consists in generating the internal image of the antigen, namely the anti-idiotype antibody, then using this new structure as an immunizing agent, rather than the antigen itself (see (1) and (2) in the Bibliographical references given at the end of the description).
  • Immunization with the antigen must first be carried out, which allows the induction of AC1 antibodies, or idiotypic antibodies, directed against this antigen.
  • immunization with the idiotype AC1 antibody should be carried out for the purpose of induction of anti-idiotype AC2 antibodies, carrying the internal image of the initial antigen.
  • This internal image of the antigen corresponds to the region of contact between it and the antibody AC1.
  • anti-idiotype AC2 makes it possible to induce anti-anti-idiotype AC3 antibodies.
  • These AC3 antibodies are capable of reacting with the original antigen (1, 2).
  • the anti-idiotype AC2 can activate the lymphocytes which would thus become capable of recognizing the initial antigen, as in the case of direct activation of these cells by the antigen.
  • the induction of the anti-idiotype AC2 can also be obtained against the receptors of sensitized T cells, responsible for cellular immunity. It is indeed possible to generate T lymphocytes whose receptors are capable of recognizing the foreign determinants expressed on the surface of a virus or of infected cells, at the same time as cellular determinants of the MHC type (major hystocompatibility complex). . They are therefore sensitized lymphocytes.
  • the R1 receptors of these lymphocytes express the structures corresponding to the idiotype, in fact performing a function similar to that of the antibody AC1.
  • lymphocytes or their membrane-bound IR receptor
  • Immunization by lymphocytes thus sensitized then allows the induction of anti-idiotype AC2 antibodies, as well as that of lymphocytes to R2 receptors, equivalent to the antibody AC2, directed against, or recognizing, the domain of the R1 lymphocyte receptor involved in contact with the antigen.
  • the R2 receptors of lymphocytes or the anti-idiotypic antibodies AC2 are capable of inducing a new response of lymphocyte cells, whose R3 receptors recognize the initial antigen (1).
  • This processing of the internal image of the antigen is particularly interesting for influencing the lymphocyte sensitization mechanism.
  • the major advantage that emerges is that the recognition of the antigen by the lymphocytes sensitized with the internal image does not depend on the genetic nature of the immunized host, since the restrictive control of identity of MHC of infected target cells, or of the virus no longer represents obstacles to their destruction (2).
  • the antibodies directed against the anti-idiotype having the ability to cross-react with the initial antigen, are capable of inducing the immunity in which both B and T lymphocytes are involved.
  • the use of anti-idiotype lymphocyte receptors or antibodies as vectors of the internal image of the antigen in the prevention or treatment of immunodeficient diseases is not without danger.
  • AIP also known as "Resonant Recognition Model” RRM
  • FFT Fast Fourier Transform
  • This AIP method as described in particular in (3) is based on a representation of the primary structure of a protein in the form of a numerical series, in assigning to each amino acid a defined parameter describing a physicochemical property involved in the biological activity of the protein.
  • Such a frequency has been detected for example in peptide hormones and their receptors.
  • the object of the invention is therefore to provide such peptides, constituting in particular analogs of the proteins of viral agents, or analogs of the antibodies formed against these proteins, that is to say capable of mimicking these proteins and antibodies in terms immunogenicity.
  • the invention also aims to provide a process for obtaining these peptides making it possible to develop a peptide sequence according to the desired immunogenicity with respect to a target antigen and to easily obtain it by synthesis.
  • the invention also relates to the biological applications of these peptides, and more particularly their use in diagnosis, prevention and therapy.
  • the invention in particular opens up possibilities for the treatment of autoimmunity by providing peptides capable of inducing antibodies, or lymphocytes capable of recognizing autoimmune antibodies and, by destroying them, autoimmune lymphocytes.
  • peptides according to the invention which are, in particular, immunologically related to the proteins of a viral agent, are characterized in that they comprise, or that they consist of an amino acid sequence
  • the peptides as ordered by the AIP method are artificial peptides whose sequences are established on the basis of that of a target antigen and so as to present in common with this antigen, one (or more) frequency (s) characteristic (s) of a biological activity.
  • target antigen designates both the protein expressed by the viral agent, or a fragment of this protein, as well as an antibody formed against this protein or a fragment of the latter, or even the antigen to be protected in the case of an autoimmune pathology, or even a peptide whose amino acid sequence was itself ordered using the digital method AIP.
  • characteristic frequency means that it is the frequency (or frequencies) in the Fourier spectra considered, which appears (appear) related to the immunoreactivity with the target antigen or to the antigen to be protected (in opposite phase) and includes (s) information relevant to the antigenicity properties.
  • the invention relates in particular to a group of peptides whose frequency (s) in their Fourier spectrum is (are) in reverse phase relative to that (s) characteristic (s) of the target antigen.
  • the invention also relates to another group of peptides, the frequency (s) of which in their Fourier spectrum is (are) in phase with that (s) characteristic (s) of the target antigen.
  • the target antigen may consist of a viral protein or a fragment of such a protein containing at least the antigenic site.
  • a group A of peptides as ordered with respect to this viral protein is characterized in that said peptides present in their Fourier spectrum, in common with the viral protein, the frequency (s) characteristic (s) of this protein, but in reverse phase, and that they are capable of inducing antibodies to cross-reactivity with members of the same group.
  • the antibodies induced by these group A peptides are analogues of the internal image of the viral protein, more precisely of the domain of this protein involved in an antigen-antibody type reaction.
  • analogs of the internal image of a protein refers to the data obtained by the AIP method used as a modeling means according to the invention.
  • the target antigen with respect to which a peptide sequence is ordered, consists of peptides of group A.
  • a group B of peptides as ordered with respect to peptides of group A is characterized in that said peptides present in their Fourier spectrum one or more frequencies common with that (s) characteristic (s) of peptides A, in phase inverse compared to that (s) of peptides A, but in phase with that (s) of the target antigen with respect to which the peptides of group A were ordered.
  • these group B peptides are capable of inducing antibodies capable of forming an immune complex, as demonstrated by the Western Blot techniques, with the protein or proteins the same (s) frequency (s) expressed by the infectious agent.
  • These antibodies may correspond to the antibodies induced by type A peptides.
  • Group B peptides are also capable of inducing antibodies, or lymphocytes, recognizing autoantibodies, or autoimmune lymphocytes, directed against CD4.
  • the peptides B described above more specifically constitute analogs of proteins expressed by negative RNA viruses, positive RNA viruses, double-stranded RNA viruses or even DNA viruses such as herpes or cytomegalovirus, and their variants.
  • the invention relates specifically to the peptides mentioned above, analogues of retrovirus proteins or of antibodies formed against these proteins.
  • retroviruses mention will be made of lentiviruses with cytopathogenic power, and very especially HIV, in particular HIV-1 and HIV-2 (responsible for pathologies in humans), SIV (in monkeys), Visna (in sheep) CAEV ( in goats), ELAV (in horses), FLV (in cats), BLV (in cattle), and their variants.
  • retroviruses include oncornaviruses such as HTLV-1 and HTLV-2, isolated from humans, and animal retroviruses associated with leukemias and cancers.
  • the invention relates to peptides comprising or consisting of an amino acid sequence as ordered according to the AIP method with respect to the sequence of a protein chosen from the proteins expressed by HIV-1.
  • proteins encoded by the GAG, POL and ENV genes common to retroviruses and more particularly gp160 (envelope precursor, ENV); gpl10 / 120 (ENV); p66 / 68 (POL reverse transcriptase), p55 (internal protective precursor, GAG); p51 / 52 (POL-protease); gp41 (ENV); p40 (GAG, internal prot. precursor), p31 / 34 (POL endonuclease), p24 / 25 (GAG, internal prot.); pl7 / 18 (GAG, internal prot.).
  • gp glycoprotein
  • p is used for the term protein
  • the figures mentioned correspond to the molecular weight established by migration of the proteins in a gel and confirmed and specified by the analysis of the sequences of the corresponding genes.
  • the amino acid sequence of the peptides of the invention as ordered by the AIP method with respect to one of the sequences of the HIV proteins above, present in common with these proteins at minus a characteristic frequency.
  • Peptides of great interest have an amino acid sequence with a Fourier spectrum with at least a frequency equal to, or close to, this value, corresponding to that which the expressed target proteins gp160 / 120 and p55 have in common. respectively by the ENV and GAG genes of HIV-1 (as well as the POL protein, p66 / 68, such as that of the LAVbru strain), or the proteins which are derived therefrom, as well as their fragments.
  • peptides also have a frequency equal to or substantially equal to 0.2188, as expressed by the fragment of gp120 involved in the binding to the CD4 receptor, hereinafter designated by the expression fragment gp120cD4. More specifically, the invention relates to peptides comprising an amino acid sequence as ordered according to the AIP method with respect to the sequence of this fragment, as illustrated in the examples.
  • Advantageous peptides are peptides of group A above and exhibit Fourier spectra with a frequency characteristic of the gp120cD4 fragment, the if necessary two frequencies, but in reverse phase with respect to that (s) of this fragment.
  • peptides are from group B and exhibit Fourier spectra whose characteristic frequency (s) is (are) in reverse phase to that of the group A peptides.
  • Such peptides have the same phase as that of gp120cD4 or frequencies characteristic of the fragment.
  • they are capable of inducing antibodies recognizing certain parts of the region of the gp120cD4 fragment under the conditions of the Elisa and / or Western Blot test as described in the examples below.
  • This viral agent can be, for example, the feline leukemia virus (FLV for short, for Feline Leukemia virus), precursors of the envelope polyprotein (10), (14), this polyprotein (11), (12 ), (13), (15), or fragments, or else the T-cell leukemia virus, HTLV-I or HTLV-II, the envelope polyprotein of these viruses (16), (17) or their fragments, or the Visna virus, especially the envelope polyprotein, its precursor (18), or fragments thereof.
  • FLV feline leukemia virus
  • precursors of the envelope polyprotein (10), (14), this polyprotein (11), (12 ), (13), (15), or fragments or else the T-cell leukemia virus, HTLV-I or HTLV-II, the envelope polyprotein of these viruses (16), (17) or their fragments, or the Visna virus, especially the envelope polyprotein, its precursor (18), or fragments thereof.
  • the invention therefore makes it possible to have group A artificial peptides, ordered according to the AIP method, with respect to a given sequence of proteins or fragments of one of the viral agents above, and group B artificial peptides, ordered compared to peptides A, as well as antibodies directed against these peptides.
  • the invention provides peptides capable of inducing antibodies against autoimmune antibodies and autoimmune lymphocytes, or also provides peptide sequences analogous to these antibodies.
  • Peptides of this type are characterized in that they comprise or are constituted by an amino acid sequence as ordered according to the AIP method with respect to the viral sequences analogous to those of HLA-DR or even to those of interleukin 2, with the phase reversed with respect to those of these sequences and that they are capable of inducing antibodies (or lymphocytes) recognizing the antibodies (or the lymphocytes) directed respectively against these HLA-DR sequences and interleukin 2.
  • the peptides of the invention may exhibit immunogenicity oriented towards the proteins of an infectious agent capable of developing pathogenicity in the context of an infection caused by a first agent.
  • HHV6 viruses which belong to the Herpes family, or mycoplasmas (bacteria without walls) have been detected in subjects infected with HIV.
  • the invention therefore especially relates to peptides, as defined above, capable of inducing antibodies capable of forming an immunological complex with the proteins of such infectious agents, or capable of reacting themselves with these proteins. It also targets the above artificial peptides capable of inducing lymphocytes capable of reacting with the proteins of infectious agents.
  • peptides of the invention are as expressed by the nucleotide sequences deduced from specific amino acid sequences, as ordered by the AIP method.
  • the invention therefore relates to polynucleotide fragments characterized in that they comprise or are constituted by a sequence coding for a peptide as defined above. It should be noted that these sequences may differ due in particular to the degeneracy of the genetic code and the coding of the same amino acid by different codons.
  • polynucleotide fragments of the invention correspond to or consist of sequences complementary to the above sequences, or nucleotide sequences capable of hybridizing with one of the nucleotide sequences complementary to those of the sequences deduced from the peptide sequences.
  • Nucleotide sequences particularly targeted by the invention correspond to those coding for sequences of group A peptides.
  • sequences specially targeted by the invention correspond to those coding for sequences of group B. Mention will in particular be made of those coding for sequences having an immunological relationship with HIV proteins.
  • nucleotide sequences complementary to the sequences capable of coding for the peptides of group A or of group B.
  • the peptides coded by these complementary sequences are new products and are also targeted by the invention.
  • peptides are designated in the description and the claims by the letter c, cA and cB thus respectively denoting peptides whose sequences are deduced from the RNA sequence complementary to that of the RNA sequence corresponding to the group peptides A or B respectively and ordered using the genetic code.
  • peptides cA constitute a subgroup of the peptides B and the peptides cB a subgroup of the peptides A.
  • peptides of type A and B coded by the sequences complementary to those which can code respectively for the peptides cA and cB are also targeted by the invention. Peptides of this type are given in the examples and designated as peptides A11 and B11.
  • nucleotide fragments further comprise a or several sequences capable of coding for one or more proteins having a biological activity of particular interest for a given application.
  • expression vectors in particular plasmid or phage, and their transformed or transfected cell hosts, commonly used in genetic engineering techniques, and which comprise the coding nucleotide sequence as defined above.
  • the peptides of the invention are capable of inducing cross-reactive antibodies between members of the same group.
  • the invention also relates to a process for obtaining the peptides defined above.
  • This process is characterized in that a synthesis of amino acids is carried out synthetically as ordered according to the AIP method, as a function of the amino acid sequence of a target antigen and of a parameter, more particularly one or the periodic element (s) characteristic (s) of the target antigen.
  • the frequency (s) common (s) to several proteins in the Fourier spectrum As a characteristic parameter, it is advantageous to determine the frequency (s) common (s) to several proteins in the Fourier spectrum.
  • a numerical analysis of the amino acid sequences of the target antigen is carried out, replacing each amino acid in the sequence with an appropriate numerical value, the FFT (to be removed from Fourier) transformation of this series of values, and multiply between them the Fourier spectra, obtained for the target antigen and proteins of the same group or family or of the same biological activity as that of the antigen, which makes it possible to identify one or more common frequencies , characteristics of a group of proteins or of a given biological activity.
  • a value representing the assortment of the energy of the delocalized electrons from each amino acid residue more specifically a value corresponding to the potential for interactions between electrons and ions.
  • the sequence of the peptide sought is ordered on the basis of the sequence of a protein or of a protein fragment of a viral agent and taking into account the characteristic frequency (s) of this protein or its fragment, which determines (s) immunoreactive recognition.
  • sequence of an ordered peptide as indicated above is used as a reference.
  • group A peptides that is to say analogs of antibodies directed against the target antigen
  • the phase of the characteristic frequency or frequencies retained is reversed compared to that of the frequency or frequencies of the target antigen.
  • a new phase inversion is carried out to prepare the group B peptides analogous to the image of the target antigen, whose sequences are ordered relative to those of the group A peptides.
  • the invention it is possible to have peptides analogous to idiotypic antibodies directed against the target antigen, or alternatively, peptides analogous to anti-anti-idiotypic antibodies, vis-à-vis the target antigen .
  • the invention provides peptides analogous to the images of the target antigens, that is to say analogues of anti-idiotypic antibodies.
  • peptide sequences are advantageously carried out according to conventional techniques. As mentioned above, these peptides are capable of inducing cross-reactive antibodies against members of the same group.
  • nucleic acid fragments of the invention are advantageously obtained in a conventional manner by synthesis, for example using commercial synthesizers involving the phosphoramide method. They can also be obtained by the PCR amplification method using primers of appropriate sequences and lengths.
  • the primers prepared from the nucleotide fragments of the invention can be used for the synthesis of the artificial peptides defined in the above.
  • such a method for synthesizing the peptides of the invention comprises the chemical synthesis of nucleotide sequences, or the amplification of nucleotide sequences capable of coding for a given peptide by bringing these sequences into contact with at least one pair appropriate primers, followed by the translation of the sequences thus amplified by operating in the usual manner.
  • This last step is advantageously carried out by transformation of the host cells used with the aid of vectors containing the amplified sequences, and recovery of the peptides produced in the host cells.
  • the invention thus makes it possible, on the one hand, to have families of peptides and antibodies capable of specifically recognizing the antibodies formed against the proteins of viral agents and, on the other hand, families of peptides and antibodies capable of specifically recognizing proteins of viral agents.
  • the first family includes antibodies induced against group A peptides, and group peptides
  • This first family also includes the peptides cA which constitute a subgroup of the peptides
  • the group A peptides against which the antibodies are formed are as ordered according to the method
  • AIP as defined above, with respect to the sequence of a protein of the viral agent involved in the pathology to be studied.
  • Group B peptides have amino acid sequences as ordered with respect to those of group A peptides above. Their common frequencies are in phase with that of the viral agent protein.
  • This first family can advantageously be used as a reagent for the diagnosis of a pathology by allowing the detection of the presence of antibodies, or of lymphocytes, capable of recognizing infectious proteins.
  • a biological sample such as a biological fluid such as serum or lymphocytes from the circulating blood into contact, or a biological tissue extract taken from the patient under study, or the animal, with antibodies induced against group A peptides, or with group B peptides as indicated above, including the peptide subgroup cA, and
  • lymphocyte reaction on contact with the antigen it is demonstrated either a reactivity of the lymphocytes of the sample with the peptides or the antibodies used as reagents, or a reactivity of the peptides or antibodies with the lymphocytes of a subject previously immunized with the peptides of the invention.
  • the lymphocytes thus sensitized constitute reagents.
  • Such immunization is carried out, for example, with the peptides cA.
  • the artificial antibodies or peptides are free or fixed on a non-immunogenic support during the contacting step.
  • lymphocytes use is made of a method conventionally used in immunology such as immunofluorescence, Elisa, Western Blot, or even lymphocyte stimulation, and possibly the test of the presence CTL ("cytotoxic T lymphocytes").
  • a method conventionally used in immunology such as immunofluorescence, Elisa, Western Blot, or even lymphocyte stimulation, and possibly the test of the presence CTL ("cytotoxic T lymphocytes").
  • the detection of a selective interaction between the antibodies in the sample and the artificial peptides or induced antibodies used, or even the reactivity of the lymphocytes, is significant for the existence of the pathology.
  • This detection method makes it possible to reveal with great sensitivity, and quickly, the presence, in a biological sample, of antibodies formed against the proteins expressed by a viral agent.
  • the second family mentioned above comprises group A peptides including the cB peptides, and antibodies or lymphocytes induced against group B peptides, the sequences of these peptides and their frequencies being as defined above.
  • This family can advantageously be used to detect the presence of proteins of a viral agent.
  • a biological sample such as a biological fluid such as serum or lymphocytes from the circulating blood, or a biological tissue extract taken from the patient under study, or the animal, into contact with antibodies or lymphocytes induced against group B peptides, or with group A peptides, as defined above, including the cB peptide subgroup and
  • said antibodies or said artificial peptides are brought into contact with the antigens of the infectious agent attached to a support and the reaction is inhibited by competition by adding the biological sample to be tested, when it contains the proteins of the viral agent.
  • lymphocytes To reveal the complex possibly formed or the reactivity of the lymphocytes, one uses one of the current methods for the detection of antibodies, or the revelation of the presence of antigen (s) stimulating the lymphocytes of the animal immunized against group B peptides.
  • the detection of a selective interaction between the proteins in the sample and the induced antibodies (or test lymphocytes) or the artificial peptides used is indicative of the existence of the pathology.
  • the proteins expressed by a viral agent can thus be easily detected and in particular localized and quantified, for example at the level of biopsies of pathological tissue, or of peripheral blood cells.
  • the invention also relates to kits for the in vitro diagnosis of viral pathologies in humans or animals.
  • the kit of the invention will advantageously include lymphocytes, for example in frozen form, from the animal immunized against given peptides.
  • the antibodies and peptides used are immobilized in a conventional manner on beads, for example latex beads, microtitration plates or even strips.
  • the invention further relates to the application of the peptides defined above for the preparation of immunogenic compositions.
  • Preferred immunogenic compositions comprise at least one group A peptide whose characteristic frequency (s) in the Fourier spectrum are in opposite phase with that (s) of the responsible antigenic sequence autoimmune pathology.
  • the amino acid sequence of these group A peptides is as ordered according to the AIP method by reference to that of the antigen to be treated.
  • peptides capable of inducing antibodies capable of neutralizing autoimmune antibodies and destroying autoimmune lymphocytes are peptides capable of inducing antibodies capable of neutralizing autoimmune antibodies and destroying autoimmune lymphocytes.
  • compositions are characterized in that they comprise at least one group B peptide, in association with a pharmaceutically acceptable vehicle for the constitution of vaccines.
  • the immunogenic peptide can also be expressed by corresponding nucleotide sequences incorporated into an infectious agent whose safety has been previously established, such as a recombinant virus such as those produced from poxvirus family.
  • a virus of this type which is widely used is constituted by vaccinia, used in living form, attenuated or killed.
  • the immunogenic composition is administered in an amount and according to a protocol allowing the host to be immunized with respect to the antigens of the infectious agent.
  • Such a composition can be used for the development of vaccines capable of inducing sensitized lymphocytes and antibodies directed against the infectious agent.
  • compositions include a group B peptide as ordered according to the AIP method with respect to a group A peptide itself, ordered with respect to the proteins of a virus, more particularly with respect to those of a retrovirus , including HIV.
  • this group includes the peptides cA.
  • a single peptide proves capable of inducing antibodies which recognize all the proteins of the viral agent which express a significant periodic element in common.
  • Such an immunogenic composition vis-à-vis HIV is dosed in antigenic peptide so as to allow the administration of a dose of 10 to 100 ⁇ g / kg.
  • the lymphocytes sensitized by artificial group B peptides, analogues of the image of the target protein correspond to the category of immune lymphocytes carrying receptors involved in the destruction of the infectious agent or the cell infected by it, thanks to the ability of its receptors to recognize the antigen.
  • the activity of CTL (“Cytotoxic T Lymphocytes”) or other types of lymphocytes not requiring the mediation of antibodies, sensitized with the antiidiotype is not subject to restrictive control of genetic identity of MHC, and therefore should not represent obstacles to the destruction of infected target cells, or of the infectious agent (2).
  • the antibodies induced by the peptide analog of the internal image can intervene as well in the function of the complement as in the activity called ADCC ("Antibody-Dependent Cell-mediated Cytotoxicity") of the cytotoxic lymphocytes (K lymphocytes) destroying the cells carrying on their surfaces the antibodies captured by the antigens.
  • ADCC Antibody-Dependent Cell-mediated Cytotoxicity
  • K lymphocytes cytotoxic lymphocytes
  • the peptides of the invention and the antibodies against these peptides can also be used in therapy, in particular as vectors for drugs or antigens.
  • antigens coupled to the peptides of the invention any bacterial or viral antigen can be used, provided that the organism is immunized (or vaccinated) beforehand against it (e.g. diphtheria toxoid, polio etc.).
  • Vectors must be designed with respect to the molecular domains of target proteins (viral antigens) that do not interact with the elements cells in the process of virus / cell recognition.
  • This provision is essential to avoid the induction by the vector of antibodies which could recognize the cellular structures with which the target protein interacts.
  • the invention relates to the use of type A peptides as drug vectors and type B peptides as inducers of antibodies or cytotoxic lymphocytes capable of intercepting antibodies / lymphocytes directed against CD4.
  • the apparent potential, or pseudopotential potential, of each of the amino acids of a given protein sequence is calculated according to the following general formula (4):
  • W 0.25Z * sin (1.04 ⁇ Z *) / 2 ⁇ , (1)
  • Z * represents the average of the number of quasivalences determined by where Zi represents the number of valence electrons of the ith atom of the molecule and N the total number of atoms in the molecule.
  • the digital series used for the Fourier transformation therefore represents discrete "determining" signals of finite length.
  • the absolute values of the Fourier transform coefficients define the amplitude of the spectrum, while their phases define the phase spectrum as follows:
  • the information spectrum which is defined as follows:
  • the minimum number of points required for an analysis is determined according to the desired resolution of the spectrum (that is to say according to the number of peaks to be highlighted).
  • the algorithm procedures are applied to the sequences of HIV gp160 (ENV), of HIV p55 (GAG), of a 44 amino acid fragment of gp120 involved in the recognition of CD4 (7) and to the murine and human CD4 protein.
  • the analysis procedure comprises the following steps:
  • each amino acid sequence is transformed into a corresponding numerical series by representing each amino acid with the corresponding value of the ion-electron interaction potential
  • the peptides obtained in this way have the same spectral characteristics as those associated with the bio-recognition of gp160 / 120 and of CD4 and can be proposed to present a similar set of properties-functional motifs.
  • the polyclonal (or monoclonal) antibodies generated against these analogs of synthetic peptides should be capable of cross-reactions with the surface region of the gp160 / 120 molecule responsible for binding to CD4.
  • This frequency f1 is also distinguished as an average signal in the spectrum of the proteins gp160 and p55 from isolates such as LAVbru as shown in Figures le and
  • This characteristic frequency f1 also appears in the fragment of gp 120 corresponding to amino acids 418 to 461 (numbering according to Neosystem Laboratory-1990, HIV-peptides, SlV-peptides) responsible for the binding to CD4 (7) of various HIV isolates and the LAVbru isolate.
  • a frequency f2 0.2188 as it appears respectively in the figures le (8 HIV isolates) and lf (LAVbru isolate).
  • sequence of the peptide A1 was ordered by following the AIP method of example 1.
  • the sequence developed responds to the following sequence:
  • Sequence A1 KQQYYWYAWCQPPQDQLIMD
  • sequence of the peptide A2 was ordered by reversing the phase of the frequency f1, but without taking account in this case of the frequency f2, this peptide being established, as regards the frequencies, only by compared to f1.
  • Sequence A2 LKRDQEPMDFHIWDDYLKRD
  • A1 and A2 have no homology at the level of their sequence, although they have the frequency f1 in common.
  • This peptide was developed so as to present the same frequencies f1 and f2 as the peptide A1 but with phases opposite to these frequencies, and therefore of the same phases as the frequencies f1 and f2 of the fragment of the sequence S.
  • Sequence B1 DDALYDDKNWDRAPQRCYYQ
  • This peptide was developed by analogy with peptides A1 and A2, and has a unique frequency f1, in the opposite phase to that of A1 and A2.
  • the AIP spectrum of peptide B2 is shown in Figure 2d. This peptide, which responds to the sequence
  • Sequence B2 DFHIWDDYLKRDQEPMDFHI
  • Example 4 Synthesis of the artificial peptides of Example 3.
  • Protected peptide resins were synthesized by the multiple peptide synthesis method developed by HOUGHTEN (8) using a methyl benzhydrylamine resin (100 to 200 mesh, 0.4 to 0.8 meq / g), and the amino acids N- ⁇ -t-butoxy carbonyl (t-boc) such as those marketed by Bachem inc (Torrence, CA).
  • the peptides were separated from the resin according to the usual method of hydrogen fluoride and anisole (9). After the synthesis, the purity of the peptides was evaluated by reverse phase HPLC on a column of PepRPC HR5 (Pharmacia).
  • the peptides represent approximately 85% of the absorbent material at DO 214 .
  • the peptide sequences were checked.
  • Example 1 Application of the method of Example 1 to the analysis of the retroviral feline leukemia virus (Feline Leukemia Virus or FLV).
  • the FLV-kn20 peptide corresponds, from the point of view of motif characteristics, to the knob-gp70 peptide. In other words, it has been found to correspond to a peptide of 20 amino acids of the knob type gp70 with the sequence:
  • the FLV-knob20 peptide corresponds to a peptide of
  • the FLV-sp73 peptide corresponds, for the characteristics of these motifs, to spike p15; it's about of a 73 amino acid peptide of the spike p15 type, having the sequence:
  • the FLV-spop50 polypeptide is in reverse phase with spike 15. It is a 50 amino acid peptide of the anti-spike p15 type, having the sequence RRRDDDDDDD 10 DDDDDDDDDR 20 RRRTCMQQQW 30 WYYAKHHPPP 40
  • the FLV-spkn90 peptide is a 90 amino acid peptide of the spike p15 protein and knob gp70 type.
  • the FLV-spknop50 peptide is a peptide of 50 amino acids whose characteristics correspond to a peptide of the anti-spike 15 and anti-knob gp70 type. This peptide has the following sequence:
  • Example 6 Nucleotide fragments capable of coding for artificial peptides related to HIV proteins and expression of these peptides.
  • nucleotide sequences deduced from the peptide sequences are synthesized.
  • the cohesive ends which frame the coding sequence are chosen so as to include the restriction sites for BamH1 (5 'end of a strand and 3' of the complementary strand) and EcoR1 (3 'and 5' ends respectively of each strand) at know :
  • the chain “+” represents the messenger RNA when the base T is replaced by the base U.
  • the messenger RNA is the result of the transcription of the chain "-" (the complementary RNA of chain "-").
  • sequences of the peptides A11 and B11, illustrated below, have been deduced from the chains complementary to those which can code for the peptides cA1 and cB1 (indicated between brackets above).
  • nucleotide fragments are inserted into the plasmid pGEX-2T, the restriction map of which is shown in FIG. 3, using the GST gene fusion system from Pharmacia (Uppsala, Sweden).
  • This system includes a tac promoter, an internal lac i7 gene and thrombin cleavage sites.
  • the expressed peptide is fused to the carboxyl end of the glutathione S-transferase from Schistosoma japonicum and purified from bacterial lysates by affinity chromatography using a column of glutathione Sepharose 4B R
  • Example 7 Study of the immunological properties of the pairs of artificial peptides.
  • Two month old rabbits (New Zealand) are immunized with peptides obtained as described above.
  • the rabbits are injected on day zero with 100 ⁇ g of peptide in the complete Freund's adjuvant.
  • the sera are tested 3 days after the last inoculation according to the ELISA test to determine the presence of antibodies against the inoculated peptide.
  • the sera titers are expressed in logarithms of the highest dilution for which the serum shows significant reactivity for an antigen.
  • Standard 96-well microtiter plates are covered with the various synthetic peptides to be studied.
  • peptide solution 100 ⁇ l are used per well of a peptide solution at 1 ⁇ g / ml in a PBS solution (saline solution buffered with phosphate), pH 7.4.
  • PBS solution saline solution buffered with phosphate
  • an anti-rabbit IgG anti-serum conjugated with horseradish peroxidase (diluted to 1/1000) is added, then the plates are incubated for another hour at 37 ° C.
  • the enzymatic activity is determined using o-phenylene diamine as a substrate.
  • the absorbance is measured at 492 nm.
  • the results obtained are reported in Table 2 according to: TABLE 2
  • the values represent the logarithms of the highest dilutions for which the serum shows significant reactivity.
  • Peptides A1 and A2 are only weakly recognized by rabbit anti-B1 polyclonal antibodies in the ELISA test.
  • the specificity of the cross-reactivity of the anti-B1 and anti-B2 sera is in agreement with the concept of a common frequency f1 for the two peptides B1 and B2, as demonstrated above with the cross-reactivity of the anti-A1 sera. and anti-A2, with the peptides respectively
  • the anti-A1 serum does not give rise to a marked cross-reaction with the peptides B1 or B2 which have opposite phases, this parameter therefore also intervening in biomolecular recognition.
  • the anti-B2 serum does not give a cross-reaction with the peptide A1.
  • the anti-B2 serum gives a weak cross-reaction with the peptide A2, while the polyclonal antibodies of the anti-A2 serum significantly recognize the peptide B2 in the ELISA test.
  • This cross-reactivity must be attributable to the homology of the sequences of A2 and B2 which have two fragments in common, namely DFHIWDDYLKRD and QEPMDFHI, although the position of these sequences is reversed.
  • the anti-B1 serum recognizes the recombinant glycoprotein (Baculovirus) gp120 of HIV-1, while the anti-A1, anti-A2 and anti-B2 sera do not react with gp120 under the conditions of the experiment.
  • Example 8 - STUDY OF THE REACTIVITY OF SERUMS WITH HIV PROTEINS IN THE IMMUNOBLOT TEST:
  • results obtained with an anti-rabbit anti-B1 serum are reported below using a commercial immunoblot kit such as that marketed by Diagnostic Pasteur containing LAVbru HIV-1 proteins.
  • the serum is diluted and incubated with the immunoblot strips with anti-rabbit goat IgG labeled with alkaline phosphatase.
  • the bands are developed using a phosphatase substrate system (Diagnostic Pasteur).
  • FIG. 5 Examination of this figure shows that the antibodies induced by the peptide B1 and present in the anti-B1 serum are capable of reacting with several proteins of HIV-1.
  • the antibodies in this serum were captured by the POL p68 protein (encoded by the virus reverse transcriptase gene), by the GAG protein p55 (the precursor of the GAG proteins), as well as by the proteins derived from this protein. precursor: p18, p24 / 25 and p40.
  • the component having a clear band of 43 kDa located in the region corresponding to the diffuse band of gp41 gives rise to a cross-reaction.
  • the p15 protein is the only protein among the GAG proteins which does not seem to be recognized by the antiB1 serum. Finally, the binding of anti-B1 antibodies to gp120 does not appear significantly in the Immunoblot test.
  • HIV-1 IgG Western blot kit marketed by Ortho Diagnostic Systems.
  • the gene containing the information for the peptide B1 was synthesized and inserted into the plasmid pGEX-2T (as described in Example 7), and then introduced into the bacterium.
  • the presence of the peptide B1 (fused with glutathione S-transferase) is revealed by the anti-B1 serum using the immunoblot, after a migration of the proteins on electrophoresis gel and their transfer to nitrocellulose.
  • the rabbits were immunized with the peptide cA1, the sequence of which is as follows: VHNQLVLRRLAPSVPWLLF. It is recalled that the peptide cA1 is part of the group of peptides B, since its chain of amino acids has been deduced from the nucleotide sequence complementary to that of the peptide A1, the latter belonging to group A (as described in Example 5). THE PROTOCOL IS AS FOLLOWS:
  • 300 ⁇ g of peptide are administered to rabbits by ID or SC injection with Freund's adjuvant (complete and incomplete).
  • One injection is given every 10 days, with at least 8 injections.
  • the antibody level is tested by the ELISA method one week after the last injection.
  • Samples are taken from the ear of the immunized rabbit (20 ml of blood are necessary). The same sample is taken from a control rabbit (not immunized).
  • One volume of whole blood is diluted in two volumes of HANKS solution, then placed on a FICOLL cushion and centrifuged at 1300 rpm for 30 minutes.
  • lymphocyte ring is removed, then rinsed twice in a HANKS solution and resuspended in 1 ml of RPMI 1640 medium.
  • lymphocytes are then counted in the MALASSEZ cell, then their number is adjusted so as to have at least 250,000 cells per well.
  • the culture is carried out in 96-well flat-bottom plates, treated for cell culture (Falcon No. 3072).
  • the culture medium is RPMI 1640 with antibiotics, glutamine and 10% rabbit serum in question, taken before immunization and decomplemented.
  • Each well receives 250,000 cells and increasing quantities of immunogenic peptide (from 10 to 75 ⁇ g per well).
  • the plates are placed in an incubator at 37 ° C. containing 5% of CO 2 for 5 days.
  • the contents of the wells are collected on glass fiber filters using a suction collector, the filters are then dried counted in a scintillating liquid.
  • Bibliographical references 1 / Hay, F.C. et al, (1984), pp 117-138. Academy Presss, Inc., Orlando, Florida.

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BR112016021395A8 (pt) * 2014-03-19 2021-07-13 Vcell Therapeutics Inc métodos de geração de uma célula pluripotente e de preparação de uma célula ou tecido ensaio, uso de uma célula pluripotente, e, composição
US11839650B2 (en) * 2017-02-02 2023-12-12 Board Of Regents, The University Of Texas System Universal influenza vaccine targeting virus/host recognition
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