WO1991018454A1 - Compositions capable of blocking cytotoxicity of viral regulatory proteins and neurotoxic symptoms associated with retroviral infection - Google Patents

Abstract

The invention relates to vaccine compositions capable of blocking cytotoxic activity, particularly cytotoxic activity, with regard to lymphocytes and/or nerve cells, exhibited by retroviral regulatory proteins having basic regions, said composition possibly being capable of also blocking retroviral proliferation characterised in that the active component of said composition 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. The invention also relates to antibodies capable of blocking the cytotoxic, activities of retroviral regulatory proteins, and to peptide analogues of the regulatory proteins.

Description

COMPOSITIONS CAPABLE OF BLOCKING CYTOTOXICITY OF

VIRAL REGULATORY PROTEINS AND NEUROTOXIC SYMPTOMS

ASSOCIATED WITH RETROVIRAL INFECTION

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.

Literature references cited hereafter are listed in the form of a bibliography.

It is now established that infection with the human immunodeficiency virus type 1 (HIV-1) is often complicated by neurological syndromes, including dementia, subacute encephalitis and vacuolar degenerescence of the spinal cord^1""". The identification and isolation of HIV-1 from the brain suggested that the retrovirus itself is responsible for the neurological disorders observed in HIV infected patients. Other lentiviruses, including Visna⁵ and Simian immunodeficiency virus⁶ are also associated with brain infections. The major cells of the central nervous system (CNS) infected by HIV are of the monocyte and macrophage lineage, but infection of some other cell types including endothelial cells, glial cells, astrocytes, oligodendrocytes and neurons, has also been discussed^7*10.

However, 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.

The critical role played by retroviral regulatory proteins Tat and Rev in viral replication has been reported^11"16'²⁸. Expression of the gene tat enhances by transactivation the synthesis of all viral proteins, including Tat. Expression of the gene rev results in a decrease in the synthesis of the regulatory proteins Tat and Rev and an increase in that of the structural proteins.

HIV-1 Tat has also been reported to encode a nuclear transport signal¹⁴.

Several studies have been carried out in order to identify the regions of HIV-1 Tat responsible for the transactivating activity. Certain authors report that Tat regions II and III (cysteine-rich and basic regions respectively - see Fig. 5) are sufficient and essential for high transactivating activity¹*, whereas others report that in addition to the cysteine rich and basic regions, the N-terminal region is also necessary²⁹. 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.

These effects were first observed by testing the neurotoxicity of multiple long synthetic fragments of

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.

Similar toxic effects were observed with fragments containing the basic regions of other regulatory proteins (Rev, Rex) of other retroviruses (HIV-2, SIV, HTLV) .

The neurotoxicity of HIV-1 Tat was further investigated by structure-activity relationships using binding experiments. Using chemically synthesised Tat, it was found that ¹²⁵I Tat and ¹²⁵I Tat₃₈.₈₆ specifically bound to rat brain synaptosomal membranes whith a medium affinity (Ko_>5 = 3 x 10^"6 M) . The interaction specificity was further demonstrated by the capacity of other Tat fragments, including the basic domain, to fully inhibit the interaction of ¹²⁵l Tat₃₈.₈₆ with the synaptosomal membranes. The results obtained suggested that the interaction ^• of Tat with the membranes is mediated primarily by its basic domain (residues 49-57) .

This interaction was further investigated by electrophysiology with isolated frog muscle fibres and cockroach giant interneuron synapses. It was shown that Tat acts on the membrane of both nervous and non-nervous cells and induces a large depolarization, accompanied by a decrease in membrane resistance modifying thus cell permeability. Cytotoxic activity on nerve cell lines was further demonstrated by culturing neuroblasto a and glioma cells in the presence of HIV-1 Tat and peptides including the basic domain. Obvious damage and growth inhibition were observed.

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 CD₄-expressing lymphoid cells (CEM line) . 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. In addition, 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 μM, respectively. Using 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.

In summary, these results show that 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.

SUBSTITUTE SHEET These results suggested that these proteins, particularly Tat and Rev, by acting on the CNS, could be involved in the pathogenic mechanisms of viral- induced encephalopathy observed in patients with retroviral infection, particularly acquired immune deficiency syndrome. In addition, the potential cytotoxicity of Tat produced in HIV-1 infected cells on CD₄-expressing lymphocytes could contribute to the depletion of these cells being directly involved in virus-induced immune dysfunction observed in HIV-1 infected patients.

The observation that the basic region of these proteins possesses cytotoxic and neurotoxic activity has been applied in the preparation of pharmaceutical compositions capable of blocking the toxic effects. The invention relates to these compositions and to their component parts. "Pharmaceutical 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.

More particularly, 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.

"Basic 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. In the context of the present invention, "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,

SUBSTITUTESHEET modification of cell membrane permeability, inhibition of cell growth and by cell death. According to a preferred embodiment of the invention, 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. According to a particularly preferred embodiment of the invention, the basic region comprises at least a fragment having the sequence

BXBBB or BBBXB wherein B — any basic amino acid residue, namely an Arginine, Lysine or Histidine residue and X = NB or BN, B having the meaning indicated above and N meaning any non-basic amino acid residue. According to further preferred embodiments of the invention, the basic region comprises at least a fragment having the sequence

BBXBBB or BBBXBB wherein B and X have the above indicated meanings ; or a fragment having the sequence

RRX'RRR or RRRX'RR wherein R = Arginine residue and X' = NB, BN or N alone, B and N having the above indicated meanings. These particular sequences occur in the basic regions of several of the retroviral regulatory proteins and have been found to be particularly cytotoxic. 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. As examples of 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.

It is also possible, within the framework of the invention, to use as "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.

As examples of particularly preferred (poly)peptides having a non-basic region in addition to the basic region, fragments of the regulatory proteins themselves, provided the fragments contain a basic region, 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.

SUBSTITUTE SHEET The natural basic regions of the regulatory proteins comprise in general more than 6 amino acids. For example, the natural basic region of HIV-1 (region III) contains 9 residues. Thus, according to the invention, 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. According to another embodiment of the invention, 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. Particularly preferred 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_Agπ) Rev 23-45 ; HTLV-1 Rex 1-17.

Each of those preferred active component peptides can be used to block both neurotoxicity and toxicity to lymphocytes. 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.

According to the invention, the active component of the pharmaceutical composition may be derived from

SUBSTITUTE SHEET the regulatory protein of the virus against which protection is sought, or on the contrary, may be derived from another of the retroviruses. For example, it has been established that amongst the basic regions of the Tat proteins tested by the inventors, 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. Furthermore, 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. Thus, according to a preferred embodiment of the invention, 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. In this way, the doses administered may be relatively high, with no danger of provoking cytotoxic effects. It is of course also possible, according to the invention, to use the more toxic basic regions, for example HIV-1 Tat or the Rev proteins as active component of the vaccine composition. In this case, 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. According to the invention, 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. Thus, for treatment of HIV infection, 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.

SUBSTITUT According to another variant of the invention, 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^"7M 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 art³⁰. However, antigenicity of particular fragments and the cytotoxic blocking properties have not been described. Thus, 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 region³⁰. Experiments carried out by the inventors and described in the following examples demonstrate the lack of major epitopes in the N-terminal region. Furthermore, it has been established by the inventors that 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. Thus the N-terminal deletion mutants (for example, HIV-1 Tatsg.^ and Tatsg.^) are particularly preferred fragments in the preparation of vaccines and vaccine composition. Moreover, it has also been observed by the inventors that 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.

SUBSTITUTE SHEET 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. In the case of polyclonal antibodies, or a cocktail of monoclonal antibodies, it is preferred that 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

SUBSTITUTE SHEET testing the capacity of basic peptides to generate protective antibodies.

Particularly preferred antibodies are those directed against a (poly)peptide whose basic region comprises at least a fragment having the sequence

BXBBB or BBBXB wherein B - any basic amino acid residue, namely an Arginine, Lysine or Histidine residue and X = NB or BN, B having the meaning indicated above and N meaning any non-basic amino acid residue. According to further preferred embodiments of the invention, the antibodies are directed to a (poly)peptide whose basic region comprises at least a fragment having the sequence

BBXBBB or BBBXBB wherein B and X have the above indicated meanings ; or

wherein R = Arginine residue and X' = NB, BN or N alone, B and N having the above indicated meanings. 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. Thus,

BSTITUTE SHEET antibodies to the less toxic proteins can protect against the more toxic proteins. 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-l_Bru Tat 38-86 ; 48-86 ; 38-72 ; 21-86 ; 21-86 CM

; 38-60 ; 47-72 ;

HIV-l_Bru Rev 34-51 ;

HIV-2_RO0 Rev 34-49 ;

SIV_nnl42 ^ReV 34-49 ;

SIV_Agm Rev 23-45 ;

HTLV-1 Rex 1-17.

These 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. The absence of cytotoxic effects, as defined above, signifies the antagonist activity of the peptide analogue under test.

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.

EXAMPLES

Example 1 : peptide synthesis

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 C₁₈ reverse phase medium pressure liquid chromatography and characterised by C₁₈ analytical high pressure liquid chromatography, amino acid content determination and slab-gel electrophoresis. Example 2 a) Determination of the specific neurotoxicity to mice of HIV-1 synthetic Tat_?.«A, Tat?.,^ CM and peptides mimicking various regions of Tat (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 (LD₅₀) using intracerebroventricular injection of Cs^BI^ mice (20 g) . The peptides tested and results obtained are shown in Table 1. The LD₅₀ (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 μg. At this dose, thirty control peptides including highly basic peptides (fragment 1-34 of human spleen H-_jb histone or full- length histone from calf thymus) were found inactive (data not shown) . The abbreviated form 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 μl of solutions containg peptides in 0.1 % (w/v) bovine serum albumin, 0.9 % (w/v) sodium chloride.

- Tryptic cleavage of peptide Ta ^.^ : peptide Tat₃₈.₈₆ (8 mg) was solubilized in 136 μl of 20 mM Tris-HCl pH 8 buffer. Two percent (w/w) trypsin (16 μg in 32 μl of the same buffer) were added twice to the solution 1 hour apart and the mixture containing 200 μg of Tat_3B_₈₆ per 5 μl solution was incubated 2 hours at 37^PC.

- Chymotryptic cleavage of peptides Tat₂ι-₈₆ CM ^and Tat₂.₈₆ CM : peptides (3 mg) were solubilized in 126 μl of 20 mM Tris-HCl pH 8 buffer. Two percent (w/w) chymotrypsin (60 μg in 12 μl of the same buffer) were added as described above and the mixtures each containing 100 μg of peptide per 5 μl solution were left for 20 hours at 37*C. Proteolytic digestions using trypsin and chymotrypsin were controlled by C₁₈ analytical high presure liquid chromatography and Edman sequencing. Samples containing only enzyme were used as negative controls.

TUTE SHEET TABLE I

SUBSTITUTE SHEET As summarized in table 1, Tat^^ is lethal to mice with clinical effects resembling the neurotoxic symptoms induced by scorpion toxins²⁴ 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. The data obtained show that both oxidized ^Tat ₂-₈₆ ^an<

containing a stretch of cysteines are about 2-fold less neurotoxic than their homologous reduced and carboxamidated methyl cysteine peptide derivatives (Tat₂.₈₆ CM and Tat₂ι-₈₆ CM) . This neurotoxicity decrease could be explained by conformational constraints and/or accessibility variations of the basic domain within the intact molecule. The conformational importance of the basic region for lethal activity is suggested by the different neurotoxic potencies (LD_SQ) of active Tat peptides : the presence of adjacent but inactive Tat regions 38-46 and 73-86 was found to increase about 6 and 14 fold, respectively, the neurotoxicity of the basic domain. Interestingly, the neurotoxic peptides were found inactive at doses tested as high as 40 x

LDso when injected in mice either subcutaneously or intravenously. b) Specific neurotoxicity to mice of peptides containing the basic region of various retroviral regulatory proteins :

Peptides containing the basic region of various retroviral regulatory proteins were synthesised using the method outlined in Example 1.

The amino acid sequence of the fragments synthesised are shown in Fig. 3. The entire sequence of HIV-1 Rev is shown in Fig. 4.

The specific neurotoxicity to mice of these peptides was tested in vivo following the procedure of Example

2(a). Results are shown in Table 2.

From these results, it can be concluded that, amongst the Tat peptides. 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

2(a).

SUBSTITUTE SHEET TABLE 2

in neurotoxiciy of HIV-1 Rev :

Peptides corresponding to various fragments of HIV-1

Rev were synthesised according to the method of

Example 1, and the neurotoxicity of these peptides was tested in vivo as above.

Results are shown in Table 3 :

TABLE 3

PEPTIDES (HIV-1 REV) LETHAL DOSE

1-16 inactive

9-26 II

18-30 II

31-44

37-50 100 μg (48.7 nmol)

52-64 inactive

75-88 It

87-100 II

96-110 II

102-116 II

Example 3 ; Specific binding of HIV-1 Tat fragments to synaptosomal membranes Iodination of at38-β₆

One hundred μg (18 nmol) of Tat^.^ peptide (prepared according to the method of example 1) in 50 μl of PBS pH 7.4 and 0.25 mC¹²⁵l Na (13-17 mCi/μg) 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 ¹⁵1 Na by filtration through a Sephadex G^ column PD 10. Synaptosomes were prepared according to the method of Gray and Whittaker¹⁷. 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 MgS0₄. 1.8 mM CaCl₂, 10 mM glucose, 25 mM Hepes and Tris-base to pH 7.2). Protein was measured by a modified Lowry method²⁷. Titration of ¹²⁵ι τat₃₈_₈₆ binding to increasing amounts of rat brain synaptosomal membranes

Synaptosomes (0 to 100 μl at 1 mg of protein/ml) were incubated for 1 hour at 37"C with 50 μl of ¹²⁵I Tat^.^ at 7.5 x 10^*8 M (2.5 x 10⁵ cpm) in a final volume of 150 μl PBS buffer pH 7.4 containing 0.5 % (w/v) BSA and 0.05 % (w/v) NaN₃. 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.

Saturable binding of ¹²⁵ι Ta ₃β-β₆ ^{to rat} brain synaptosomes

Synaptosomes (30 μl at 1 mg of protein/ml) were incubated with variable concentrations of ¹⁵I

[3 x 10-⁷ to 3 x 10-⁸ M] with the protocol described above. Results are shown in Fig IB.

Inhibition of binding of ¹²⁵ι Tat₃a-₈₆ ^{to r t} brain synaptosomes by unlabeled Tat^^, Tat^^ CM (carboxamide methyl-cysteine derivative of Tat ₂.₈₆) ,

^Tat ₃₈-₈₆ (⁵⁰ l) ^at 1.2 x 10^"8 M and 50 μl of synaptosomes at 1 mg of protein/ml were incubated with increasing concentration of competitors (10^'8 to 10"³ M) in a final volume of 150 μl. The pellets were washed with 3 x 1 ml of the same bufer and then counted for radioactivity. Results are shown in Fig. 1C. Bo is the binding of ¹²⁵1 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 ¹⁵I Tat^.^, the most active peptide in vivo (table 1) , bound to synaptosomal membranes in a dose-dependent manner and

HEET with a saturable effect. Binding specificity is demonstrated by the capacity of increasing amounts of unlabeled Tat^.^ to fully inhibit this interaction (Fig 1C) . The concentration of unlabeled Tat^.^ able to inhibit 50 % of ¹²⁵I Tat^.^ binding Ko_-5) was about 3 x 10^"6 M. This Ko_₅ value shows the medium range affinity of the interaction between Tat^.^ and synaptosomal membranes. The interaction specificity was further demonstrated by the capacity of full- length synthetic Tat2-86, Tat2-86 CM (carboxamide methyl-cysteine derivative of Tat2-86) , Tat21-86, Tat21-86 CM, Tat47-72 and Tat48-86 to fully inhibit the interaction of 1251 Tat38-86 with synaptosomal membranes in a binding assay. The K0.5 values obtained with these Tat derivatives were 2.5 to 4.5 x 10-6 M and were comparable to that of Tat38-86, indicating a comparable affinity of Tat peptide derivatives for the synaptosomal binding site. In addition, a binding assay carried out using full- length 1251 Tat2-86 CM gave results similar to those obtained with 1251 Tat38-86 (data not shown) . Interestingly, 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. These data, suggest that the interaction of Tat with synaptosomal membranes is mediated primarily by its basic domain. As a negative control, a highly basic peptide, but unrelated to Tat (fragment 1-34 from human spleen Hlb histonelδ) was tested as a competitor for 1251 Tat38-86, but no binding inhibition was obtained (Fig. 1C) . These

SUBSTITUTE SHEET results argue for the presence of Tat binding site in rat CNS.

Example 4 : Electrophysiological activity of HIV-1 Tat fragments

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 technique¹⁹. 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. The action potentials evoked under current clamp conditions in (a) were recorded on a digital oscilloscope, and the resting potential in (b) on chart pen-recorder. In (b) the small vertical deflections correspond to the lower part of the action potential. The experiments were performed at room temperature (18°C) on skeletal muscle fibres isolated from the semitendinosus muscle of the frog Rana esculanta. E_H : membrane potential ; NR : normal Ringer.

In isolated frog muscle fibres, application of 5 x 10^"6 M Tat^^ CM induced a rapid and large membrane depolarization of 66.2 ⁺ 2.1 mV (mean ⁺ SE of the mean, n = 6) (Fig. 2A) . Consequently, the action potential (AP) elicited with a brief depolarization under current clamp conditons decreased in amplitude and disappeared without any modification in its shape. Depolarization was accompanied by a decrease of membrane resistance. Tetrodotoxin (TTX at 10"⁶ M) which blocks Na⁺ entry through the Na⁺ chanel²² did not

SUBSTITUTE SHEET antagonize this effect. Similarly, the use of Cd⁺ ions (1 mM) , which block Ca²⁺ permeability²³, did not inhibit the depolarization effect induced by Tat^^ CM. A similar depolarization effect was obtained with the peptide at^.^. In accordance with binding assay results, peptides Tat^.^ or 1-34 of human spleen b histone at 10"⁴ M had no effect on membrane polarization or on the AP.

B. Effect of Tat₃β-₈₆ ^{on the} cereal-afferent giant interneuron synapses in the sixth abdominal ganglion of the cockoach Periplaneta americana : In addition, at^.^ was tested on the cereal-afferent giant interneuron synapses in the neuropile of the sixth abdominal ganglion of the cockroach Periplaneta americana. The electrophysiological method used was the single-fibre oil-gap method²¹. The Tat peptide was applied close to the synapses with a micropressure ejection system. Synaptic events were monitored on an oscilloscope, digitized and stored with a signal analyser. The composition of the buffer used was 208 mM NaCl, 3.1 mM KCl and 10 mM CaCl₂. The pH was adjusted to 7.4 with Hepes.

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 μl 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

SUBSTITUTE SHEET 50 %) and a resulting decrease of the composite excitatory postsynaptic potential evoked by electrical presynaptic stimulation. The amplitude of the action potential triggered by block stimulation was somewhat modified. Tat^.^ at 10^"5 M produced a mean amplitude depolarization of 10 mV, varying with the localization of the tip of the injecting miσropipette. This depolarization was totally reversible within 30 min after Tat action.

Example 5 : immunological properties of peptide fragments

Polyclonal anti-Tat (HIV-1) was prepared by injecting rabbits with 100 μg purified synthetic Tat (residues

2-86) three times, 2 weeks apart.

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

ELISA.

Results are shown in Table 4 :

TABLE 4

HIV-1 Tat fragments HIV-1 Tat fragments recognised not recognised

46-60 2-23

38-60 11-24

47-72 13-48

57-86 34-50 38-72 56-70

These results indicate the absence of epitope in the NH₂ terminal region.

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.

Chemical synthesis of Tat^^ and Tat peptides was performed according to the method of Example 1. Recombinant Tat was obtained from Transgene, Strasbourg, France.

CD₄ ⁺-lymphoϊd cells of the CEM line, clone 13 (American Type Culture Collection, Roσkville, MD) , were cultured at 37^βC 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 % C0₂. i) CEM cell binding of synthetic FITC-labeled Tat2-₈₆ analyzed by direct fluorescence assay : Tat₂.₈₆ 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 μl of 100 mM sodium bicarbonate buffer, pH 9.5, and incubated in darkness with FITC (125 μg) for 2 hours at 25^βC. The reaction mixture was then loaded on a Sephadex G₂₅ 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) .

Varying amounts of FITC-labeled Tat₂.₈₆ (0.2 to 20 μM) or FITC-labelled Tat peptides were incubated for 1 h at 37^βC with 10⁶ CEM cells in 50 μl PBS, pH 7.4,

SUBSTITUTESHEET containing 0.5 % (wt/vol) bovine serum albumin and 0.05 % (vt/vol) NaN₃. After two washes, cells were resuspended in 500 μl of PBS containing 1 % (wt/vol) paraformaldehyde. Membrane fluorescence intensity was measured by flow cytometry using the FACS analyzer (Becton-Dickinson, Montain View, CA) .

The flow cytometry analysis showed that labeled

could interact with cells below the μM concentration (about 0.2 μM) in a dose-dependent manner. Cell fluorescence could be related to intracellular labeling due to cell uptake of FITC-labeled Tat^^ and/or to surface labeling of the cell membrane. The results also show that Tat peptides (about 5 μM) including the basic domain from 49 to 57 (Tat₃₈.₆₀, ^Tat ₃₈-₇₂ ^{and Tat} ₃₈-_8ό) bound to CEM cells while

and Tats_/.^ at 100 μM had no significant binding activity. These fragments were used to delineate the Tat binding site on the cell membrane (Fig. 6A) . Similar results were obtained when using E. coli expressed recombinant Tat. ii) CEM cell binding of synthetic or recombinant HIV-1 Tat analyzed by indirect immunofluorescence assay :

Varying amounts of synthetic or recombinant Tat (0.04 to 5 μM) or Tat peptides were incubatd for 1 h at 37^βC with 10⁶ CEM cells in 25 μl PBS, pH 7.4, containing 0.5 % (wt/vol) bovine serum albumin and 0.05 % (wt/vol) NaN₃ (final volume 50 μl) . The cells were washed and resuspended in 25 μl 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. After washing, cells were resuspended in 25 μl of streptavidin-phycoerythrin conjugate (1:25) for 30 min at 4^βC. After two washes, cells were resuspended in 500 μl of PBS containing 1

SUBSTITUTE SHEET % (wt/vol) paraformaldehyde. Membrane fluorescence intensity was measured by the FACS Analyzer. Dead cells were excluded by volume and scatter gating. Flow cytometry analysis showed that Tat, stained with rabbit anti-Tat antibodies, could interact with lymphoid cells in a dose-dependent manner. A significant interaction was detected with synthetic Ta^.^ at the concentration of 0.4 μM. Similar results were obtained when using E. coli expressed recombinant Tat. As a control, no shift of cell fluorescence was observed when incubation was with rabbit preimmune serum instead of anti-Tat antibodies (data not shown) .

Tat peptides were also used in these indirect binding experiments to delineate the Tat binding site on the cell membrane (Fig. 6B) . As with the direct binding experiments, the results show that Tat peptides (about 5 μM) including the basic domain bound to CEM cells whilst those devoid of the basic domain showed no significant binding activity. iii) Binding of HIV-l Tat to human peripheral blood lymphocytes :

A similar specific binding of Tat was obtained when incubation was done with human PBL (data not shown) . 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 ⁵¹Cr release assay from Tat^^-treated labeled CEM cells. CEM cells (2xl0⁶ cells/ml) were radiolabeled for 12 h at 37^βC with lOOμCi chromium-51 sodium chromate in RPMI 1640 containing 1 % fetal calf serum. Labeled cells were washed and suspended in medium at 10⁶ cells/ml and dispensed in 100 μl aliquots into a

SUBSTITUTE SHEET flat-bottom 96-well microtiter plate. Various amounts of synthetic Tat₂.₈₆ (0.18 to 18 μM) and Tat peptide derivatives (0.2 to 60 μM) were added to each well. After 4 h incubation at 37°C in 5 % C0₂ humidified atmosphere, cell-free supernatants were harvested and counted in a gamma counter. Wells containing labeled cells in RPMI alone or in RPMI plus 1 % (vol/vol) Triton X-100 served as controls for spontaneous and maximal cellular ⁵¹Cr release, respectively. The results clearly show the protein effect on cell permeability (Fig. 7A-B) . Significant celular release of ⁵¹Cr was observed below the μM concentration of ^Tat ₂-₈₆ (°-^{2 to 1} M) and 66 % ⁵¹Cr release was obtained at 18 μM. The synthetic Tat peptides containing the Tat basic domain (Tat₂₁.₈₆, Tat₃₈.₆₀, Tat^. and Tat^.^) produced a similar effect on these cells. Tat^.^ (24 μM) and Tat^.^ (23 μM) induced 70 and 90% ⁵¹Cr release, respectively (Fig. 7A) . Tat^^ (60 μM) and Tat^.^ (52 μM) did not elicit significant ⁵¹Cr release from labeled cells (Fig. 7) , nor did the highly basic control fragment 1-34 of human spleen Hlb histone at a similar concentration. These results suggest that the basic domain of Tat may be involved in both interaction and permeabilization of lymphoid cell membranes. ii) Cell viability : The effect of Tat on cell viability was also investigated by flow cytometry of propidium iodide treated CEM cells. Synthetic Tatg.^ (0.14 to 70 μM) incubated with cells modified cell viability (about 0.2 μM) in a concentration-dependent manner. Incubation of CEM cells with Tat^^ at 14 μM resulted in the labeling of about 50 % propidium iodide treated cells, corresponding to 50 % cell death. Similar results were obtained with recombinant Tat. The cell viability analysis by trypan blue exclusion

SUBSTITUTE SHEET of CEM cells or human PBL treated with recombinant Tat (0.1 to 100 μM) clearly showed the cytotoxicity of Tat, with about 40 % of cell death at 10 μM Tat concentration (Fig. 8) .

In addition, the effect of Tat was investigated on HeLa cells. When HeLa cells were cultured in the presence of synthetic Tat^^, the cells exhibited obvious damage and growth inhibition. Culture of these cells in the presence of various concentrations of synthetic Tat^^ (1.3 x 10^"7 to 1.3 x 10^"5 M) induced cell death within 24 to 48 h. Similar cytotoxicity was observed in cell culture with Tat peptides including the basic domain (data not shown) . These results show that the significant and 50 % effects of Tat^^ on both membrane permeability and cell viability experiments were obtained at similar concentrations of the protein.

Furthermore, these results enable the mechanism of Tat cell uptake to be explained. 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. However, 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 :

It has been reported that Tat from HIV-1 could inhibit antigen-induced, but not mitogen-induced, human peripheral blood mononuclear cell (PBL) proliferation (Viscidi et al., 1989). In the light of

SUBSTITUTE SHEET the results concerning the mode of action of Tat with CD₄ ⁺-CEM cells, the effect of synthetic Tat^^ and Tat peptide derivatives on human PBL proliferation was investigated.

Human PBL (10⁵) in a final volume of 200 μl were incubated in microtiter plate wells in the presence of various concentrations of synthetic Tat^^ (0.18 to 35 μM) or Tat peptide derivatives (0.5 to 60. μM) , with or without tuberculin antigen (PPD ; 12.5 μg/ml) or mitogens [phytohemagglutinin P (PHA ; 1:400), pokeweed mitogen (PWM ; 1:20)]. On days 3 or 6, cultures were pulsed for 8 h with 1 μCi or [³H]- thymidine. Cells were harvested and [³H]-thymidine incorporation in DNA was determined in a liquid scintillation spectrometer.

Stimulation of PBL (10⁵) with PPD antigen in the presence of increasing amounts of Tat^^ (0.18 to 8.8 μM) or Tat peptides (3.2 to 32 μM) resulted in a dose-dependent inhibition of PPD-induced lymphocyte proliferation (Fig. 9A) . Under the experimental conditions described, 50 % inhibition was obtained with a Tat^^ concentration of 0.9 μM. Tat^.^ and Tat₃₈.₇₂ also inhibited PBL proliferation by 60 and 58 % at concentrations of 2.3 and 3.2 μM, respectively. The effect of Tat₂.₈₆ 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 μM) . In contrast to results reported by Viscidi et al, the presence of Tat₂.₈₆ resulted in a dose-dependent inhibition of mitogen-induced PBL proliferation. From the four PBL samples tested, Tat₂.₈₆ inhibited lymphocyte proliferation by 10, 30, 50 and 100 % at concentrations of about 0.35, 3.5, 8 and 20 μM. The

SUBSTITUTE SHEET capacity of various Tat peptides to inhibit PHA- induced lymphocyte proliferation (Fig. 9C) 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 μM. 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.

Similar effects of Tat^^ and Tat peptides were obtained when using pokeweed mitogens (data not shown) .

Thus, 50 % inhibition of PHA-induced stimulation was observed at a Tat^^ concentration 9-fold higher (8 μM) 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.

These results clearly show that 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. In addition, these data suggest the possible implication of Tat in the depletion of CD₄-expressing lymphocytes by a direct cytolytic action of the protein produced within HIV- infected cells.

SUBSTITUTE SHEET BIBLIOGRAPHY

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SUBSTITUTE SHEET

Claims

CLAIMS 1. 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, said composition possibly being capable of also blocking retroviral proliferation characterised in that the active component of said composition 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.

2. Vaccine composition according to claim l characterised in that the amino acid sequence of the basic region is identical to at least a part of that of the basic region of a retroviral regulatory protein having such a basic region, particularly the Rev/Rex or Tat protein of the viruses HIV-1, HIV-2, SIV, HTLV-1.

3. Vaccine composition according to claim 1 or 2 characterised in that a non-basic region is also present, said non-basic region being identical to or presenting at least 80 % homology with at least a part of the non-basic regions of a retroviral regulatory protein having a basic region, particularly those parts which are immediately adjacent to the basic region.

4. Vaccine composition according to any one of claims 1 to 3 characterised in that the basic region comprises at least a fragment having the sequence

BXBBB or BBBXB

SUBSTITUTE SHEET wherein B = any basic amino acid residue, namely an Arginine, Lysine or Histidine residue and X = NB or BN, B having the meaning indicated above and N meaning any non-basic amino acid residue.

5. Vaccine composition according to claim 4 characterised in that the basic region comprises at least a fragment having the sequence

BBXBBB or BBBXBB wherein B and X have the above indicated meanings.

6. Vaccine composition according to claim 4 or 5 characterised in that the basic region comprises at least a fragment having the sequence

RRX'RRR or RRRX'RR wherein R = Arginine residue and X' = NB, BN or N alone, B and N having the above indicated meanings.

7. Vaccine composition according to any one of the preceding claims characterised in that the active component is a peptide comprised of at least a fragment of Tat or Rev/Rex of a virus chosen from HIV-1, HIV-2, SIV or HTLV-1, said fragment including the basic region of the protein.

8. Vaccine composition according to claim 7, characterised in that the active component is a peptide comprised of at least a fragment of Tat of HIV-1 including the basic region, or of at least a fragment of Tat of SIV_Agm.

9. Vaccine composition according to claim 7 or 8, characterised in that the peptide is comprised of a HIV-1, HIV-2 or SIV Tat -or Rev- NH_z-terminal deletion mutant and preferably extends upto and includes the carboxy terminal amino acids.

10. Vaccine composition according to claim 8 or 9 characterised in that the peptide is chosen from the following fragments :

HIV-l_Bru Tat 38-86 ; 48-86 ; 2-86 ; 38-72 21-86 ; 46-60 ; 38-60 47-72 ;

SUBSTITUTE SHEET HIV-l_Bru Rev 37-50 ; 34-51 ; HIV-2_R00 Rev 34-49 ; SIV^uz Rev 34-49 ; SIV_Agm Rev 23-45 ; HTLV-1 Rex 1-17.

11. Vaccine composition according to any one of the preceding claims characterised in that the active component is a synthetic peptide, a proteolytic fragment of a regulatory protein, a recombinant peptide, or a peptide expressed in vivo by a live viral vector.

12. Monoclonal or polyclonal antibodies capable of blocking the toxic activity associated with retrovirus regulatory protein possessing a basic region and possibly 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.

13. Monoclonal or polyclonal antibodies according to claim 10 directed against a polypeptide whose basic region has an amino acid sequence identical to at least a part of that of the basic region of a retroviral regulatory protein having such a basic region, particularly the Rev/Rex or Tat protein of the viruses HIV-1, HIV-2, SIV, HTLV-1.

14. Monoclonal or polyclonal antibodies according to claim 10 or 11 directed against a (poly)peptide also having a non-basic region, said non-basic region being identical to or presenting at least 80 % homology with at least a part of the non-basic

SUBSTITUTE SHEET regions of a retroviral regulatory protein having a basic region, particularly those parts which are adjacent to the basic region.

15. Monoclonal or polyclonal antibodies according to any one claims 12 to 14, directed against a (poly)peptide whose basic region comprises at least a fragment having the sequence

BXBBB or BBBXB wherein B = any basic amino acid residue, namely an Arginine, Lysine or Histidine residue and X = NB or BN, B having the meaning indicated above and N meaning any non-basic amino acid residue.

16. Monoclonal or polyclonal antibodies according to claim 15, directed against a (poly)peptide whose basic region comprises at least a fragment having the sequence

BBXBBB or BBBXBB wherein B and X have the above indicated meanings.

17. Monoclonal or polyclonal antibodies according to claim 15 or 16 directed against a (poly)peptide whose basic region comprises at least a fragment having the sequence

RRX'RRR or RRRX'RR wherein R = Arginine residue and X' = NB, BN or N alone, B and N having the above indicated meaning.

18. Monoclonal or polyclonal antibodies according to any one of claims 12 to 17, directed against a peptide comprised of at least a fragment of Tat or Rev/Rex of a virus chosen from HIV-1, HIV-2, SIV or HTLV-1, said fragment including the basic region of the protein and particularly of at least a fragment of Tat of HIV-1 including the basic region.

19. Monoclonal or polyclonal antibodies according to claim 18 directed against a peptide comprising a HIV-1, HIV-2 or SIV Tat -or Rev- NH_z-terminal

SUBSTITUTE SHEET deletion mutant said peptide preferably extending upto and including the carboxy terminal amino acids.

20. Monoclonal or polyclonal antibodies according to claim 18 or 19 directed against any 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 ;

34-51 ;

21. Monoclonal or polyclonal antibodies according to any one of claims 12-20 which recognise the basic region of HIV-1 Tat.

22. Antiidiotype antibodies directed against the idiotype of any of the antibodies of claims 12 to 21 and capable of playing the role of a vaccine protecting against the proliferation of retroviruses and against the cytotoxic activity, for example neurotoxicity, of retroviral regulatory proteins having a basic region.

23. Peptide possessing cytotoxic activity, particularly with regard to nerve cells or lymphocytes, said peptide being selected from the group consisting of :

HIV-l_Bru Tat 38-86 ; 48-86 ? 38-72 ; 21-86 ; 21-86 CM

; 38-60 ; 47-72 ;

HIV-l_Bru Rev 34-51 ;

HIV-2_R00 Rev 34-49 ;

SI- V_1Brt42 ^V 34-49 ;

SIV_Agm Rev 23-45 ;

HTLV-1 Rex 1-17 .

24. Method of detecting the presence of neurotoxic retroviral regulatory protein in samples of

SUBSTITUTE SHEET cerebrospinal fluid characterised by the following steps : i) contacting the sample of cerebrospinal fluid with an antibody according to any one of claims 12 to 21 ; ii) detecting the presence of antibody-bound retroviral regulatory protein by appropriate detection methods.

25. Method of detecting the presence of antibodies directed against neurotoxic retroviral regulatory proteins in samples of cerebrospinal fluid, characterised by the following steps : i) contacting the sample of cerebrospinal fluid with a peptide according to claim 23 ; ii) detecting the presence of antibody-bound retroviral regulatory protein by appropriate detection means.

26. Peptide according to claim 23 for use as a medicament in the treatment or prevention of retroviral infections, particularly in the blocking of retroviral proliferation and/or cytotoxic activity particularly neurotoxic activity associated with retroviral regulatory proteins having basic regions.

27. Use of a peptide according to claim 18 for the manufacture of a medicament in the treatment or prevention of retroviral infections, particularly in the blocking of retroviral proliferation and/or cytotoxic activity particularly neurotoxic activity associated with retroviral regulatory proteins having basic regions.

28. Vaccinating composition for immunisation against infection by a retrovirus, said composition being composed of chemical entities capable of inducing an immunological response which blocks infection by the retrovirus, characterised in that, in addition to these chemical entities, the composition contains the

SUBSTITUTE SHEET vaccine composition according to any one of claims l to 11.

29. Peptide analogue of a neurotoxic peptide according to claim 23 presenting at least 60 % and preferably at least 70 % homology with said neurotoxic peptide, characterised in that the peptide acts as an antagonist with respect to the cytotoxic activity of the toxic peptide, particularly the neurotoxic activity and toxicity with regard to lymphocytes, this antagonism including competitive inhibition.

30. Peptide analogue according to claim 29 for use as a medicament in the treatment or prevention of retroviral infection, particularly by HIV-1.

31. Pharmaceutical composition comprising at least one peptide analogue according to claim 29 in combination with a pharmaceutical excipient.

32. 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 nerve cells, associated with retroviral regulatory proteins, for example HIV-1 Tat.

SUBSTITUTE SHEET