WO1991018454A1 - Compositions capables de bloquer la cytotoxicite de proteines regulatrices de virus et les symptomes neurotoxiques associes aux infections par retrovirus - Google Patents

Compositions capables de bloquer la cytotoxicite de proteines regulatrices de virus et les symptomes neurotoxiques associes aux infections par retrovirus Download PDF

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WO1991018454A1
WO1991018454A1 PCT/EP1991/000928 EP9100928W WO9118454A1 WO 1991018454 A1 WO1991018454 A1 WO 1991018454A1 EP 9100928 W EP9100928 W EP 9100928W WO 9118454 A1 WO9118454 A1 WO 9118454A1
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tat
basic
hiv
peptide
basic region
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PCT/EP1991/000928
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English (en)
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Jean-Marc Sabatier
Elmostafa Bahraoui
Jurphaas Van Rietschoten
Kamel Mabrouk
Eric Vives
Hervé Rochat
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Centre National De La Recherche Scientifique
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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
    • 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
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention concerns compositions capable of blocking cytotoxicity of viral regulatory proteins, particularly cytotoxicity with regard to lymphocytes and/or nervous cells, associated with retroviral infection.
  • HIV-1 human immunodeficiency virus type 1
  • CNS central nervous system
  • the pathogenic mechanism by which the viruses cause encephalopathy remains unknown. It is an object of the present invention to identify factors involved in the virus-induced neurological disorders observed in patients with retroviral infections, and to provide compositions capable of blocking these disorders. It is also an object of the present invention to identify factors involved in lymphocyte cytotoxicity associated with retroviral infection, and to provide compositions capable of blocking this toxicity.
  • HIV-1 Tat has also been reported to encode a nuclear transport signal 14 .
  • Tat regions II and III are sufficient and essential for high transactivating activity 1 *
  • others report that in addition to the cysteine rich and basic regions, the N-terminal region is also necessary 29 .
  • These authors have identified the essential regions by synthesising Tat fragments spanning various Tat domains and testing the transactivating activity of the fragments in vitro. An involvement of the regulatory proteins in neurological disorders and in lymphocyte cytotoxicity has not been reported.
  • the present invention is based on the discovery by the present inventors that retroviral regulatory proteins possessing basic regions, for example Tat and Rev, show potent cytotoxic activity and lethal neurotoxic activity in vivo. Furthermore, the basic region of these proteins is necessary and sufficient for toxicity.
  • SUBSTITUTE SHEET gpl60, p25, p27 nef and Tat of HIV-1 The present inventors discovered that intracerebroventricular injection of Tat and some Tat fragments caused toxic effects in mice. Gpl60, p25, p27 and nef fragments showed no toxic effects.
  • the cytotoxic effects of the regulatory proteins on lymphocytes have also been demonstrated by the present inventors.
  • Various length Tat peptides (including the basic domain) from HIV-l Bru were shown to bind to CD 4 -expressing lymphoid cells (CEM line) .
  • CEM line CD 4 -expressing lymphoid cells
  • Studies on the post-binding effect of Tat showed that Tat can modify lymphoid cell membrane permeability and viability in a dose-dependent manner. This cytotoxic activity was confirmed by culturing CEM cells, human peripheral blood lymphocytes, or HeLa cells with Tat, which revealed the Tat-induced damage and growth inhibition of these cell types.
  • Tat was found not only to inhibit antigen- induced human peripheral blood lymphocyte proliferation in vitro, as previously reported (Viscidi et al., 1989), but also the lymphocyte proliferative response to mitogen, with 50 % inhibition obtained at 0.9 and 8 ⁇ M, respectively.
  • Tat peptides it was found that those containing the Tat basic region from 49 to 57 could bind to the cell membrane and exhibit cytotoxic activity.
  • HIV-1 Tat and other retroviral regulatory proteins and their peptide derivatives containing the basic domain can : i) bind specifically to rat brain synaptosomal membranes and to lymphoid cells ; ii) generate a membrane depolarization by modifying the cell membrane permeability in both vertebrate and invertebrate biological systems ; iii) induce lethal neurotoxicity to mice ; iv) manifest potential cytotoxic activity in nerve cell and lymphoid cell lines and ; v) induce transient damage to lymphoid cell membranes at subcytolytic concentrations via a direct interaction of the basic domain with the cell, giving rise to pore formation and internalisation of Tat.
  • compositions in the present context means prophylactic compositions such as vaccines capable of inducing the formation of protective antibodies, and also therapeutic compositions for use in cases where retroviral infection is already established.
  • the pharmaceutical compositions of the invention may be used in prevention or therapy of retroviral infection and immunodepressive states, as well as associated disorders for example sub-acute encephalitis and dementia.
  • the invention relates to a vaccine composition capable of blocking cytotoxic activity, particularly cytotoxic activity with regard to lymphocytes and/or nerve cells, exhibited by retroviral regulatory proteins having basic regions, and possibly of blocking retroviral proliferation, characterised in that the active component is a (poly)peptide composed of : i) at least one basic region, said basic region comprising at least 6 amino acids, at least 4, and preferably at least 5 of any six consecutive amino acids of this region being basic amino acids ; ii) optionally a non-basic region or regions wherein "non-basic" means less than 4 amino acids of any 6 consecutive amino acids being basic.
  • Base region in the context of the invention means a region comprising at least 6 amino acids, at least 4 or 5 of any six consecutive amino acids being basic.
  • the basic amino acids are arginine, lysine and histidine.
  • Vaccine composition in the context of the invention means an immunogenic composition comprising an active component devoid of cytotoxic activity at the required dose, and capable of inducing an immunological response which blocks cytotoxic and neurotoxic effects of the regulatory proteins, together with any necessary adjuvants and/or excipients. This type of composition may be used to prevent cytotoxic symptoms, and to treat them once infection is established.
  • cytotoxic means capable of affecting the viability or the function of the cells in question, for example, nerve cells or lymphocytes.
  • Neurotoxic means cytotoxic to nervous cells. Neurotoxicity is manifested in vitro by depolarisation of the cell membrane, accompanied by a decrease in membrane resistance leading to a modification of cell permeability ; by binding of the protein to synaptosomal membrane preparations, by damage and growth inhibition or by cell death. In vivo, the neurotoxicity is manifested by muscular tremors, convulsion and death.
  • Cytotoxicity to lymphoid cells is manifested by binding of the protein to the cell membrane
  • the amino acid sequence of the basic region of the active component is identical to at least a part of the basic region of a retroviral regulatory protein possessing such a basic region, for example Rev or Tat of HIV-1, HIV-2, SIV and Rex of HTLV-1.
  • the basic region comprises at least a fragment having the sequence
  • the basic region comprises at least a fragment having the sequence
  • the vaccines of the invention may have as active component, a (poly)peptide which, in addition to the essential basic region, also contains a non-basic region, wherein "non-basic" means a region wherein less than 4 amino acids of any six consecutive amino acids are basic.
  • non-basic regions sequences which are capable of increasing the toxicity-blocking and/or im unogenic character of the
  • SUBSTITUTE SHEET polypeptide may be cited, or transport signals to assist in the targetting of the vaccine, or sequences which confer resistance to certain enzymes in certain parts of the body. It is, of course, essential that the non-basic region of the polypeptide does not detract from the immunizing or toxicity-blocking character of the basic region.
  • non-basic regions sequences which are identical to, or which present at least 80 % homology with, the non-basic regions found naturally in the regulatory proteins and which are adjacent to or which flank the natural basic regions. It is also possible according to the invention to use a basic region which corresponds to the natural basic region of a particular regulatory protein in combination with non-basic flanking regions derived from a different regulatory protein. In this way, the immunogenic response to the polypeptide can be tailored to take advantage of particularly immunogenic and neutralising basic regions, which may be derived from a retrovirus other than the one against which vaccination is required. For example, an SIV Agm Tat or Rev basic region could be substituted in an HIV Rev or Tat protein.
  • fragments of the regulatory proteins themselves may be cited. Surprisingly, such fragments have been shown by the inventors to be more toxic than the whole proteins. Particularly preferred fragments are those consisting of the basic region and the amino acids upto and including the carboxy terminal. In this case, the N-terminal of the regulatory protein is missing.
  • the natural basic regions of the regulatory proteins comprise in general more than 6 amino acids.
  • the natural basic region of HIV-1 (region III) contains 9 residues.
  • the presence of the whole natural basic region as it occurs in the regulatory protein is not necessary in the vaccine.
  • a stretch of any six of the residues of the natural basic region, provided they fulfil the criteria of at least 4 or 5 of the residues being basic, is sufficient to allow induction of antibodies susceptible of blocking the neurotoxic symptoms and/or cytotoxicity.
  • the active component of the vaccine may comprise a polypeptide containing multiple basic regions, linked head to tail. This type of structure generally improves the immunogenic response to the vaccine. Additional non-basic regions may also be present.
  • active component peptides to be used in any of the above variants are : HIV-l Bru Tat 38-86 ; 48-86 ; 2-86 ; 38-72 ; 21-86 ; 46-60 ; 38-60 47-72 ; HIV-l Bru Rev 37-50 ; 34-51 ; HIV-2 R00 Rev 34-49 SIV ⁇ H J , Rev 34-49 ; SIV Ag ⁇ ) Rev 23-45 ; HTLV-1 Rex 1-17.
  • Lymphocyte protection is particularly effective using the above mentioned HIV-1 Tat fragments.
  • Nerve cell protection may, in addition, be particularly effective using the Rev and Rex fragments.
  • the active component of the pharmaceutical composition may be derived from
  • the regulatory protein of the virus against which protection is sought may be derived from another of the retroviruses.
  • Tat protein basic regions for instance that of HIV-1 is the most cytotoxic.
  • Other Tat protein basic regions for instance that of SIV Agm are not cytotoxic or are only cytotoxic at extremely high doses.
  • similarities in sequence and secondary structure between the basic regions of the different Tat proteins, and of the different Rev proteins indicate that antibodies generated by the basic region (or basic region-containing fragment) of one retroviral protein cross react with that of a different retrovirus.
  • the less toxic regulatory protein basic regions for example those illustrated in Table 2 below, particularly SIV Agm Tat or Rex HTLV-1, are used as active components in vaccine compositions to raise protective antibodies against the more toxic regulatory proteins, particularly HIV-1 Tat and HIV-1 Rev respectively.
  • the doses administered may be relatively high, with no danger of provoking cytotoxic effects.
  • the more toxic basic regions for example HIV-1 Tat or the Rev proteins as active component of the vaccine composition.
  • the doses administered to the patient are low and progessive in order to avoid manifestation of cytotoxic effects.
  • the vaccine of the present invention nay be a recombinant vaccine wherein the active component is first produced by expression of an appropriate nucleic acid sequence by an engineered host, or may be a live recombinant vaccine, such as a vaccinia
  • SUBSTITUTE SHEET virus engineered to express the active component in vivo. It is also possible to use a chemically synthesised (poly)peptide or a proteolytic fragment of the regulatory protein as active components.
  • the active component is formulated together with any necessary adjuvants and physiologically acceptable excipients, for example muramyl dipeptide. Excipients which are suitable for central nervous system administration (for example by spinal cord injection) can be used in cases where a localised central nervous system treatment is necessary.
  • the vaccine compositions of the invention are thus prepared by combining the active component as defined above with suitable adjuvants and/or excipients.
  • the vaccine according to the invention is particularly advantageous in that not only the cytotoxic properties, for example neurotoxicity, associated with the regulatory proteins are blocked, but, in addition the replication of the retrovirus may be blocked, thus halting proliferation.
  • This is particularly true in the case of infection by HIV since the basic region of HIV Tat is essential in transactivation.
  • Induction of antibody formation by the vaccines of the invention involves production of antibodies against the basic region. Since the basic region is involved in the interaction of the protein with the cell membrane, and in uptake by the cell of Tat, antibodies blocking the basic region prevent Tat from penetrating into the infected cell and thus prevent transactivation.
  • the vaccines whose active components are derived from HIV Tat, or which induce formation of antibodies cross-reacting with the basic region of HIV-1 Tat, are particularly preferred.
  • the vaccine of the invention may be used in a vaccinating "cocktail" or composition.
  • This composition is composed of chemical entities capable of initiating an immunological response which blocks infection by the retrovirus, and, in addition, the vaccine of the invention which blocks cytotoxic activity and proliferation.
  • Chemical entities susceptible of blocking retroviral infection are those known in the art, for example entities which induce an immunological response leading to a blockage of retroviral receptors.
  • the vaccinating cocktail of the invention is particularly advantageous in that the toxicity -and proliferation- blocking effect of the peptide having the basic region acts as reinforcement of the immunity against the retrovirus and limits or prevents development of infection should the infection-blocking agent prove insufficient.
  • the cytotoxic blocking capacity of the vaccines of the invention can be tested in vitro and in vivo using the cytotoxicity experiments outlined below in the examples. More particularly, the basic peptide under test is used to generate antibodies, which are then incubated with a cytotoxic regulatory protein, for example HIV-1 Tat. Then, following the protocols described in the examples for detecting cell binding, membrane depolarisation, effect on cell viability or in vivo toxicity, the effects of the regulatory protein previously incubated with the anti-basic region antibodies is tested. The basic peptides giving rise to antibodies which block the cytotoxic symptoms, for example those preventing tremors, convulsion and death following intracerebroventricular injection of the regulatory protein, are selected. In such tests, the concentrations of the cytotoxic regulatory proteins
  • SUBSTITUTE SHEET used are those indicated in the examples, for example, in vitro : 10 "7 to 10 " ⁇ for binding, 10 "5 to 10 "7 M for depolarisation or permeability changes and for effects on cell viability.
  • In vivo neurotoxicity tests can be carried out using the orders of concentrations given in tables 1 and 2.
  • the antigenic nature of the whole regulatory proteins has been recognised in the prior art 30 .
  • antigenicity of particular fragments and the cytotoxic blocking properties have not been described.
  • screening of antibodies to Tat(1-72) 31 to select those antibodies presenting these properties has been neither reported nor suggested.
  • the vaccinating and immunogenic properties of the peptides of the invention are surprising, especially for Tat-derived peptides in view of the fact that the major antigenic epitope of HIV-1 Tat was thought to be present in the N-terminal region 30 .
  • Experiments carried out by the inventors and described in the following examples demonstrate the lack of major epitopes in the N-terminal region.
  • fragments of the regulatory proteins which are missing the N- terminal amino acids ("N-terminal amino acids" is to be construed as meaning residues 1-37 for Tat HIV-1) are more toxic than the same fragments containing the N-terminal amino acids.
  • N-terminal deletion mutants for example, HIV-1 Tatsg. ⁇ and Tatsg. ⁇
  • N-terminal deletion mutants are particularly preferred fragments in the preparation of vaccines and vaccine composition.
  • fragments of the regulatory proteins containing, in addition to the basic region, the C terminal amino acids are particularly toxic. This type of fragment is also particularly advantageous in vaccine compositions.
  • the invention also concerns monoclonal or polyclonal antibodies capable of blocking the cytotoxic activity associated with retrovirus regulatory proteins possessing a basic region and possibly of blocking retroviral proliferation, said antibodies being directed against a (poly)peptide composed of : i) at least one basic region, said basic region comprising at least 6 amino acids, at least 4, and preferably at least 5 of any six consecutive amino acids of this region being basic amino acids ; ii) optionally a non-basic region or regions wherein "non-basic" means less than 4 amino acids of any 6 consecutive amino acids being basic.
  • these antibodies are capable of blocking cytotoxic activity, for example neurotoxic activity, and possibly also virus proliferation as described above.
  • Preparation of the antibodies is effected according to conventional techniques.
  • the antibodies be devoid of antibodies which do not possess the desired blocking activity, that is, in a polyclonal serum, a careful screening is effected to eliminate antibodies which are not capable of neutralising cytotoxic activity. Screening of the antibodies for the desired blocking activity can be carried out in vivo by administration of the antibodies together with administration of toxic retroviral regulatory proteins. Alternatively, infection by whole retrovirus could be effected.
  • the neurotoxic symptons of the regulatory proteins such as muscular tremors, convulsions and death within two hours of administration are blocked by antibodies exhibiting the desired properties.
  • the antibodies can be tested in vitro by blocking of membrane depolarisation by blocking of synaptosomal binding or by effect on cell viability, as described earlier for
  • antibodies are those directed against a (poly)peptide whose basic region comprises at least a fragment having the sequence
  • the antibodies are directed to a (poly)peptide whose basic region comprises at least a fragment having the sequence
  • Most particularly preferred antibodies are those directed against one of the following fragments : HIV-l Bru Tat 38-86 ; 48-86 ; 2-86 ; 38-72 ; 21-86 46-60 ; 38-60 ; 47-72 ; HIV-l Bru Rev 37-50 ; 34-51 ; HIV-2 R0O Rev 34-49 ; SIV ⁇ uz Rev 34-49 ; SIV Agm Rev 23-45 ; HTLV-1 Rex 1-17.
  • Particularly preferred antibodies are those capable of recognising the basic regions of the most strongly toxic regulatory proteins, for example HIV-1 Tat and the Rev proteins of HIV-l Bru , HIV-2 R00 , SIV ⁇ and SIV Agm .
  • Immunological cross-reactivity between the basic domains means that antibodies raised to SIV Agm Tat may react, for example, with HIV-1 Tat.
  • the invention therefore also relates to a pharmaceutical composition containing antibodies which recognise the basic region of HIV-1 Tat, said composition being capable of blocking the cytotoxic effects, particularly cytotoxic effects with regard to lymphocytes and/or nervous cells, associated with retroviral regulatory proteins, for example HIV-l Tat.
  • the antibodies of the invention may be used in passive immunity by direct administration, and may also be used in the preparation of anti-idiotype vaccines. Furthermore the antibodies of the invention may be used in a method of detecting the presence of neurotoxic retroviral regulatory protein in samples of cerebrospinal fluid characterised by the following steps : i) contacting the sample of cerebrospinal fluid with an antibody according to the invention ; ii) detecting the presence of antibody-bound retroviral regulatory protein by appropriate detection methods.
  • the invention also relates to peptides possessing cytotoxic activity, for example neurotoxic activity, selected from the group consisting of :
  • HIV-2 RO0 Rev 34-49 HIV-2 RO0 Rev 34-49 ;
  • peptides may be used in a method of detecting the presence of antibodies directed against neurotoxic retroviral regulatory proteins in samples
  • SUBSTITUTE SHEET of cerebrospinal fluid characterised by the following steps : i) contacting the sample of cerebrospinal fluid with a peptide according to the invention ; ii) detecting the presence of antibody-bound retroviral regulatory protein by appropriate detection means.
  • the invention further relates to a peptide analogue of any of the above-described cytotoxic peptides according to the invention, said analogue presenting at least 60 % and preferably at least 70 % or even at least 80 % homology with said cytotoxic peptide, characterised in that the peptide acts as an antagonist with respect to the cytotoxic activity of the cytotoxic peptide, particularly neurotoxicity or toxicity with regard to lymphocytes.
  • the antagonist action may be competitive.
  • These analogues may be selected by substitution, deletion or insertion of amino acids in the sequences of the cytotoxic peptides.
  • Particularly preferred antagonists are those having a size approximately the same as the basic regions of the regulatory proteins. The antagonists exert their effect by blockage of the cellular sites, for example the sites on lymphocytes or on nervous cells, where the basic regions of the regulatory proteins normally act, rather than by antibody formation.
  • the antagonist activity can therefore be screened by incubation of the peptide analogue under test with cells sensitive to the cytotoxic effects of the regulatory proteins, for example nerve cell lines or lymphoid cell lines, followed by administration of a cytotoxic regulatory protein in the doses defined above.
  • SUBSTITUTE SHEET The invention therefore further relates to these peptide analogues for use as a medicament in the treatment or prevention of retroviral infection, and to pharmaceutical compositions comprising the peptide analogue(s) in combination with a suitable pharmaceutical excipient.
  • Peptides for use in the following examples were chemically synthesised on an Applied Biosystems peptide synthesiser (model 430 A) according to the stepwise solid phase method 25"26 .
  • the peptides were purified by C 18 reverse phase medium pressure liquid chromatography and characterised by C 18 analytical high pressure liquid chromatography, amino acid content determination and slab-gel electrophoresis.
  • Tat peptides HIV-l Bru Tat peptides (synthesised according to the method of Example 1) were tested in vivo for their toxicity by determining the 50 % lethal dose (LD 50 ) using intracerebroventricular injection of Cs ⁇ BI ⁇ mice (20 g) . The peptides tested and results obtained are shown in Table 1. The LD 50 (right column) is expressed in both micrograms and nanomol of injected peptides. The peptides were considered inactive when neither lethal effect nor neurotoxic symptoms were observed after injecting 200 ⁇ g.
  • LD 50 50 % lethal dose
  • CM corresponds to the carboxamide methyl-cystein peptide derivatives.
  • Lethal dose 50 % determination Intracerebroventricular injections were carried out on groups of eight mice per dose using 5 ⁇ l of solutions containg peptides in 0.1 % (w/v) bovine serum albumin, 0.9 % (w/v) sodium chloride.
  • 86 CM Chymotryptic cleavage of peptides Tat 2 ⁇ - 86 CM and Tat 2 .
  • 86 CM peptides (3 mg) were solubilized in 126 ⁇ l of 20 mM Tris-HCl pH 8 buffer. Two percent (w/w) chymotrypsin (60 ⁇ g in 12 ⁇ l of the same buffer) were added as described above and the mixtures each containing 100 ⁇ g of peptide per 5 ⁇ l solution were left for 20 hours at 37*C. Proteolytic digestions using trypsin and chymotrypsin were controlled by C 18 analytical high presure liquid chromatography and Edman sequencing. Samples containing only enzyme were used as negative controls.
  • Tat ⁇ is lethal to mice with clinical effects resembling the neurotoxic symptoms induced by scorpion toxins 24 such as apathy followed by preliminary muscular tremors (about 10 in after injection) , convulsions, wasting and spastic paralysis just before death generally occurring between 15 min and two hours (depending on the peptide dose injected) .
  • This neurotoxic activity is specific, since numerous control peptides (derived from gpl60, p27 nef and p25) injected at higher doses did not induce neurotoxic symptoms and lethality.
  • the use of a panel of Tat peptides (see table 1) , delimited the minimal neurotoxic region of Tat.
  • the neurotoxicity of Tat in vivo is related to its basic domain (sequence 49-57) , since Tat ⁇ . ⁇ which mimics this region, or all the peptides which the entire basic domain, are lethal to mice while other Tat peptides are inactive (regions 2-23, 11-24, 13-48, 36-50, 56-70, 57-86, 65-80 and 67-86).
  • Neurotoxic activity is further demonstrated to be specifically related to the basic domain of Tat since tryptic digestion within this arginine/lysine-rich region completely abolished neurotoxicity while chymotryptic cleavage of CM (at specific positions 8, 11, 26, 32, 38, 43, 47 and 69), preserving the integrity of the basic domain, did not affect the Tat lethality.
  • mice when injected in mice either subcutaneously or intravenously.
  • mice b) Specific neurotoxicity to mice of peptides containing the basic region of various retroviral regulatory proteins :
  • Tat HIV-l Bru is the most toxic. All the Rev peptides tested, but not Rex HTLV-1, showed strong toxicity. "Active" or “inactive” peptides were assessed according to the criteria applied in example
  • Example 1 Example 1, and the neurotoxicity of these peptides was tested in vivo as above.
  • Example 3 Specific binding of HIV-1 Tat fragments to synaptosomal membranes Iodination of at38- ⁇ 6
  • Tat ⁇ . ⁇ peptide prepared according to the method of example 1 in 50 ⁇ l of PBS pH 7.4 and 0.25 mC 125 l Na (13-17 mCi/ ⁇ g) were added to a tube precoated with 100 nmol iodogen and incubated for 10 min at room temperature.
  • the iodinated peptide was desalted from free 15 1 Na by filtration through a Sephadex G ⁇ column PD 10.
  • Synaptosomes were prepared according to the method of Gray and Whittaker 17 .
  • Whole brains were dissected from Wistar rats and homogenized with a Teflon pestle in a 0.32 M sucrose solution (0.1 g of tissue/ml).
  • the homogenate was centrifuged for 10 min at 750 g and the resulting supernatant was further centrifuged for 25 min at 10 000 g.
  • the pellet was suspended in Hepes-choline buffer (140 mM choline chloride, 5.4 mM KCl, 0.8 mM MgS0 4 . 1.8 mM CaCl 2 , 10 mM glucose, 25 mM Hepes and Tris-base to pH 7.2). Protein was measured by a modified Lowry method 27 . Titration of 125 ⁇ ⁇ at 38 _ 86 binding to increasing amounts of rat brain synaptosomal membranes
  • Synaptosomes (0 to 100 ⁇ l at 1 mg of protein/ml) were incubated for 1 hour at 37"C with 50 ⁇ l of 125 I Tat ⁇ . ⁇ at 7.5 x 10 *8 M (2.5 x 10 5 cpm) in a final volume of 150 ⁇ l PBS buffer pH 7.4 containing 0.5 % (w/v) BSA and 0.05 % (w/v) NaN 3 . After three washes and centrifugations (5 min at 3000 g) , the radioactivity in the pellet was counted in a gamma counter. The results are shown in Fig. 1A.
  • Bo is the binding of 125 1 Tat ⁇ . ⁇ in the absence of competitors and, B the binding in presence of the indicated concentration of unlabeled peptides.
  • the values are means of four identical experiments. Non specific binding, less than 10 % of the total binding, was determined in the presence of a large excess (10 *4 M) of unlabeled Tat ⁇ . ⁇ peptide.
  • Figures 1A and IB show that 15 I Tat ⁇ . ⁇ , the most active peptide in vivo (table 1) , bound to synaptosomal membranes in a dose-dependent manner and
  • peptide Tat67-86 did not compete with 1251 Tat38-86 for binding, while Tat48-86 fully inhibited this interaction, indicating that region 48-66 includes the binding site of Tat.
  • This region contains a highly basic domain (49-57) previously found critical for efficient Tat transactivation, and it was presumed to be a nuclear targeting signal or a nucleic acid binding site.
  • A. Evolution of (a) the action potential and (b) the resting potential in frog muscle fibre The effects of 5 x 10 "6 M of Tat ⁇ CM on isolated frog muscle fibres were determined by studying, the modification of membrane potentials measured under current clamp conditions, with the double mannitol- gap technique 19 .
  • the normal physiological solution contained 12 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl and 5 mM glucose.
  • the solution was buffered with 6.5 mM Tris-HCl and pH was adjusted to 7.2 with Tris-base.
  • the mannitol solution was isotonic with the physiological solution. Results are shown in Fig. 2A.
  • Fig. 2B shows the evolution of the action potential (a) , the excitatory postsynaptic potential (b) , the resting postsynaptic potential (c) and membrane resistance (d) during microinjection of 1 ⁇ l 10 "5 M of Tat ⁇ . ⁇ within the cockroach sixth abdominal ganglion.
  • Injection of Tat ⁇ . ⁇ (arrow) triggers a sudden depolarization (c) accompanied by a brief firing of the giant axon.
  • the membrane resistance measured as the downward (about 50 %) deflection of hyperpolarizing pulses and the excitatory postsynaptic potential are strongly decreased whereas the action potential is only slightly modified.
  • the sudden depolarization was accompanied by a decrease in postsynaptic membrane resistance (about
  • HIV-1 Polyclonal anti-Tat (HIV-1) was prepared by injecting rabbits with 100 ⁇ g purified synthetic Tat (residues).
  • Tat fragments were synthesised according to the method of example l and then contacted with the polyclonal anti-Tat serum, and recognition or otherwise of the individual fragments was detected by
  • SUBSTITUTESHEET Example 6 Interaction of synthetic or recombinant HIV-1 Tat with lymphoid cells (CD -CEM cell line) : To investigate the mechanism of Tat uptake at the molecular level by lymphoid cells in culture, the interaction of synthetic or recombinant Tat (Fig. 5) with the CEM cell line was studied by direct fluorescence assay, and by indirect immunofluorescence assay.
  • CD 4 + -lympho ⁇ d cells of the CEM line, clone 13 (American Type Culture Collection, Ro ⁇ kville, MD) , were cultured at 37 ⁇ C in RPMI 1640 medium (Flow Laboratories Inc. , Irvine, Scotland) supplemented with 10 % fetal calf serum, 1 % glutamine, and 1 % streptomycin-penicillin antibiotics in a humidified atmosphere with 5 % C0 2 .
  • RPMI 1640 medium Flow Laboratories Inc. , Irvine, Scotland
  • 10 % fetal calf serum 1 % glutamine
  • streptomycin-penicillin antibiotics in a humidified atmosphere with 5 % C0 2 .
  • CEM cell binding of synthetic FITC-labeled Tat2- 86 analyzed by direct fluorescence assay Tat 2 .
  • Tat 2 -86 and Tat peptide derivatives were labelled with Fluorescein isothiocyanate (FITC) as follows : Tat 2 -86 and Tat peptide derivatives (5 mg) were solubilized in 250 ⁇ l of 100 mM sodium bicarbonate buffer, pH 9.5, and incubated in darkness with FITC (125 ⁇ g) for 2 hours at 25 ⁇ C. The reaction mixture was then loaded on a Sephadex G 25 PD10 column equilibrated with 0.5 M acetic acid, and target fractions were collected and lyophilized. Quantitation of the fluorescent peptide derivatives was by amino acid content determination after hydrolysis (6 N HC1, 1 h, 150 • C) .
  • FITC Fluorescein isothiocyanate
  • Varying amounts of FITC-labeled Tat 2 . 86 (0.2 to 20 ⁇ M) or FITC-labelled Tat peptides were incubated for 1 h at 37 ⁇ C with 10 6 CEM cells in 50 ⁇ l PBS, pH 7.4,
  • SUBSTITUTESHEET containing 0.5 % (wt/vol) bovine serum albumin and 0.05 % (vt/vol) NaN 3 . After two washes, cells were resuspended in 500 ⁇ l of PBS containing 1 % (wt/vol) paraformaldehyde. Membrane fluorescence intensity was measured by flow cytometry using the FACS analyzer (Becton-Dickinson, Montain View, CA) .
  • Varying amounts of synthetic or recombinant Tat (0.04 to 5 ⁇ M) or Tat peptides were incubatd for 1 h at 37 ⁇ C with 10 6 CEM cells in 25 ⁇ l PBS, pH 7.4, containing 0.5 % (wt/vol) bovine serum albumin and 0.05 % (wt/vol) NaN 3 (final volume 50 ⁇ l) .
  • the cells were washed and resuspended in 25 ⁇ l of serum (1:1,000) from rabbit immunized with Tat ⁇ . After 30 min at 37°C, cells were washed and incubated again for 30 min at 4°C with 1:25 sheep anti-rabbit IgG coupled to biotin.
  • Tat peptides were also used in these indirect binding experiments to delineate the Tat binding site on the cell membrane (Fig. 6B) .
  • the results show that Tat peptides (about 5 ⁇ M) including the basic domain bound to CEM cells whilst those devoid of the basic domain showed no significant binding activity.
  • Example 7 Effect of synthetic or recombinant HIV-1 Tat on CD -CEM, human PBL, and HeLa cell membrane permeability and cell viability : i) Membrane permeability : Post-binding effect of HIV-1 Tat on lymphoid cell membrane permeability was investigated with the 51 Cr release assay from Tat ⁇ -treated labeled CEM cells. CEM cells (2xl0 6 cells/ml) were radiolabeled for 12 h at 37 ⁇ C with lOO ⁇ Ci chromium-51 sodium chromate in RPMI 1640 containing 1 % fetal calf serum. Labeled cells were washed and suspended in medium at 10 6 cells/ml and dispensed in 100 ⁇ l aliquots into a
  • Tat ⁇ (60 ⁇ M) and Tat ⁇ . ⁇ (52 ⁇ M) did not elicit significant 51 Cr release from labeled cells (Fig.
  • Tat uptake may be initiated by a direct interaction of the basic residue-rich region of the protein with negatively charged phospholipids of the membrane bilayer, causing transient damage to the cell mambrane with pore formation leading to cellular internalization of Tat.
  • this effect is observed at subcytolytic concentrations of Tat, since the protein is cytolytic at high concentrations, as demonstrated by culturing HeLa cells, CEM cells or human PBL in the presence of increasing concentrations of Tat.
  • Example 8 Inhibition of antigen- and mitogen- induced human lymphocyte (PBL) proliferation in the presence of synthetic Tat 2-86 :
  • Tat from HIV-1 could inhibit antigen-induced, but not mitogen-induced, human peripheral blood mononuclear cell (PBL) proliferation (Viscidi et al., 1989).
  • PBL peripheral blood mononuclear cell
  • SUBSTITUTE SHEET the results concerning the mode of action of Tat with CD 4 + -CEM cells, the effect of synthetic Tat ⁇ and Tat peptide derivatives on human PBL proliferation was investigated.
  • Tat ⁇ (0.18 to 8.8 ⁇ M) or Tat peptides (3.2 to 32 ⁇ M) resulted in a dose-dependent inhibition of PPD-induced lymphocyte proliferation (Fig. 9A) .
  • 50 % inhibition was obtained with a Tat ⁇ concentration of 0.9 ⁇ M.
  • Tat ⁇ . ⁇ and Tat 38 . 72 also inhibited PBL proliferation by 60 and 58 % at concentrations of 2.3 and 3.2 ⁇ M, respectively.
  • the effect of Tat 2 . 86 and Tat peptides on mitogen- induced lymphocyte proliferation was further investigated using PHA and PWM on PBL samples from four HIV-1 seronegative donors.
  • Figure 9B shows the results of PHA stimulation in the presence of varying amounts of Tat ⁇ O.SS to 35 ⁇ M) .
  • the presence of Tat 2 . 86 resulted in a dose-dependent inhibition of mitogen-induced PBL proliferation.
  • Tat 2 . 86 inhibited lymphocyte proliferation by 10, 30, 50 and 100 % at concentrations of about 0.35, 3.5, 8 and 20 ⁇ M.
  • SUBSTITUTE SHEET capacity of various Tat peptides to inhibit PHA-induced lymphocyte proliferation shows that peptides Tat ⁇ . ⁇ , Tat ⁇ . ⁇ and Tat ⁇ . ⁇ , containing the entire basic domain, fully inhibited the PHA-induced proliferative response. These Tat peptides induced about 10, 50 and 100 % inhibition at concentrations ranging from 2-5, 10-20 and 20-50 ⁇ M. In contrast, Tat 2 - 23 an ⁇ * Tat 57 - 86 ' or fragment 1-34 of human spleen Hlb histone, has a negligible inhibitory effect at the mM concentration.
  • Tat ⁇ concentration 9-fold higher (8 ⁇ M) that that required for 50 % inhibition of PPD- induced stimulation.
  • This Tat ⁇ concentration is lower but comparable to those corresponding to 50 % of both cell membrane permeability and cell viability.
  • Tat ⁇ and Tat peptides including the basic domain are able to inhibit both the antigen- and mitogen-induced lymphocyte proliferation in vitro by a cytotoxic activity of the protein.
  • these data suggest the possible implication of Tat in the depletion of CD 4 -expressing lymphocytes by a direct cytolytic action of the protein produced within HIV- infected cells.

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Abstract

L'invention se rapporte à des compositions sous forme de vaccins capables de bloquer l'activité cytotoxique, particulièrement l'activité cytotoxique relative aux lymphocytes et/ou aux cellules nerveuses, développée par des protéines régulatrices de rétrovirus possédant des régions de base; ladite composition est éventuellement capable de bloquer également la prolifération de rétrovirus et caractérisée par le fait que le composant actif de celle-ci est un (poly)peptide composé de: i) au moins une région de base, ladite région de base comprenant au moins 6 acides aminés, au moins 4 et de préférence au moins 5 sur ces six acides aminés consécutifs étant des acides aminés de base; ii) éventuellement une ou des région(s) non basique(s) où le terme ''non basique'' signifie que moins de 4 acides aminés sur 6 acides aminés consécutifs sont basiques. L'invention se rapporte également à des anticorps susceptibles de bloquer les activités cytotoxiques de protéines régulatrices de rétrovirus et à des peptides analogues des protéines régulatrices.
PCT/EP1991/000928 1990-05-18 1991-05-17 Compositions capables de bloquer la cytotoxicite de proteines regulatrices de virus et les symptomes neurotoxiques associes aux infections par retrovirus WO1991018454A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
FR2773156A1 (fr) * 1997-12-26 1999-07-02 Biovacs Inc Nouveaux immunogenes anti-retroviraux (toxoides), nouveaux procedes de preparation et application a la prevention et au traitement du sida
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them
DE4407621B4 (de) * 1994-03-08 2005-03-10 Hawo S Kornmuehlen Gmbh Mahlwerk für eine Kornmühle
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US9206239B2 (en) 2009-03-23 2015-12-08 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV Tat derivative polypeptides
US9663556B2 (en) 2013-10-04 2017-05-30 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV tat derivative polypeptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002989A1 (fr) * 1985-11-06 1987-05-21 United States Of America, Represented By The Unite Expresion du gene du virus du sida
EP0233764A1 (fr) * 1986-02-14 1987-08-26 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Plasmides inhibant la réplication de virus lymphotropique type III de T-cellules humaines
US4910132A (en) * 1987-05-12 1990-03-20 Centocor Virus-free in vitro assay for anti-HIV agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002989A1 (fr) * 1985-11-06 1987-05-21 United States Of America, Represented By The Unite Expresion du gene du virus du sida
EP0233764A1 (fr) * 1986-02-14 1987-08-26 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Plasmides inhibant la réplication de virus lymphotropique type III de T-cellules humaines
US4910132A (en) * 1987-05-12 1990-03-20 Centocor Virus-free in vitro assay for anti-HIV agents

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMIAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 162, no. 3, 15 August 1989, pages 963-970, Academic Press, Inc., S. KUBOTA et al.: "Functional similarity of HIV-I rev and HTLV-I rex proteins: Identification of a new nucleolar-targeting singal in rev protein"i, see discussion *
CELL, vol. 58, no. 1, 14 July 1989, pages 215-223, (Cambridge, MA, US), M. GREEN et al.: "Mutational analysis of HIV-1 Tat minimal domain peptides: Identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression", see summary; page 222: "Development of an AIDS therapy" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 86, no. 19, October 1989, pages 7397-7401, (Washington, DC, US), A.D. FRANKEL et al.: "Activity of synthetic peptides from the Tat protein of human immunodeficiency virus type 1", see abstract; figure 1 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4407621B4 (de) * 1994-03-08 2005-03-10 Hawo S Kornmuehlen Gmbh Mahlwerk für eine Kornmühle
US6132721A (en) * 1995-03-08 2000-10-17 Neovacs Non-Toxic immunogens derived from a retroviral regulatory protein, antibodies, preparation method therefor, and pharmaceutical compositions containing same
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them
US6420141B1 (en) 1997-12-26 2002-07-16 Neovacs Anti-HIV immunogens (toxoids), preparation methods and use for preventing and treating aids
EP1041888A4 (fr) * 1997-12-26 2000-10-18 Zagury Jean Francois Immunogenes antiretroviraux, preparation et utilisation de ceux-ci
EP1041888A1 (fr) * 1997-12-26 2000-10-11 ZAGURY, Jean-François Immunogenes antiretroviraux, preparation et utilisation de ceux-ci
WO1999033872A1 (fr) * 1997-12-26 1999-07-08 Neovacs Nouveaux immunogenes anti-hiv (toxoides), procedes de preparation et application a la prevention et au traitement du sida
FR2773156A1 (fr) * 1997-12-26 1999-07-02 Biovacs Inc Nouveaux immunogenes anti-retroviraux (toxoides), nouveaux procedes de preparation et application a la prevention et au traitement du sida
US7022326B1 (en) 1997-12-26 2006-04-04 Biovacs, Inc. Carboxymethylated retroviral regulatory proteins and interferon-α
EP1975173A1 (fr) 1997-12-26 2008-10-01 Neovacs Immunogènes anti-rétroviraux, préparation et utilisation
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US9206239B2 (en) 2009-03-23 2015-12-08 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV Tat derivative polypeptides
US9663556B2 (en) 2013-10-04 2017-05-30 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV tat derivative polypeptides
US10159707B2 (en) 2013-10-04 2018-12-25 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV Tat derivative polypeptides

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