Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-02-16
2004-03-02
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S204100, C435S007100
Reexamination Certificate
active
06699481
ABSTRACT:
BACKGROUND OF THE INVENTION
Herpes Simplex Virus (HSV) entry into mammalian cells is a complex process requiring interaction of multiple viral envelope proteins with several host cell membrane receptors. Virion glycoproteins, including gB and gC, appear to mediate initial virus binding to cell surface heparan sulfate glycosaminoglycans. However, this attachment is not sufficient to mediate entry, since some cell types such as swine testis (ST) or Chinese hamster ovary (CHO) cells bind HSV but are not susceptible to infection. Entry of virus into cells requires binding of yet other glycoprotein(s) to one or more cell surface receptors. Glycoproteins gD, gB, and the complex formed by gH and gL are believed to act separately or in concert to promote pH-independent fusion of the viral envelope with the cellular membrane.
Herpesvirus entry mediator protein, a cellular protein designated as HveA (also designated HVEM in some literature sources), is a member of the tumor necrosis factor receptor (TNFR) superfamily. This protein has been described as a target cellular receptor capable of mediating post-attachment entry of HSV into host cells. HveA was identified by expression cloning of several HeLa cell products which, when expressed in otherwise nonpermissive CHO cells rendered the CHO cells susceptible to entry by many HSV strains. A recombinant form of HveA (HveA:Fc) blocked HSV-1 entry into CHO cells which were stably transformed to express HveA. Additionally, antibodies to HveA inhibited HSV-1 entry into some susceptible cell types. Furthermore, a recent study suggests that HveA participates not only in entry of free virus into cells but also in cell-to-cell spread of infection. These studies suggest that HveA mediates virus entry into mammalian cells (Terry-Allison et al., 1998, J. Virol. 72:5802-5810; Montgomery et al., 1996, Cell 87:427-436). The HSV protein which mediates HSV binding with HveA has been shown to be glycoprotein D (gD), which binds with a soluble form of HveA, designated HveA(200t) (Whitbeck et al., 1997, J. Virol. 71:6083-6093) in a specific and saturable manner and inhibits binding of HSV to HveA-expressing cells (Nicola et al., 1997. J. Virol. 71:2940-2946; Nicola et al., 1996, J. Virol. 70:3815-3822; Sodora et al., 1991, J. Virol. 63:5184-5193; Sodora et al., 1991, J. Virol. 65:4424-4431; Tal-Singer et al., 1994, Virology 202:1050-1053; Whitbeck et al., 1997, J. Virol. 71:6083-6093).
Several studies suggest that HveA is involved in activation of the host immune response. For example, HveA is predominantly expressed in lymphocyte-rich tissues, and binding of HveA to several members of the TNFR-associated factor (TRAF) family of proteins activates transcriptional regulators such as nuclear factor &kgr;B (NF-&kgr;B), Jun N-terminal kinase, and AP-1. Moreover, HveA binds to lymphotoxin-alpha (LT-&agr;) and to a membrane-associated protein designated LIGHT. Lymphotoxin-alpha is a cytokine that is sometimes designated tumor necrosis factor &bgr; (TNF&bgr;) (Imboden, 1997, In: Medical Immunology, 9th ed., pp. 150-152, Stites et al., eds., Appleton and Lange Press, Stamford, Conn.). The LT-&agr; cytokine molecule mediates an influx of effector cells such as natural killer cells, large granular lymphocytes, and eosinophils which, in turn, mediate antibody-dependent cellular cytotoxicity (ADCC) activity as described in Gillies et al. (1991, Hybridoma 10:347-356), such that binding of LT-&agr; to HveA, a member of the TNFR family, is associated with these immune processes.
LIGHT is a lymphotoxin homolog, and is expressed by T cells upon induction with phorbol 12-myristate 13-acetate (PMA) and a Ca
2+
ionophore (Mauri et al., 1998, Immunity 8:21-30; Marsters et al., 1997, J. Biol. Chem. 272:14029-14032; Hsu et al., 1997, J. Biol. Chem. 272:13471-13474).
Interestingly, LIGHT competes with HSV gD for binding to HveA, suggesting that gD can modify HveA signaling activities during entry or egress of HSV, thus modulating the immune response of the host. Indeed, a recent study using recombinant proteins expressed in the baculovirus system, demonstrated that among HSV glycoproteins involved in entry, only gD was capable of binding directly with HveA (Whitbeck et al., 1997, J. Virol. 71:6083-6093). Further, Whitbeck et al., supra, demonstrated that fluid-phase gD bound directly and in a specific and saturable manner with HveA at a 2:1 (HveA:gD) molar ratio. This interaction was dependent on the native conformation, but not on N-glycosylation, of gD.
Previous studies implicated gD as an HSV receptor-binding protein. For example, soluble forms of gD ectodomain blocked virus infection of cells as well as expression of gD at the cell surface (i.e., gD-mediated interference). Moreover, UV-inactivated wild type HSV, but not UV-inactivated gD-deficient HSV, were able to inhibit infection (Johnson et al., 1989, J. Virol. 63:819-827). However, three infectious strains of HSV (Rid1, Rid 2 and ANG) which contain point mutations in the gD ectodomain, failed to bind to HveA, suggesting that proteins other than HveA may have a role in HSV entry into cells. Subsequently, two additional cell surface proteins, both members of the immunoglobulin (Ig) superfamily, have been identified which facilitate HSV entry into CHO cells. These proteins are the Poliovirus Receptor Related Protein 1 (HveC, formerly Prr1) and Poliovirus Receptor Related Protein 2 (HveB, formerly Prr2). Moreover, HveA is not the sole receptor for gD; rather, gD has also been identified as the viral ligand for HveC. In contrast to HveB, which enhances entry of a limited number of HSV mutant strains, HveC mediates entry of several alphaherpesviruses (HSV-1, HSV-2 PRV and BHV-1) into cells.
Given the frequency and severity of HSV infections in humans, there is a need to develop compounds which inhibit HSV replication. To date, anti-HSV therapeutics have been directed primarily at inhibiting HSV DNA replication, an event which occurs following entry of the virus into cells. Inhibition of entry of virus into cells prior to DNA replication has significant advantages over therapies directed at events subsequent to virus entry, in that such inhibition guarantees that no progeny virus will be generated (because the virus is rendered incapable of infecting the cell). The present invention provides compounds which inhibit entry of HSV into cells and also provides methods of making such compounds and of using them as inhibitors of HSV entry into cells.
BRIEF SUMMARY OF THE INVENTION
The invention relates to cyclic peptides that bind with HveA and inhibit interaction of HveA with its ligands. Binding of HveA with one or more of the peptides inhibits interaction of the receptor with HSV gD such that virus entry into cells is inhibited. Furthermore, binding of HveA with one or more of the peptides inhibits HveA interaction with LT-&agr;.
Thus, the invention includes a cyclic peptide selected from the group consisting of BP-1, BP-2, and mutants, homologs, derivatives, and variants thereof, wherein the peptide is capable of binding with HveA. In one aspect, the peptide inhibits binding of herpes simplex virus gD with HveA. In another aspect, the peptide is BP-1 and it inhibits binding of lymphotoxin-alpha (LT-&agr;) with HveA. In a further aspect, the peptide inhibits entry of a HSV e.g. HSV-1 or HSV-2, into a cell.
The invention also includes an isolated nucleic acid encoding a cyclic peptide selected from the group consisting of BP-1, BP-2, and mutants, homologs, derivatives, and variants thereof, wherein the peptide binds HveA. In one aspect, the peptide inhibits binding of HSV gD with HveA. In another aspect, the peptide is BP-1, and the peptide inhibits binding of LT-&agr; with HveA.
The invention further includes a method of inhibiting the ability of HveA to bind with HSV gD. The method comprises contacting HveA with a peptide selected from the group consisting of BP-1, BP-2, and mutants, homologs, derivatives, and variants thereof. In one aspect, the peptide is added to a preparation of HSV gD and HveA.
The invention includes a
Cohen Gary H.
Eisenberg Roselyn J.
Lambris John D.
Montgomery Rebecca I.
Sarrias Maria Rosa
Morgan & Lewis & Bockius, LLP
Salimi Ali R.
The Trustees of the University of Pennsylvania
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