OA10615A - Immunogens devoid of toxicity derived from a retroviral regulatory protein antibody preparation method and pharmaceutical compositions containing them - Google Patents

Description

See '1' 010615

Ionunogens devoid of toxicity derived from atrotreclonal regulatory protein, antibodies, process for their preparation and pharmaceutical compositions containing them

The present invention relates to novel retroviral immunogens using retroviral regulatory proteins whose essential biological properties have been previously inactivated in such a way that,. Whether they maintain or increase their immunogenic properties, their preparation process and pharmaceutical compositions containing them, such "inactivated" immunogens can be used to induce active immunization in man capable of preventing or correcting the effects of deregulation that the native proteins of which they are present. come from can contribute to produce. Also, the present invention relates to novel antibodies obtained by the use of such "inactivated" immunogens, their preparation methods and pharmaceutical compositions containing them for passive immunization.

The Tat molecule is an HIV regulatory protein that is not found in the viral particle but is encoded by the HIV-1 genome. Within the infected cells, this protein encoded by proviral DNA plays a transactivating role of viral or cellular genes. However, this genetic regulatory protein can also be found externally in the extracellular circulating medium, excreted in native or fragmented dead cell debris or by secretory process. Its presence in the circulating medium explains the existence of anti-detectable antibodies in certain seropositive subjects. In this extracellular context of Tat, the C-terminal segment bearing the RGD residues recognized by the cell surface integrins and the basic region of the molecule (residues 45-70) will allow it, as do the bacterial toxins, to act on the non-cellular cells. infected with different tissues. Thus, the circulating Tat protein acting as a true viral toxin can exert adverse effects on the endothelial cells and, in association with the BFGF growth factor, contribute to the neoangiogenesis of Kaposi's sarcoma characterizing the AIDS disease. The circulating Tat protein may also aggravate immunosuppression, which gradually sets in to AIDS, either by direct immunosuppressive action on T cells or by helping to disrupt the production of interferon a by antigen-presenting cells called APC (macrophages or dendritic cells).

The acquired immunodeficiency syndrome (AIDS) is clinically defined by opportunistic diseases due to immunosuppression or by Kaposi's sarcoma. Acquired immunosuppression, which is progressively established during the course of HIV infection, is biologically manifested by the loss of immunological activity of T cells (cytostasis and decreased IL2 production) after stimulation by the antigen of the caller first, then by alloantigens and finally mitogens (PHA). This immunosuppression is associated with the overproduction of α and γ interferons.

The cytopathic mechanisms that induce immunosuppression are complex and involve various factors of viral origin acting either directly on the cells of immunity, T cells and APC, or indirectly via the cytokine network. Thus, the envelope protein gp120 carried by the HIV-1 particle and whose extracellular presence is measured by the serum viral load can induiredirectly the T cell anergy of CD4 phenotype. For their part, the HIV regulatory proteins, in particular Tat, in its extracellular configuration of circulating viral toxin, also appear to induce direct immunosuppression of T cells or other pathogenic effects, for example in vitro antigen-activated T cells. In addition, the circulating Tat protein seems to facilitate the overproduction by the APCs of interferon, a cytostatic and apoptogenic cytokine capable of amplifying the immunosuppression and the apoptosis observed in the presence of the anti-CD3 antibody. in AIDS illness. Indeed, the secretion of interferon a by macrophages no longer appears, in the presence of TAT, stopped by a feedback control which in the normal state controls this production of interferon a (refractory period).

Kaposi's Sarcoma is clinically manifested by the appearance of vascular nodules representing neoangiogenesis consisting of endothelial cells. These cells, activated by inflammatory processes that produce cytokines (interferon γ, IL1 and IL6), produce BFGF (Basic Fibroblastic Growth Factor) and multiply. Proliferation of activated endothelial cells in vitro is enhanced by the presence in the medium of the Tat protein. Anti-Tat antibodies block invitro proliferative effects of the Tat protein. The action of Tat on the endothelial cells bearing on their adhesin surface of the Integrin 'family explains by the presence of the RGD sequence recognized by these molecules.

The neoangiogenesis that underlies Kaposi's sarcoma in vivo would therefore be favored by the Tat regulatory protein in its extracellular configuration, which could be recognized, by virtue of its C terminal fragment containing the RGD sequence, by the endothelial cell integrins. Deplus Tat may also act by its basic region rich in K and R residues, and therefore likely to bind heparin sulfate of the extracellular matrix which concentrates the BFGF growth factor. Thus growth of endothelial cells induced by growth factors is enhanced by the presence of Tat and engenders neoangiogenesis. This effect on the growth of endothelial cells is reduced in vitro by the action of antianti-Tat. It therefore appears desirable to block the harmful activity of the retrovirus regulatory proteins, in particular Tat circulating in the extracellular tissues (blood, lymph, interstitial media, etc.), true viral toxins.

With regard to the HIV viruses, attempts to vaccinate with structural proteins or structural protein fragments of these viruses have heretofore been made, but never with regulatory proteins or protein fragments. regulating these viruses.

It has surprisingly been found that, just like bacterial toxins (tetanus, diphtheria or botulinum) whose toxic activity is neutralized by specific antibodies, the harmful effects of viral regulation proteins, and in particular Tat, a true toxin of HIV in its extracellular configuration - whether by T-cell immunosuppression, disruption of α-interferon production by APC or by neoangiogenesis underlying Kaposi's sarcoma - are abolished in the presence of anti-Tat specific antibodies, as will be seen below in the experimental part.

It is the same with other virustal regulatory proteins than V1H-1, V1H-2, HTLV-1 or HTLV-2. It would therefore be desirable to provide immunogens administrable to humans capable of producing such antibodies, as well as to have such antibodies for both curative and preventive purposes. Indeed, the compounds of the prior art used as immunogens may be toxic to humans (see for example WO-A-9118454), especially those comprising the basic regions. These are unmodified fragments of Tat, Nef or Rev-natives, in contrast to the present invention based on the inactivation of these proteins or protein fragments. This is why the present application relates to immunogens which can be administered to humans because they are devoid of toxicity, characterized in that they derive from a protein that regulates an HIV-1, HIV-1, or 2, HTLV-1 or HTLV-2, by chemical treatment using a coupling agent such as an aldehyde, or a carrier protein activated by a pre-treatment with an aldehyde, preferably formaldehyde or glutaraldehyde, allowing them to be recognized by antibodies directed against said proteinprotein, and retaining immunogenic properties sufficient tocreate antibodies neutralizing or blocking said native protein, while having lost at least 50%, including at least 80 %, especially more than 95% of the toxic biological properties of said native protein.

These compounds, by analogy to bacterial toxoids such as lethanol toxoid, will be hereinafter referred to as "toxoids".

Indeed, just like conventional bacterial toxoids, they are devoid of their own toxicity, but are nevertheless capable of causing immunization by administration in a subject. - 5 - 010615

The chemical treatment below can be completed by physical treatment such as irradiation and especially U.V. irradiation, in order to reduce the residual toxicity of the immunogenic compounds according to the invention.

The above toxoids, for example, may be prepared from a peptide having a sequence identical or similar to a peptide sequence of a regulatory protein, such as Tat, and may be obtained, for example, by conventional peptide resin synthesis or genetic engineering. All these methods are well known from the state of the art.

In order to verify that the modified regulatory protein or its modified fragment according to the invention is well recognized by antibodies directed against said native regulatory protein, it is possible, for example, to immunologically verify by Elisa in the presence of specific antibodies, the formation of antigen-antibody complexes such as we will see it later in the experimental part.

In order to determine whether the immunogenic properties of the regulatory protein have been sufficiently conserved to create antibodies neutralizing said native protein, it is possible, for example, to immunize mammals (rabbits, rats, mice) with the aid of an immunogenic compound according to the invention and to verify that the antibodies produced neutralize the toxic activities of the regulatory protein, as will be seen for Tat in the experimental part.

In order to determine if the modified regulatory protein has lost at least 50% of its toxic biological properties, one can for example study the effect of the regulatory protein such as inactivated Tat on T cell immunosuppression or on the production of interferon by activated peripheral blood mononuclear cells or on laneoangiogenesis induced by the regulatory protein. The inactivation of the Tat regulatory protein is verified, for example, by the "Tat Rescue Assay" using a non-infectious HIV mutant.

Deficient Tat grown on the HLM-1 cell line whose replication depends on an exogenous supply of native Tat. 010615

The immunogenic compound may be derived from any of the virus regulatory proteins. HIV-1, HIV-2, HTLV-1 or HTLV-2 and especially Vif, Rev, Nef and Tat of HIV-1 and HIV-2; we particularly remember Rev, Nef, and Tat, more particularly Rev and Tat, and preferably the latter.

We can also mention the Tax protein of HTLV-1 or HTLV-2.

It is also particularly possible to mention the regions outside the basic Tat and Rev regions that are external to regions 49 to 57 of Tat and 35 to 50 of Rev, or overlapping these regions on at most 4 amino acids, preferably at most 2 amino acids, particularly HQVSLSKQPTSQPRGD peptide.

By "drifting" or "deriving" from a regulatory protein of an HIV1, HIV2, HTLV1 or HTLV2 virus, it is meant that the immunogenic compound may consist of all or a fragment of the regulatory protein and may comprise one or more modifications in the amino acids of this protein or fragment such as deletions, substitutions, additions, or functionalizations such as amino acid acylation, insofar as these modifications remain within the framework specified above (absence of toxicity, characters immunological). For example, in general the replacement of a leucine residue with an isoleucine residue does not modify such properties; the modifications must generally concern less than 30% of amino acids, preferably less than 20% and especially less than 10% on homology segments to the regulatory protein of at least 8 amino acids, in particular at least 12 amino acids . A fragment may comprise from 8 to 60 amino acids for example, preferably from 12 to 40 amino acids, and especially from 25 to 40 amino acids. For example, C-terminal residues 65-80 of HIV-1 Tat can be mentioned.

Under preferential conditions, the immunogenic compounds of the invention comprise at least 50% of the totality of the regulatory protein, preferably at least 70%, particularly at least 90%, and most preferably all or substantially -totality of said protein.

In general, with regard to the modifications, the homology or the similarity between the modified immunogen and the protein or part of the native protein, as well as the dimensions of the immunogenic compound, as well as the modalities of use, or coupling of the compound immunogenic according to the invention to an immunogenic protein such as tetanus toxoid, it may in particular refer to WO-A-86/06414 or EP-A-0.220.273 or PCT / US.86 / 00831, equivalent, whose teaching is incorporated icipar reference.

The immunogenic compounds according to the invention can be used as follows:

A patient is administered an immunogenic compound according to the present invention, for example subcutaneously or intramuscularly, sufficient to be therapeutically effective, to a subject in need of such treatment. The dose administered may range, for example, from 100 at 1000 μg subcutaneously, once a month for three months, then periodically depending on the level of serum antibodies induced, for example every 2-6 months.

A composition according to the invention may be administered by any conventional route in use in the field of vaccines, particularly subcutaneously, intramuscularly, intravenously or orally. The administration may take place as a single dose or repeated one or more times after a certain time interval. This is why the present application also relates to a pharmaceutical, curative or preventive composition, characterized in that it comprises as active ingredient, an immunogenic compound as defined above. The immunogenic compound can be packaged alone or admixed with a pharmaceutically acceptable excipient such as an adjuvant. The invention also relates to medicaments characterized in that they consist of immunogenic compounds as defined above, namely immunogenic compounds above for their use in a method of therapeutic treatment of the body human or animal, as well as the use of such an immunogenic compound for the preparation of a curative or preventive medicament for the treatment or prevention of harmful effects of the above regulatory proteins, and in particular Tat. In fact, the compounds according to the invention have lost their toxic properties and can therefore be administered in humans as will be seen hereinafter in the experimental part. The administration of immunogenic compounds according to the invention corresponds to active immunotherapy. It may also be advantageous to carry out passive immunotherapy, that is, to provide a patient directly with the antibodies that he needs to neutralize the harmful effects of the regulatory proteins of the V1H-1, HIV-2, HTLV-1 viruses. or HTLV-2.

These anti-regulatory protein antibodies can be obtained conventionally and, by way of example, after immunization of a mammal, human or animal, with the aid of an immunogenic compound as defined above, by cloning human B lymphocytes. transformed by the Epstein-Barr virus

and then recovering the expected antibodies secreted by said transformed B lymphocytes, or else by genetic recombination from a dephasing library. Therefore, the present application also relates to such methods for the preparation of anti-viral regulatory protein antibodies to HIVV-1, HIV-2, HTLV-1 or HTLV-2, and in particular anti-HIV antibodies. A toxoid state, and especially a process for the preparation of an above antibody characterized by immunizing a mammal with an immunogenic compound as defined above.

The subject of the present application is also an anti-protein for the regulation of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus and, in particular, polyclonal or monoclonal antibodies obtained from human subjects or from mammals immunized with the implementation of the methods described above.

These specific antibodies may come from: 1. the subject itself, induced by an active immunization (vaccination) with a biologically inactivated but immunogenic regulatory protein, in particular the Tat Such an immunogenic compound, in the case, for example, of Tat It is called Tat-toxoid in analogy to bacterial toxoids, or 2. a foreign organ, allo- or xenogeneic, administered to the subject by passive immunization (serotherapy). The allogeneic antibodies (in humans) can be generated in uninfected subjects voluntarily after active immunization (vaccination) with a protein immunogen or its derivatives according to the invention, in particular Tat (Tat-toxoid or peptide fragments of Tat according to the invention) . These passively administered antibodies, whether allogeneic (human) or xenogeneic (animal), may be complete monodonal or polyclonal antibodies or F (ab *) 2 or Fab fragments of the antibody.

By "anti-regulatory protein antibodies" is meant monoclonal or polyclonal antibodies or F (ab *) 2 or Fab fragments of these antibodies or anti-regulatory protein antibodies obtained from a genetic library. phages.

The allogeneic antibodies of human origin are: - either polyclonal - obtainable in volunteer seronegative subjects immunized with an immunogenic compound according to the invention, in particular Tat-toxoid or peptide fragments of Tat. either monoclonal from clones specific for B cells transformed by the EBV virus of these immunized individuals (EBV specific EBV lines).

The xenogeneic antibodies come from animals hyperimmune with an immunogenic compound according to the invention, especially Tat or its derivatives (Tat-toxoid, peptide fragments of Tat detoxified according to the invention), and are - either polyclonal from hyperimmunized animals, - or monoclonal , obtained after hybridization according to the Kohler and Milstein technique of spleen cells or of adenocytes with a myelomaous type 10 - 010615 line, type x63, in particular x63AG3. In this case, the equine or rabbit antibodies are preferred. The subject of the present application is also a process for the preparation of anti-regulatory protein antibodies to an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, characterized in that a mammal is immunized, man or animal, with an immunogenic compound as defined above.

The present invention also relates to a process for the preparation of monoclonal antibodies against the regulatory protein of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, characterized in that B-variant cells are used. of individuals immunized with an immunogenic compound according to the present invention, said B cells being transformed by the EBV virus and producing specific anti-regulatory protein antibodies of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus.

The above EBV + cells can be cultured to produce the expected antibodies. These cells, as we have seen, come especially from patients immunized with a native regulatory protein, orby an immunogenic compound according to the invention.

The present application also relates to a method for the preparation of monoclonal antibodies according to the invention, directed against a protein for the regulation of an inactivated or non-inactivated HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, characterized in that mammalian hybridomas, in particular mice, are prepared from splenocytes or adenocytes, especially mice immunized with a native regulatory protein or an immunogen compound according to the invention, and myeloma cells, preferably delineated x63, according to the well-known methods. known from the state of the art (Kohler and Milstein).

The subject of the present application is also a process for obtaining antibodies to the regulatory protein of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus by genetic recombination technology, characterized in that an immunogenic compound as defined above is used as an immunogen. - 11 - 010615

The present application also relates to F (ab ') 2 or Fab fragments of said antibodies; these can be obtained by enzyme digestion for example.

Another subject of the present invention is a passive immunization method for subjects contaminated with the HIV virus, using specific anti-virion protein-regulating antibodies to the virus virus and especially anti-Tat neutralizing or blocking the harmful effects of this protein in its configuration. extracellular and prepared as indicated above, or fragments F (ab ') 2 or F (ab) of these antibodies.

The present application also relates to an active immunization process against HIV or AIDS using an immunogenic compound described above, associated with a constitutive protein (or structure) of an HIV virus, in particular the gp 120 envelope glycoprotein or gp 160 or a modified or unmodified fragment thereof, or the inactivated HIV virus, or the HIV virus expanded from its genomic RNA for example by alkaline hydrolysis. The subject of the invention is also a method for activating HIV or AIDS immunization using an immunogenic compound described above, combined with one or more immunogens based on inactivated cytokines and especially α interferon, TGFβ or TNF. Indeed, as will be seen hereinafter in the experimental part, a combination of the effects of the antibodies according to the invention against regulatory proteins and anti-interferon antibodies in particular, completely restores HIV-induced immunosuppression. Therefore, the present application also relates to a composition comprising two immunogenic compounds, namely an immunogen compound of the above-described, and an immunogenic compound capable of inducing antibodies against a cytokine, such as interferon or TNF, as described for example in WO-A-9118454, as well as a composition closing an antibody directed against a cytokine including interferon a and an antibody above directed against a regulatory protein. - 12 - 010615

The present application also relates to an active immunization process characterized in that an immunogenic compound as defined above is used as an immunogen combined with an adjuvant of mineral, oily or synthetic immunity, or an immunogenic compound. as defined above coupled or associated with a protein that increases its immunogenicity.

These immunizations can be carried out both as a curative and as a preventive measure. As an immunogen for all the above processes and hereinafter, a derivative of the Tat protein is preferably used.

The subject of the present invention is also a process for hyperimmunisation of seronegative or seropositive subjects HIV-1, HIV-2, HTLV-1 or HTLV-2, characterized in that an immunogen as defined above is used, for the production of hyperimmune human sera, which may be particularly intended for passive serotherapy by administration of purified specific antibodies or their F (ab ') 2 or Fab fragments. The invention furthermore relates to a pharmaceutical composition comprising, as a curative or preventive active principle, at least one anti-virus protein for regulation of a virus as defined above or obtained according to the above methods. Finally, a subject of the invention is the use of an above-mentioned immunogen or antibody compound for the preparation of a medicament for the treatment of the harmful effects of a virus-regulating proteinVIH-1, HIV-2 , HTLV-1 or HTLV-2.

In summary, and in particular, the subject of the present invention is preventive or curative use in the subject who is HIV positive or has specific ARC / Sidad antibodies so as to block the harmful action of a protein that regulates a virus. HIV-1, HIV-2, HTLV-1 or HTLV-2 and especially the Tatcirculant These specific antibodies may come from. 1. the subject himself, induced by an active immunization (vaccination) with biologically inactivated but immunogenic Tat, called Tat-toxoid by analogy to toxoids 13 - 010615 bacterial or 2. a foreign organism, allo- or xenogeneic, administered subject by passive immunization (serotherapy). Allogeneic antibodies (in man) may be generated in non-infected volunteers after active immunization (vaccination) with a regulatory protein such as Tat or its derivatives (Tat-toxoid or peptide fragments of Tat according to the invention). These passively administered antibodies, whether allogeneic (human) or xenogeneic (animal), may be complete monoclonal or polyclonal antibodies or F (ab *) 2 or Fab fragments of the antibody.

By Tat-toxoid, as indicated above is meant a peptide or Tat protein treated with a chemical agent. This treatment caused the molecule to lose the toxic biological properties of circulating Tat (immunosuppression of T cells, induction of interferon production by interferon-producing cells, endothelial cell neoangiogenesis, blocking of the antiviral effect of interferon on macrophages) but have preserved the properties likely to induce the formation of the antibodies, when presented and appropriately prepared, whether or not coupled to a "carrier", aggregated or not, in the presence or absence of adjuvant.

The concept of Tat-toxoid has been extended to the immunogenic peptide fragments of Tat, that is to say a peptide sequence of Tat capable of inducing the formation of anti-Tat antibodies when presented in the appropriate manner, coupled to a carrier or not in the presence or adjuvant The invention also relates to pharmaceutical compositions. a) A pharmaceutical composition comprising, as a preventive or curative agent, a viral toxoid or a fragment or analog of a regulatory protein, in particular Tat according to the invention. b) A pharmaceutical composition comprising, as a preventive or curative agent, anti-Tat antibodies produced from organisms immunized against said protein or its F (ab *) 2 or Fab fragments, according to the invention.

In addition the invention also provides a kit comprising a vaccine pharmaceutical composition which in addition to the active ingredient (Tat-toxoid or its derivatives or anti-Tat antibodies) may comprise an adjuvant and / or another immunogen with anti-retrovirus properties.

Finally, the invention provides a pharmaceutical composition in a conventional dosage form. In particular, the active ingredient according to the invention is combined in an amount sufficient to be therapeutically effective with a pharmaceutically acceptable diluent or carrier. EXPERIMENT 1: Pathogenic Effects of Circulating Tat Protein

Immunosuppression of T cells in the presence of Tat: The effect of Tat on activated T cells of peripheral blood was investigated. Mononuclear cells (PMBC) and immunoglobulin isolated T cells (HLA35 DR-) were activated by anti-CD3 antibodies in the presence or absence of the Tat molecule. After 5 days of culture, cell proliferation was measured by incorporation. thymidine H3. The results showed that Tat inhibited the proliferation of HLA DR-T cells (85% inhibition at the concentration of 1.5 μg per ml). This inhibition disappears the presence of anti-Tat specific antibody (Intracel, U.K.). EXPERIMENT 2: Pathogenic Effects of the Circulating Tat Protein:

Inhibition of the effect of interferon on macrophages in the presence of Tat: The effect of Tat on the antiviral action of interferon a was measured by the conventional biological test using the measurement of the potency of VSV virus on MDBK cells .

Effect of Tat on exogenous interferon

Increasing doses of Tat have been shown to be able to inhibit, as soon as low concentrations are present, a dose-response curve for Tat-15-010615 concentrations ranging from 1 to 20 μg / ml, the anti-viral potency of the exogenous interferon. In a representative experiment, the protective effect of interferon was apparent until the 1/4800 dilution (corresponding to 150 U.l.) was reduced to 1/300 dilution for Tat concentrations of 10 μg / ml. Thus at the concentration of 10 μg / ml Tat, at the 4800 dilution of the exogenous interferon the VSV titer increased from 10 μg (interferon samples without Tat) to 105.5 (interferon samples in the presence of Tat). In these experiments, the inhibition of the anti-viral effect of interferon by the Tat molecule is suppressed in the presence of anti-Tat specific antibodies (Intracel, UK), as well as by the horse antibodies which we have prepared (see examples below). EXPERIMENT 3: Demonstration in subjects infected with HIV-1 of the Tat protein in its extracellular configuration.

Presence of anti-Tat antibodies in the serum of seropositive patients: a) An Elisa study of serum anti-Tat antibodies using the native Tat molecule as antigen was performed in 50 seropositive and 15 seronegative subjects. All sera from seronegative subjects and 20 sera from seropositive did not reveal the presence of anti-Tat antibodies with an optical density below the threshold (eut off) (OD = 0.250). Of the 30 sera of HIV-infected individuals who exceeded the threshold, 6 had an OD greater than 0.500. No apparent correlation could be made between the presence of anti-Tat antibodies on the one hand, the clinical state and the number of CD4 cells per mm3 of blood on the other hand. Serum Tat using different antigens as Tat peptides was performed in subjects with seropositive stages at different stages of their HIV infection - asymptomatic subjects; patients with pre-AIDS clinical manifestations (ARC) and patients with AIDS. The results of this study are as follows: ## STR1 ## 1) The peptide sequences of Tat .. MEPVDPRLEPWKHPG (residues 1 - 15 in N terminal) HQVSLSKQPTSQPRGD (residues 65-80 in C terminal) have been recognized by b large majority of the sera seropositive and no seronegative serum (controls). Reactions were strongly positive, but no correlation could be shown with HIV infection and CD4 counts in subjects. 2) The other sequences studied were not appreciably recognized by the sera whether they were seropositive individuals or controls (see Table 1). Only a few rare sera for each sequence showed an optical density greater than the threshold (2 times the average control sera). TABLE 1

Infected Subjects * (72 sera) Control Subjects (10 sera) Peptide Residues of Tat Positive Serums ** Mean OD Positive Serums ** OD Average 1-15 67 0.43 0 0.08 9-20 0 0.26 0 22 - 37 7 0.78 0 0.41 36-50 5 0.86 0 0.65 46-60 6 0.53 0 0.40 52-60 2 0.32 0 0.38 56-70 0 0.19 0 0.20 65-80 70 1.01 0 0.08 15 * HIV-infected individuals are at various stages; asymptomatic subjects, ARC patients and AIDS patients. ** The reaction is considered positive (threshold) when the optical density is 2 times higher than the average of the seronegative. 3) It is interesting to note that the most reactive sequence (AA 65-80) is that containing the RGD residues recognized by the integrins. EXAMPLE 1 Preparation of the immunogenic Tat protein (Tat-toxoid) - 17 - 010615

Inactivation of the Tat Protein in the Presence of Formaldehyde To a solution of Tat (1 mg / ml) in 70 mM disodium phosphate, pH 8.0, formaldehyde was added to the final concentration of 33 mM.

The mixture is incubated at 37 ° C for 1,3,5,7 and 9 days. At the end of each incubation period, a sample is taken and the reaction with formaldehyde blocked by addition of glycine to the final concentration 100 mM .

Each sample is dialyzed for 1 night at 4 ° C against 100 times its volume of PBS (phosphate buffered saline). EXAMPLE 2 Preparation of the immunogenic Tat protein (Tat-toxoid) Inactivation of the Tat protein in the presence of glutaraldehyde To a solution of Tat (1 mg / ml) in 70 mM disodium phosphate, pH 8.2, glutaraldehyde is added to the final concentration of either 0.026M or 0.0026M. The reaction with glutaraldehyde is allowed to continue for various times varying from 1 minute to 3 hours. After the reaction time, at the laboratory temperature, the reaction is blocked by addition of glycine to the final concentration of 100 mM. The various samples are dialyzed for 16 hours at 4 ° C. compared to 100 times their volume of PBS. EXAMPLE 3 Preparation of the immunogenic Tat protein (Tat-toxoid) Inactivation and simultaneous coupling to the tetanus toxin of TatA a mixture of tetanus toxin and Tat in a molar ratio of 1 to 15, in a 70 mM-100 mM phosphate buffer (pH ranging from 6.8 to 8.2) glutaraldehyde was added at the final concentration (ranging from 0.0026M to 0.026M) and the inactivation and coupling reaction was allowed to proceed for varying times (3- 15 minutes) at the laboratory temperature. The reaction is blocked by adding glycine to the final concentration of 100 mM and the various samples are dialyzed for 16 hours, at 4 ° C, against 100 times their volume of PBS. EXAMPLE 4 Immunogenic Potency and Antigenicity of Tat-toxoids

Immunogenic Tat-toxoid in mice: Antibodies by Elisa

The immunogenic power of the various preparations of Tatinactivé, either by formaldehyde (Tat-toxoid), or by glutaraldehyde (Tat-polan), or after coupling with tetanus toxin (conjugate Tat-toxinetétanique), ie compounds immunogens of Examples 1 to 3, was determined in the mouse.

Swiss mice of 18-20 g were immunized by two subcutaneous injections of the immunogen of Examples 1, 2 and 3 in the presence of Freund's complete adjuvant, the second one carried out 3 weeks after the first day with 20 μg of emulsion preparation in presence of incomplete Freund's adjuvant. 15 days after the booster injection, blood samples were taken intracardially. The anti-Tat antibodies were determined in the sera by Elisa. The optical density was measured at 480 nm.

The results obtained, summarized in Table 2, show that all preparations of Tat are immunogenic to varying degrees, with the Tat-TT conjugate being treated for a short time (3 minutes) with laglutaraldehyde, which was toxic and killed the patients. mouse in 24 hours (TT too weakly inactivated). It should be noted that sera from non-immunized mice respond below 0.200 (D.O) to native Tat as well as to Tat-toxoid. Moreover, these results show that the native Tat is recognized by the antibodies in the same way as the toxoid, which confirms their equivalent antigenicity and still justifies the use of Tat-toxoid to immunize. - 19 - 01 Ο 6 î 5 TABLE 2

Preparations Native Anti-Tat Antibody Rates Optical Density (OD) Anti-Toxoid Tat Levels (3 Days) Optical Density (OD) Tat-toxoid (1 day) 0.320 0.331 Tat-toxoid (3 days) 0.465 0.494 Tat-toxoid ( 5 days) 0.998 0.901 Tat-toxoid (9 days) 0.807 0.780 Tat -polan (1 min;) 0.026 M 0.718 0.706 Tat -polan (15 min;) 0.026 M 1.564 1.452 Tat -polan (60 min;) 0.0026 M 1,065 1,113 Tat -polan (3h;) 0,0026 M 0,194 0,210 Conjugate Tat-TT (3 min, 0.0026M) Toxic Toxic Conjugate Tat-TT (15 min, 0.0026M) 1,987 1,897 conjugate Tat-TT (6 min; 0.026 M) 0.824 0.795 Tat-TT conjugate (15 min, 0.026M) 1.642 1.556 EXAMPLE 5 Acute toxicity

The toxicity of the Tat-toxoid and Tat-polan preparations was determined by subcutaneous injection into the Swiss 18-20g mouse. The preparations of Tat-toxoid (7 days) and Tat-polan (15 min, 0.026 M) were administered at a dose of 100 μg per mouse. The animals were observed for 7 days.

No overt signs of toxicity were observed. The weight of the animals continued to grow. Macroscopic examination of the organs at autopsy did not reveal any abnormalities. EXAMPLE 6 Anti-Tat Antibodies

Anti-Tat antibodies were prepared as follows: The IgG fragment was isolated on a G protein column from mouse sera immunized with Tat-polan. The F (ab ') 2 fragments were also prepared from the isolated IgG fraction by pepsic digestion. The immunological reactivity of these fractions was verified by Elisa. EXAMPLE 7: Biological Inactivity of Tat-Toxoid

The various inactivated Tat-toxoids 1, 3, 5, and 9 days of Example 1 are tested in a reporter gene expression activity test.

Chloramphenicol-Acetyl-Transferase (CAT) dependent on "Long Terminal

Repeat "(LTR) of HIV-1.

The principle of this assay is as follows: Line cells containing the CAT gene under control of the HIV LTR are placed in contact with the native Tat or toxoids for 6 hours. After 24h culture, tescellules are lysed and the amount of CAT produced is measured by a CAT activity test which measures the percentage of acetyl-CoA hooked to chloramphenicol. If the Tat molecule is active, there will be a high percentage of acetylated chloramphenicol, otherwise this percentage will be low. The culture of these adherent cells is standard in RPMI medium, 10% FCS. Laconcentration of Tat added to have a dose effect ranges from 1 μg to 10 μg per ml of supernatant.

The results presented here show that Tat-toxoid is completely inactivated after 3 days, with low residual activity at 1 day.

Preparations Acetylation of chloramphenicol (%) Native Tat 100 Tat-toxoid (1 day) 18 Tat-toxoid (3 days) 3 Tat-toxoid (5 days) 2 Tat-toxoid (9 days) 3 EXAMPLE 8: Absence of pathogenic effects of Tat-toxoid a) Absence of immunosuppression of T cells in the presence of

Tat-toxoid (contrary to experiments 1 and 2). The effect of Tat-toxoid (5 days) on activated T cells of peripheral blood was investigated. Immunomagnetically isolated mononuclear cells (PMBC) and T cells (HLA 35 DR-) were activated by anti-CD3 antibodies in the presence or absence of immunogenic compounds of Example 1. After 5 days of culture, cell proliferation was measured by incorporation of H3 thymidine. The results showed that, unlike native Tat, the Tat-toxoid did not inhibit the proliferation of HLA DR-T cells. Thus, at the dose of 5 μg / ml in the culture, in the presence of

Tat-toxoid, the proliferation of cells was identical to that of control. b) Absence of inhibition of the effect of interferon α on macrophages in the presence of Tat-toxoid: The effect of Tat-toxoid on the antiviral action of interferon a was measured by the conventional biological test using the measurement of the cytokathogenicity of the VSV virus on MDBK cells.

Effect of Tat-toxoid on exogenous interferon

In contrast to native Tat, equivalent doses of Tat-toxoid from Example 1 failed to inhibit the anti-viral potency of exogenous interferon. In a representative experiment, the protective effect of interferon apparent up to 1/4800 dilution (corresponding to 150 IU) is maintained in the presence of 50 μg of Tat-toxoid, whereas it is decreased to 1/300 with Native Tat. At the 4800 dilution of the exogenous interferon, the titre of VSV is maintained at the level of the 10 -8 control (interferon samples with Tat-toxoid) while it increases to 10 -5 (interferon samples in the presence of Tat). . EXAMPLE 9 Protective Role of Anti-Tat-Toxoid Antibodies Facing the Effects of Native Tat

Anti-Tat-toxoid antibodies of experiment 1 (3-day treatment with formaldehyde) were prepared in hyperimmune horses. From these antibodies, F (ab *) 2 fragments were also prepared according to the procedure described in Example 6.

Antibodies, like F (ab ') 2 fragments, inhibit the different biological activities of native Tat in the various tests: transactivation of HIV LTR (CAT assay) (see Example 7), immunosuppression (see experiment 1), inhibition of the effect of exogenous interferon on culture (see experiment 2). Experiments 1, 2, and Example 7 were taken up with native Tat and preincubated or not this native Tat 1h at 37 ° with the antibodies or fragments FCab, at a dose of 40 μg antibody for 1 μg of native Tat. The results showed that antibodies inhibit the action of

Native Tat. EXAMPLE 10: Systematic recovery of cell proliferation by virtue of the combination of anti-Tat antibodies and anti-interferon antibodies: synergy of anti-Tat and anti-interferon a antibodies.

It has been observed that PBLs (peripheral blood mononuclear cells) of healthy subjects infected in vitro with HIV-1 and cultured 6 days exerted an immunosuppressive activity.

Indeed, if one adds such cells infected for 6 days, or not infected) irradiated in proportion of 1 for 5 to autologous cells activated by the protein Staphylococcus Enterotoxin B (SEB), one observes at the end of 4 days that the proliferation autologous cells is reduced by 80% compared to controls (uninfected cells).

In this model, if infected cells are added to the culture invitro anti-interferon antibodies, these cells then lose their suppressor effect in 50% of subjects. In 20% of cases, the cellulessuppressives lose 60% of their suppressive effect, and in 30% this suppressor is significantly maintained.

If anti-interferon antibodies are added to anti-Tat antibodies (monoclonal antibodies described in experiment 1, or polyclonal horses of Example 9), it is in 100% of cases that cells infected in vitro lose their suppressor effects.

These experiments show the combined toxic role of Tat and interferon-a in HIV-1 induced immunosuppression and that a combination of anti-interferon and anti-Tat antibodies restore normal immunity. EXAMPLE 11 Preparation of Immunogenic Nef Protein (Nef-toxoid)

Inactivation of the Nef protein in the presence of formaldehyde. To a solution of Nef protein (1 g / ml) in 70 mM disodium phosphate, pH 8.0, formaldehyde is added to the final concentration of 33 mM.

The mixture is incubated at 37 ° C for 1, 3, 5, 7 and 9 days. At the end of each of the incubation periods, a sample is taken and reaction with formaldehyde blocked by the addition of glycine to the final 100 mM concentration.

Each sample is dialyzed for 1 night at 4 ° C against 100 times its volume of PBS (phosphate buffered saline), and the Nef-toxoid isolated. EXAMPLE 12 Preparation of the immunogenic Nef protein (Nef-polan)

Inactivation of the Nef protein in the presence of glutaraldehyde. To a solution of Nef (1 mg / ml) in disodium phosphate 70 mM, pH 8.2, is added glutaraldehyde at the final concentration, that is to say 0.026 M, or 0.0026 M. The reaction with glutaraldehyde is allowed to dry again during Various times varying from 1 min to 3 h. After the selected reaction time, at the laboratory temperature, the reaction is blocked by adding glycine to the final concentration of 100 mM. the various samples are dialyzed for 16 hours at 4 ° C, compared with 100 times their volume of PBS, and the isolated Nef-polan. EXAMPLE 13 Immunogenic Potency and Antigenicity of Nef-Toxoids

Immunogenic nef-toxoid in mice: Antibodies by Elisa.

The immunogenicity of the various inactivated Nef preparations, either by formaldehyde (Nef-toxoid) or by glutaraldehyde (Nef-polan), ie the immunogenic compounds of Examples 12 and 13, was determined in mice. . 24 010615

Swiss mice of 18-20 g were immunized by 2 subcutaneous injections of the immunogen of Examples 12 and 13 in the presence of Freund's adjuvant, the second one performed 3 weeks after the first, with 20 μg of emulsion preparation in the presence Incomplete adjuvant of Freund. 15 days after the booster injection, the blood samples were taken intracardially. Anti-Nef antibodies were determined in sera by Elisa. The optical density was measured at 490 nm.

The results obtained, summarized in the table below, show that all Nef preparations are immunogenic to varying degrees. It should be noted that sera from unimmunized mice give a response level of less than 0.2 (in OD) in these Elisa assays.

These results show that the native Nef protein is recognized by the antibodies in the same way as the toxoid, which confirms their equivalent antigenicity and still justifies the use of Nef-toxoid to immunize.

Preparations Native Anti-Nef Antibody Rates Optical Density (OD) Anti-Toxoid Antibody Rate (3 days) Optical Density (OD) Nef-toxoid (1 day) 0.56 0.5 Nef-toxoid (3 days) 0, 78 0.8 Nef-toxoid (5 days) 0.99 1.01 Nef-toxoid (9 days) 0.65 0.75 Nef-polan (1 min .;) 0.026 M 1.12 0.98 Nef-polan ( 15 min;) 0.026 M 1.69 1.56 Nef -polan (60 min;) 0.0026 M 1.31 1.41 Nef -polan (3h;) 0.0026 M 0.35 0.41 - 25 - 010615

SEQUENCE LIST (1) GENERAL INFORMATION: (i) DEPOSITOR:

(A) NAME: NEOVACS

(B) STREET: 117, rue Vieille du Temple (C) CITY: PARIS

(E) COUNTRY: FRANCE (F) POSTAL CODE: 75003 (ii) TITLE OF THE INVENTION: New immunogens, new antibodies, process for their preparation and pharmaceutical compositions containing them. (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: (A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) ) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEQUENCE ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid · (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) MOLECULE TYPE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1:

His Gin Ser Val Ser Ser Lys Pro Gin Ser Ser Gin Pro Arg Gly Asp 1 5 10 15

Claims (21)

- 26 - 010615 CLAIMS 1. An immunogenic compound that can be administered to humans because it lacks detoxicity, characterized in that it derives from a regulatory protein of a virusVIH-1, HIV-2, HTLV-1 or HTLV-2, by chemical treatment with using a coupling agent such as an aldehyde, or a carrier protein activated by a pre-treatment with an aldehyde, allowing it to be recognized by antibodies directed against said regulatory protein, and to retain immunogenic properties sufficient to create neutralizing antibodies that omit said native protein, while having lost at least 50%, toxic biological properties of said native protein. 2. Immunogenic compound according to claim 1, characterized in that it derives from one of the following regulatory proteins of the viruses V1H-1 or V1H-2, Nef, Tat, Vif or Rev. 3. Immunogenic compound according to claim 2, characterized in that it derives from the Tat protein An immunogenic compound according to claim 1, which is derived from a Tax protein, a HTLV-1 or HTLV-2 virus. 5. Pharmaceutical composition characterized in that it comprises an immunogenic compound as defined in one of claims 1 to 4. 6. An immunogenic compound as defined in one of Claims 1 to 4 for use in a method of treating human or animal body. 7. Process for the preparation of an immunogenic compound as defined in one of claims 1 to 4, characterized in that a regulatory protein of an HIV-1, HIV-2, HTLV-1 or HTLV-1 virus is subjected. 2 to a chemical treatment with an aldehyde, then selects and purifies the compound awaited. 8. Process according to claim 7, characterized in that the chemical treatment comprises a treatment with an aldehyde followed by a coupling to a carrier protein. . 9. Process for the preparation of an anti-protein antibody which is a regulator of a virus V1H-1, HIV-2, HTLV-1 or HTLV-2, characterized in that a mammal is immunized with an immunogenic compound as defined in one of claims 1 to 4. 10. Process for the preparation of an anti-protein antibody which is a regulation of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus characterized in that the cloning of B cells transformed with the Epstein-1 virus is carried out. Barr, then recovering the expected antibodies secreted by said B-transformed lymphocytes. 11. Process for the preparation of an anti-HIV protein of an HIV-1 or HIV-2 virus, characterized in that cloned E-mutated B cells transformed with the Epstein-Barr virus, then recovering the expected antibodies secreted by said transformed B lymphocytes. 12. Process for the preparation of an anti-protein Tax antibody of a HTLV-1 or HTLV-2 virus, characterized in that the Epstein-Barr virus-transformed B cells are cloned and then the expected antibodies secreted by said Epstein-Barr virus are cloned. transformed B cells. 13. Process for the preparation of an anti-protein antibody which is a regulation of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, characterized in that said antibody is prepared by genetic recombination, starting from a librairiede phages. 14. Process for the preparation of an F (ab ') 2 or F (ab) fragment of an antibody as prepared in one of Claims 9 to 13, characterized in that the enzymatic digestion of a such an antibody. 15. An anti-regulatory protein antibody of an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus obtained by immunization of a mammal with an immunogenic component as defined in one of the following: Claims 1 to 4. 16. An F (ab ') 2 or F (ab) fragment of an antibody as defined in claim 15. 17. A pharmaceutical composition characterized in that it comprises an antibody as defined in one of claims 15 and 16. 18. Pharmaceutical composition characterized in that it comprises an immunogenic compound as defined in one of claims 1 to 5, combined with a Gag, Pol or Env protein of an HIV-1, HIV-1, HTLV-1 J virus. 5 or HTLV-2. "19. Pharmaceutical composition characterized in that it comprises an immunogenic compound as defined in one of claims 1 to 5, combined with a gp120 / gp160 protein, native or inactivated by physical, chemical, genetic or immunological treatment or to a peptide fraction of this protein, modified or not. 20. An immunogenic compound having the following formula HQVSLSKQPTSQPRGD or MEPVDPRLEPWKHPG or corresponding to the HIV1 or KIV2 Tnnatifatif after inactivation with an aldehyde. 21. A vaccine composition comprising an immunogenic compound as defined in one of claims 1 to 4. an immunogenic compound which is a cytokine in a form different from its native and inactivated form or an inactive analogue or a fragmentinactive or inactivated cytokine. 22. A composition comprising - an anti-HIV protein regulating antibody, HIV-1, HIV-2, HTLV-1 or HTLV-2 - an anti-cytokine antibody such as a human anti-interferon antibody.