Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-05-15
2001-03-13
Stucker, Jeffrey (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S207100, C514S002600, C530S324000, C530S350000, C530S826000
Reexamination Certificate
active
06200575
ABSTRACT:
The present invention relates to new retroviral immunogens which use retroviral regulatory proteins, the essential biological properties of which will have been inactivated beforehand such that their immunogenic properties are retained or increased, a process for their preparation and pharmaceutical compositions comprising them. These new “inactivated” immunogens can be used to induce active immunization in humans which is capable of preventing or correcting the deregulatory effects which the native proteins from which they are produced may help to produce.
The present invention also relates to new antibodies obtained by using these “inactivated” immunogens, processes for their preparation and pharmaceutical compositions for passive immunization which comprise them.
The Tat molecule is an HIV regulatory protein which is not found in the viral particle but is coded by the HIV-1 genome. Inside infected cells, this protein coded by the proviral DNA plays a transactivating role on viral or cell genes. However, this genetic regulatory protein can also be found outside cells in the circulating extracellular medium, excreted within the debris of dead cells in the native or fragmented state or by a secretion process. Its presence in the circulating medium explains the existence of anti-Tat antibodies detectable in some seropositive subjects. In this extracellular context of Tat, the terminal C segment which carries the RGD residues recognized by the integrins of the cell surface and the base region of the molecule (residues 45-70) enable it, as do bacterial toxins, to act on the non-infected cells of different tissues. The circulating Tat protein thus acts like a true viral toxin, can exert harmful effects on endothelial cells and, in combination with the growth factor BFGF, can contribute to the neoangiogenesis of Kaposi's sarcoma, which characterizes the AIDS disease. The circulating Tat protein can also aggravate the immunosuppression which becomes established progressively with AIDS, either by direct immunosuppressive action on the T cells or by helping to deregulate the production of interferon-&agr; by the cells presenting the antigen, called APC (macrophages or dendrite cells).
Acquired immunodeficiency syndrome (AIDS) is defined clinically by opportunistic diseases due to immunosuppression or by Kaposi's sarcoma. Acquired immunosuppression, established progressively in the course of HIV infection, manifests itself biologically by the loss of immunological reactivity of T cells (cytostasis and the reduction in the production of IL2), after stimulation in the first instance by memory antigens, and then by alloantigens, and finally by mitogens (PHA). This immunosuppression is associated with excessive production of interferon-&agr; and -&ggr;.
The cytopathogenic mechanisms which induce immunosuppression are complex and involve various factors of viral origin, which act either directly on the immunity cells, T lymphocytes and APC, or indirectly via the cytokine system. The envelope protein gp120 which is carried by the HIV-1 particle and of which the extracellular presence is measured by the serum viral charge can thus induce anergy of T cells of phenotype CD4 directly. On their part, the HIV regulatory proteins, in particular Tat in its extracellular configuration of circulating viral toxin, also seem to induce a direct immunosuppression of T cells or other pathogenic effects, for example by the fact that in vitro T cells activated by a memory antigen or by anti-CD3 antibodies no longer proliferate in the presence of the Tat molecule. In addition, the circulating Tat protein seems to facilitate excessive production by the APC of interferon-&agr;, a cytostatic and apoptogenic cytokine which is capable of amplifying the immunosuppresion and apoptosis observed with the AIDS disease. In fact, the secretion of interferon-&agr; by macrophages no longer seems halted in the presence of Tat by a retroregulation (feedback), which in the normal state controls this production of interferon-&agr; (refractory period).
Kaposi's sarcoma manifests itself clinically by the appearance of vascular nodules representing neoangiogenesis starting from endothelial cells. These cells, which have been activated by inflammatory processes generating the production of cytokines (interferon-&ggr;, IL1 and IL6), produce BFGF (basic fibroblastic growth factor) and multiply. The proliferation of activated endothelial cells in vitro is increased by the presence in the medium of the Tat protein. The anti-Tat antibodies block the proliferative effects of the Tat protein in vitro. The action of Tat on the endothelial cells which carry adhesin from the family of integrins on their surface is explained by the presence of the RGD sequence recognized by these molecules.
The neoangiogenesis which underlies Kaposi's sarcoma in vivo was thus said to be promoted by the Tat regulatory protein in its extracellular configuration, which could be recognized, thanks to its terminal C fragment containing the RGD sequence, by the integrins of endothelial cells. Furthermore, the Tat may also act by its base region rich in residues K and R, and consequently be capable of bonding to heparin sulphate of the extracellular matrix, which concentrates the growth factor BFGF. The proliferation of endothelial cells induced by the growth factors is thus increased by the presence of Tat and generates neoangiogenesis. This effect on the growth of endothelial cells is reduced in vitro by the action of anti-Tat antibodies.
It thus appears desirable to block the harmful activity of retroviral regulatory proteins, in particular of Tat circulating in the extracellular media (blood, lymph, interstitial media . . . ), the true viral toxins.
As regards the HIV viruses, up to the present time attempts at vaccination have been made with the aid of structure proteins or fragments of structure proteins of these viruses, but never with the aid of regulatory proteins or fragments of regulatory proteins of these viruses.
Thus, it has been found, surprisingly, that like the bacterial toxins (tetanic, diphtheric or botulic), the toxic activity of which is neutralized by specific antibodies, the harmful effects of regulatory viral proteins, and in particular of Tat, a true toxin of HIV in its extracellular configuration—which these exert by immunosuppression of T cells, by deregulation of the production of interferon-&agr; by the APC or by the neoangiogenesis which underlies Kaposi's sarcoma—are abolished in the presence of specific anti-Tat antibodies, as will be seen below in the experimental part.
The same applies to other regulatory proteins of viruses such as HIV-1, HIV-2, HTLV-1 or HTLV-2. It would thus be desirable to have available immunogens which can be administered to humans and are capable of producing such antibodies, and also to have available such antibodies, for both curative and preventive purposes. In fact, the compounds of the prior art used as immunogens may be toxic to humans (see, for example, WO-A-9118454), in particular those containing base regions. These are non-modified native fragments of Tat, Nef or Rev, in contrast to the present invention, which is based on the inactivation of these proteins or protein fragments.
The present Application thus relates to immunogenic compounds which can be administered to humans, since they are non-toxic, characterized in that they are derived from an HIV-1, HIV-2, HTLV-1 or HTLV-2 virus regulatory protein by chemical treatment with the aid of a coupling agent such as an aldehyde, or from a carrier protein activated by pretreatment with the aid of an aldehyde, preferably formaldehyde or glutaraldehyde, enabling them to be recognized by antibodies to the said regulatory protein, and to retain sufficient immunogenic properties to create antibodies which neutralize or block the said native protein, while having lost at least 50%, in particular at least 80%, more particularly 95%, of the toxic biological properties of the said native protein.
These compounds, by analogy to bacteri
Bizzini Bernard
Zagury Daniel
Zagury Jean-François
Browdy and Neimark
NEOVACS
Stucker Jeffrey
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