WO2001013910A2 - Pharmaceutical preparations for the inhibition of herpes simplex virus 1 entry - Google Patents
Pharmaceutical preparations for the inhibition of herpes simplex virus 1 entry Download PDFInfo
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- WO2001013910A2 WO2001013910A2 PCT/US2000/023288 US0023288W WO0113910A2 WO 2001013910 A2 WO2001013910 A2 WO 2001013910A2 US 0023288 W US0023288 W US 0023288W WO 0113910 A2 WO0113910 A2 WO 0113910A2
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- ost
- hsv
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- polysaccharide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
Definitions
- the present invention relates generally to the field of virology.
- the present invention relates to methods and pharmaceutical preparations for inhibiting viral penetration and entry of herpes simplex virus 1 into mammalian cells via heparan sulfate binding.
- Heparan sulfate is widely expressed in animal and human tissues and has diverse roles in development, differentiation, and homeostasis.
- HS and other glycosaminoglycans are unbranched polymers covalently attached to the protein cores of proteoglycans, which are ubiquitously expressed as integral membrane proteins, glycerol phosphatidyl inositol-linked membrane proteins, and proteins of the extracellular matrix.
- the HS polymer is assembled by sequential addition of D-glucuronic acid (GlcA) alternating with N-acetyl glucosamine
- GlcNAc glucosamine
- IdoA iduronic acid
- HS protein binding to HS may serve to sequester the protein at a particular site or to activate the protein. For example, the binding of antithrombin to a specific pentasaccharide sequence in HS results in activation of its anticoagulant activity (reviewed by Lindahl et al., 1998; Rosenberg et al., 1997).
- HSV Herpes simplex viruses
- HSV-1 and HSV-2 establish latent infections in neurons of peripheral ganglia and may reactivate to cause recurrent lesions.
- HSV-1 and HSV-2 establish latent infections in neurons of peripheral ganglia and may reactivate to cause recurrent lesions.
- the virus spreads to the central nervous system to cause meningitis or encephalitis (reviewed by Spear, 1993).
- viruses including the human herpesviruses of the neurotropic alpha herpesvirus subfamily, use sites on HS as receptors for binding to cells (Byrnes and Griffin, 1998; Chung et al., 1998; Compton et al., 1993; Jackson et al., 1996; Shieh et al., 1992; Summerford and Samulski, 1998). Viral entry may require interactions with other cell surface receptors as well (Geraghty et al., 1998; Montgomery et al., 1996; Summerford et al., 1999; Warner et al., 1998).
- Infection involves (1) virus attachment to the cell surface membrane, followed by (2) virus penetration and entry into the cells. These two steps can be experimentally dissociated.
- the virus binds to cells through interactions of cell surface HS with viral envelope glycoproteins gB and/or gC (reviewed by Spear, 1993).
- viral envelope glycoproteins gB and/or gC are resistant to viral entry, even though the viral binding to these cells appears normal.
- a third viral envelope glycoprotein, gD interacts with one of multiple specific receptors, resulting in viral entry by fusion of the virion envelope with the target cell membrane. This fusion reaction requires the concerted action of three additional viral glycoproteins, gB, gH and gL (reviewed by Spear, 1993), and appears to be triggered by the binding of gD to its cognate receptors.
- the human gD-binding receptors that have been identified include a member of the TNF receptor family, designated HVEM (Montgomery et al., 1996; Whitbeck et al., 1997) or herpesvirus entry protein A (HveA), and officially named TNFRSF14, and two members of the immunoglobulin superfamily (Geraghty et al., 1998; Krummenacher et al., 1998; Warner et al., 1998).
- HveC Garnier et al.
- HveB Warner et al, 1998) or nectin 1 and nectin 2 (Takahashi et al., 1999), respectively.
- HveA and HveC serve as gD-binding entry receptors for wild-type HSV-1 and HSV-2 strains
- HveB serves as an entry receptor for only a subset of HSV strains, (Geraghty et al., 1998; Montgomery et al., 1996; Warner et al., 1998).
- These gD-binding cell surface receptors are expressed at different levels in various human tissues and cell lines, suggesting a specific susceptibility to HSV in certain tissues.
- 3-OSTs heparan sulfate 3-O-sulfotransferases
- 3-OST-3 enzymes are present in two highly similar forms (3-OST-3A and 3-OST-3B).
- 3-OST-3A and 3-OST-3B different 3-OST isoforms sulfate glucosamine residues with different saccharide sequences around the modification site.
- 3-OST-l, 3-OST-2, and 3-OST-3 generate different saccharide sequences (Liu et al., 1999a).
- 3-OST-3A and 3- OST-3B have been shown to sulfate identical saccharide sequences.
- the differences in sulfation of saccharide sequences are thought to correlate with differences in function.
- heparan sulfates which have been 3-O-sulfated by 3-OST-l, possess anticoagulant activity
- heparan sulfates which have been 3-O-sulfated by 3-OST-2 and 3-OST-3A do not possess anticoagulant activity.
- the present invention depends, in part, on the discovery that specific saccharide sequences in HS can serve as a gD-binding receptor for HSV-1 entry into cells.
- the present invention provides pharmaceutical preparations for inhibiting HSV-1 infection, and methods of inhibiting HSV-1 infection in a mammal using the pharmaceutical preparations of the invention.
- the present invention provides a pharmaceutical preparation for inhibiting HSV-1 infection in a mammal, comprising a polysaccharide preparation enriched for 3-O-sulfated glucosamine residues (GlcN3S and/or GlcN3S6S).
- the polysaccharide preparation is enriched for polysaccharide sequences capable of specific binding with HSV-1 gD viral glycoprotein, for example, IdoA2S-GlcN3S6S and/or IdoA2S-GlcN3S.
- the polysaccharide preparation is enriched for a polysaccharide sequence that includes the disaccharide IdoA2S-GlcNH 3S6S.
- the polysaccharide preparation is enriched for the trisaccharide GlcNS-IdoA2S- GlcNH 2 3S6S, the tetrasaccharide UA2S-GlcNS-IdoA2S-GlcNH 2 3S6S the pentasaccharide GlcNS6S-UA2S-GlcNS-IdoA2S-GlcNH 2 3S6S, the hexasaccharide UA-GlcNS6S-UA2S-GlcNS- IdoA2S-GlcNH 2 3S6S, heptasaccharideGlcNAc-UA-GlcNS6S-UA2S-GlcNS-IdoA2S- Gl
- the polysaccharide preparation is enriched for the octasaccharide UA-GlcNAc-UA-GlcNS6S-UA2S-GlcNS-IdoA2S-GlcNH 2 3S6S.
- the polysaccharide sequence specifically binds to HSV-1 gD with a high degree of affinity.
- the polysaccharide preparation has a binding affinity (expressed in terms of the disassociation constant (Kd) and measured by affinity co-electrophoresis as described herein) of less than 30 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of less than 20 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of less than 10 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of approximately 2 ⁇ M-10 ⁇ M for HSV-1 viral protein gD.
- the present invention provides a pharmaceutical preparation for inhibiting HSV-1 infection in a mammal, including a polysaccharide that has been enriched for saccharide structures modified by a 3-OST-3 enzyme.
- the 3-OST-3 enzyme may be human 3-OST-3A or 3-OST-3B, or murine 3-OST-3B.
- Such enriched preparations may be produced by contacting a polysaccharide with a 3-OST-3 enzyme in the presence of a sulfate donor, under conditions suitable for 3-0 sulfation of the polysaccharide by a 3-OST-3.
- the polysaccharide preparation includes 3-O-sulfated heparan sulfate.
- the 3-O-sulfated heparan sulfate includes disaccharide sequences of IdoA2S-GlcN3S6S and/or IdoA2S-GlcN3S.
- the enriched preparation maybe further enriched by purification of the 3-OST-3 modified polysaccharide by a purification technique that is known in the art, for example HPLC.
- the pharmaceutical preparations of the present invention include a pharmaceutically acceptable carrier.
- the carrier may be a lotion, cream, jelly, liniment, ointment, salve, oil, foam, gel, wash, suppository, slow-releasing polymer, or coating.
- the preparation of the present invention may include at least one skin penetrating enhancer.
- the skin penetrating enhancer may be dimethylsulfoxide (DMSO), propylene glycol, isopropanol, ethanol, oleic acid, and/or N- methylpyrrolidone.
- the present invention provides for methods of inhibiting HSV-1 infection in a mammal, using the pharmaceutical preparations of the invention.
- Figure 1 depicts a sequence alignment of the amino acid sequences of m3-OST-3B, h3-OST-3B, and the sulfotransferase domain of h3-OST-3A (amino acids 148-406).
- Figure 2 depicts the IdoA2S-GlcNH2 ⁇ 6S3S substrate specificity of heparan sulfate D- glucosaminyl 3-O-sulfotransferase (3-OST-3).
- the term "enriched" means that the proportion of the polysaccharide structure in a polysaccharide preparation is statistically significantly greater than the proportion of the polysaccharide structure in naturally-occurring, untreated polysaccharide preparation.
- the polysaccharide preparations of the invention are enriched for 3-OST-3-sulfated approximately 10-100 fold. Whereas the percentage of 3-OST-3-sulfated polysaccharide in a typical, unenriched preparation is less that 0.1%, the percentage of 3-OST-3-sulfated polysaccharide in the enriched polysaccharide preparations of the invention is about 5-7 %.
- Heparan sulfate or the abbreviation "HS” means a polysaccharide made up of repeated disaccharide units D-glucuronic acid or L-iduronic acid linked to N-acetyl or N-sulfated D-glucosamine.
- the polysaccharide is modified to a variable extent by sulfation of the 2-O-position of GlcA and IdoA residues, and the 6-O- and 3- O- positions of GlcN residues and acetylation or de-acetylation of the nitrogen of GlcN residues.
- this definition encompasses all of the glycosaminoglycan compounds variously referred to as heparan(s), heparan sulfate(s), heparin(s), heparin sulfate(s), heparitin(s), heparitin sulfate(s), heparanoid(s), heparosan(s).
- the heparan molecules may be pure glycosaminoglycans or can be linked to other molecules, including other polymers such as proteins, and lipids, or small molecules such as biotin.
- Heparan Sulfate D-Glucosaminyl 3-O-Sulfotransferases are responsible for the addition of sulfate groups at the 3-OH position of glucosamine in HS. These enzymes are present as several isoforms expressed from different genes at different levels in various tissues and cells, as described in WO 99/22005.
- the 3-OSTs act to modify HS late in its biosynthesis (reviewed by Lindahl et al., 1998) and each isoform recognizes as substrate glucosamine residues in regions of the HS chain that have specific, but different, prior modifications, including epimerization and sulfation at other nearby positions (Liu et al., 1999). Thus, different 3-OSTs generate different potential protein-binding sites in HS.
- the 3-OST-3 proteins have 3-O-sulfotransferase activity on polysaccharide sequences including the sequences IdoA2S-GlcN and IdoA2S-GlcN6S, and convert these polysaccharide sequences to IdoA2S-GlcN3S or IdoA2S-GlcN3S6S, respectively.
- the term "3- OST-3" is meant to include the 3-OST-3A and 3-OST-3B proteins, allelic or conservative substitution variants, and combinations thereof, and/or recombinantly produced fusions or chimeras thereof, as described in WO 99/22005.
- Figure 1 shows the sequence alignment of m3-OST-3B in comparison with the sequence of h3-OST-3B and h3-OST-3A.
- Single-underlined residues include the predicted membrane- spanning region of m3-OST-3B and the double-underlined residues indicate the potential sites for addition of N-linked glycans.
- the filled triangle marks the beginning of the conserved sulfotransferase domain.
- the position in this conserved domain of the amino acid substitution in h3-OST-3A (K162A) that results in loss of enzymatic activity is indicated by the shading.
- m3-OST-3B is 91% identical to both human 3-OST-3s; the remaining N-terminal portion of m3-OST-3B is 81% identical to that of h3-OST-3B and unrelated to h3-OST-3A.
- FIG. 2 shows the substrate specificities of two 3-OST-3 isoforms.
- Each enzyme isoform transfers sulfate to the 3-OH position of glucosamine residues in different disaccharide and oligosaccharide sequences.
- the arrows indicate the sulfated position within glucosamine by the enzyme, and the 3-O-sulfation is highlighted by rectangles.
- the abbreviated names for the disaccharides are listed under each chemical structure, where R represents H or SO 3 .
- binding affinity means that the binding affinity between a ligand and a receptor is sufficient to initiate a specific biological response.
- the binding affinity is considered specific when the binding affinity of viral entry protein to cell surface polysaccharides is sufficient to induce fusion.
- binding of viral protein gD and 3-OST-3 modified polysaccharides binding is considered non-specific where the disassociation constant K d has a value of greater than 30 ⁇ M as measured by affinity co-electrophoresis.
- binding is considered specific where the disassociation constant Kd has a value of less than 30 ⁇ M as measured by affinity co-electrophoresis.
- substantially pure means a preparation which contains at least 60% (by dry weight) the polysaccharide of interest, exclusive of the weight of other intentionally included compounds.
- the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by dry weight the polysaccharide of interest, exclusive of the weight of other intentionally included compounds.
- Purity can be measured by any appropriate method, e.g., column chromatography, gel electrophoresis, amino acid compositional analysis or HPLC analysis.
- a "substantially pure" preparation means a preparation in which the total dry weight of the polysaccharide of the invention is at least 60% of the total dry weight, exclusive of the weight of other intentionally included compounds.
- the total weight of the polysaccharides of the invention should be at least 75%, more preferably at least 90%, and most preferably at least 99%, of the total dry weight of the preparation, exclusive of the weight of other intentionally included compounds.
- the polysaccharides of the invention are mixed with one or more other compounds (e.g., diluents, detergents, excipients, salts, sugars, lipids) for purposes of administration, stability, storage, and the like, the weight of such other compounds is ignored in the calculation of the purity of the preparation.
- the polysaccharide is a proteoglycan
- the protein component of the proteoglycan is excluded for purposes of calculating purity.
- Inhibiting generally refers to the ability of a pharmaceutical preparation to prevent entry of a virus in a cell. Inhibition may be measured by monitoring the expression of viral genes in cells or the number of viruses produced.
- the viral entry assay described herein is an example of an in vitro method of measuring viral entry, and hence, viral inhibition.
- the term “inhibiting” means that the amount of virus entering a cell is decreased by a statistically significant measurable amount in a cell, culture, or organism treated with the polysaccharide preparations of the invention relative to an untreated cell, culture, or organism.
- the present invention depends, in part, on the discovery that specific saccharide sequences in HS can serve as gD-binding receptors for HSV-1 entry into cells.
- These saccharide sequences include one of the disaccharides, -IdoA2S-GlcN3S6S- or -IdoA2S-GlcN3S-.
- the present invention provides for substantially pure pharmaceutical preparations for use in the treatment of HSV-1 infection.
- Such pharmaceutical preparations include a substantially pure preparation of glycosaminoglycans or proteoglycan polysaccharides, which have been enriched for saccharide sequences capable of specifically binding to the viral envelope protein gD.
- the preparation includes glycosaminoglycans or proteoglycan polysaccharides enriched for GlcN3S and/or GlcN3S6S.
- the preparation includes glycosaminoglycans or proteoglycan polysaccharides enriched for IdoA2S-GlcN3S6S and/or IdoA2S-GlcN3S.
- the polysaccharide preparation is enriched for a polysaccharide sequence that includes the disaccharide IdoA2S-GlcNH 2 3S6S.
- the polysaccharide preparation is enriched for the trisaccharide GlcNS-IdoA2S- GlcNH 2 3 S6S, the tetrasaccharide UA2S-GlcNS-Ido A2S-GlcNH 2 3 S6S the pentasaccharide
- GlcNS6S-UA2S-GlcNS-IdoA2S-GlcNH 2 3S6S the hexasaccharide UA-GlcNS6S-UA2S-GlcNS- IdoA2S-GlcNH 2 3S6S, heptasaccharideGlcNAc-UA-GlcNS6S-UA2S-GlcNS-IdoA2S- GlcNH 2 3S6S, and/or the octasaccharide UA-GlcNAc-UA-GlcNS6S-UA2S-GlcNS-IdoA2S- GlcNH 2 3S6S.
- the polysaccharide preparation is enriched for the octasaccharide UA-GlcNAc-UA-GlcNS6S-UA2S-GlcNS-IdoA2S-GlcNH 2 3S6S.
- the enriched polysaccharide sequence is capable of specifically binding to the viral envelope protein gD.
- the binding affinity between the polysaccharide sequence and the gD is sufficient to initiate a specific biological response.
- the binding affinity is considered specific when the binding affinity of gD to the enriched polysaccharides is sufficient to induce fusion.
- the binding is considered non-specific where the disassociation constant K d has a value of greater than 30 ⁇ M, as measured by affinity co- electrophoresis.
- binding is considered specific where the disassociation constant K d has a value of less than 30 ⁇ M, as measured by affinity co-electrophoresis.
- the polysaccharide sequence specifically binds to HSV-1 gD with a high degree of affinity.
- the polysaccharide preparation has a binding affinity (expressed in terms of the disassociation constant (Kd) and measured by affinity co- electrophoresis as described herein) of less than 30 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of less than 20 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of less than 10 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of less than 5 ⁇ M for HSV-1 viral protein gD.
- the polysaccharide structure has a binding specificity of approximately 2 ⁇ M to 10 ⁇ M for HSV-1 viral protein gD.
- the enriched polysaccharide preparations of the invention may be produced by contacting a polysaccharide preparation with a 3-OST-3 enzyme, for example, 3-OST-3A and/or 3-OST-3B, in the presence of a sulfate donor under conditions that permit 3-OST-3 enzymatic activity.
- a 3-OST-3 enzyme for example, 3-OST-3A and/or 3-OST-3B
- the 3-OST-3 proteins may be m3-OST-3B, h3-OST-3A, h3-OST-3B, allelic or conservative substitution variants, combinations thereof, and/or recombinantly produced fusions or chimeras thereof, as described in WO 99/22005.
- the sulfate donor is 3'-phospho-adenosine 5'-phosphosulfate (PAPS).
- the enriched polysaccharide preparations of the present invention may be further enriched by one of the many purification techniques currently known in the art.
- one of ordinary skill in the art could affinity purify the polysaccharide preparations of the invention by applying the preparation to a column or other substrate to which 3-OST-3 protein or gD has been immobilized, washing away the unbound fraction of HS, and thereafter eluting the binding fraction.
- the proportion of the 3-OST-3-modified residues in a polysaccharide preparation is statistically significantly greater than the proportion of the polysaccharide structure in naturally-occurring, untreated polysaccharide preparation. In a preferred embodiment, the proportion 3-OST-3-modified residues are greater than 7 %.
- the enriched polysaccharide preparations of the present invention may be combined with one or more other compounds (e.g., diluents, detergents, excipients, stabilizers, salts, sugars, lipids, or proteins) for purposes of administration, stability, and/or storage.
- the pharmaceutical preparations of the present invention are incorporated into conventional carriers, such as, for example, lotions, creams, jellies, liniments, ointments, salves, oils, foams, gels, washes, suppositories, slow-releasing polymers, coatings, and the like so that they can be easily applied topically in the present methods.
- the carriers may also include other ingredients such as, for example, pH modifiers, stabilizers, buffers, surfactants, moisturizers, colorants, thickeners, flavorings, fragrances, perfumes, and the like.
- the pharmaceutical preparations of the present invention may also be used with conventional birth-control or safe-sex devices.
- the pharmaceutical preparations could be incorporated into or used in conjunction with condoms (i.e., via lubricants applied to the interior and/or exterior surfaces), diaphragms, cervical caps, or similar products.
- the pharmaceutical preparations of the present invention could also, for example, be introduced into a bodily orifice by hand, via suppositories, or by using conventional tampon or syringe techniques.
- the method of administering or delivering the pharmaceutical preparation to the potential HSV-transmission site is not critical if an effective amount of the pharmaceutical preparation is delivered to the site in a timely manner.
- the formulations and/or method of delivering the pharmaceutical preparations allows the pharmaceutical preparations to remain in the appropriate area during HSV-1 contact in order to maximize effectiveness.
- the pharmaceutical preparations of the invention may also be combined with a skin penetrating enhancer, for example, dimethylsulfoxide (DMSO), propylene glycol, isopropanol, ethanol, oleic acid, and N-methylpyrrolidone.
- DMSO dimethylsulfoxide
- the pharmaceutical preparations of the invention are combined with substance(s) that is/are appropriate for topical application to mucocutaneous tissues (i.e., tissues of the nasal, oral, vaginal, and anal orifices).
- the present invention is directed to methods of treating a mammal, (e.g., a human) diagnosed with or at risk of contracting HSV-1 infection.
- the present invention provides for therapeutic intervention of or prophylaxis for HSV-1 infection by reducing entry of HSV-1 into a target cell.
- 3-OST-3 modified HS binds to viral envelope protein gD and may promote the fusion of the viral coat with the target cell.
- Treatment or prophylaxis of HSV-1 infection may be achieved by administering a therapeutically effective amount of the pharmaceutical preparations of the invention including enriched gD-binding polysaccharide preparations to a mammal diagnosed with or at risk of HSV-1 infection.
- the pharmaceutical preparations of the present invention are preferably provided to a mammal directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus).
- the preferred mode of administration of pharmaceutical preparations of the invention is topical.
- the pharmaceutical preparations of the invention may be used to prevent the primary (first-episode) HSV-1 infection.
- the pharmaceutical preparation of the invention may be topically applied to tissue that is at risk of exposure to HSV- 1.
- the present invention provides for methods of treating HSV-1 infection by administering a therapeutically effective amount of the pharmaceutical preparations of the invention to an area that may be exposed to HSV-1.
- the topical administration of the pharmaceutical preparations of the present invention occurs prior to the potential exposure to HSV-1.
- the likelihood of HSV-1 infection may be reduced by using the pharmaceutical preparations of the invention to competitively inhibit the binding of the viral envelope protein gD to 3-OST-3 modified HS on a target cell surface.
- the pharmaceutical preparations of the invention may be used to treat persistent (recurring) HSV-1 infection.
- the present invention provides for methods of treating HSV-1 infection by administering a therapeutic amount of the pharmaceutical preparations of the invention to an affected area, such as a lesion, to ameliorate the clinical manifestations of HSV-1 infection.
- the topical administration of the pharmaceutical preparations of the present invention occurs at the first sign of a prodromal syndrome, a detectable subclinical manifestation (e.g., prodromal syndrome or HSV-1 shedding on mucosal surfaces detectable by PCR) and/or clinical manifestation (e.g., lesion formation) of recurrent HSV-1 infection.
- the administration of the pharmaceutical preparations of the present invention occurs at the first sign of a detectable subclinical (e.g., HSV-shedding on mucosal surfaces detected by PCR) and/or clinical manifestation (e.g., lesion formation) of recurrent HSV-1 infection and continues until the subclinical and/or clinical manifestations have subsided.
- a detectable subclinical e.g., HSV-shedding on mucosal surfaces detected by PCR
- clinical manifestation e.g., lesion formation
- the frequency, intensity and/or duration of HSV-1 recurring lesions may be reduced by using the pharmaceutical preparations of the invention to competitively inhibit the binding of gD to 3-OST-3 modified HS.
- Wild-type virus strains used were HSV-l(KOS), HSV-l(Patton), HSV-l(F) and HSV-2(333). Mutant strains included HSV-l(KOS)Ridl and Rid2 (Dean et al, 1994).
- Antibodies used included rabbit antiserum R7 specific for HSV gD (Isola et al., 1989), the anti-gD mAb DL6 (Isola et al., 1989) and an anti-myc mAb (cat. no. R950-25, Invitrogen, Carlsbad, CA).
- Purified recombinant proteins included m3-OST-l secreted from L cells (Liu et al., 1996), a secreted N-truncated form of h3-OST-3A (Liu et al.
- gD-1 two secreted C- truncated forms of gD-1 designated gD-l(306t) and gD-l( ⁇ 290-299t) (Nicola et al., 1996).
- the h3-OST-3A, gD-l(306t), and gD-l( ⁇ 290-299t) were produced in insect cells from baculovirus vectors and purified as described in the references cited.
- the hybrid protein gD-1 :Fc was produced by CHO cells transfected with plasmid pBG64.
- the hybrid protein was harvested from the medium (Ham's F12 supplemented with 3% fetal bovine serum depleted of IgG) and the protein was purified by Protein G-Sepharose chromatography. Screening assay for cDNAs encoding HSV-1 entry proteins Wild-type Chinese hamster ovary (CHO) cells are resistant to HSV-1 entry (Shieh et al.,
- a 15.5 days gestation mouse embryonic cDNA expression library (Gibco-BRL, Gaithersburg, MD) was screened for plasmids capable of rendering transfected CHO cells susceptible to HSV-1 entry, as previously described (Montgomery et al., 1996).
- This plasmid had an insert of 1666 base pairs, including the polyA tail with one major open reading frame encoding the protein.
- GenBank accesion No. AF168992
- Plasmid pDS43/myc-His was generated by in-frame fusion of the C-terminus of the m3- OST-3B open reading frame, through conversion of the stop codon to a BamHI site, with the myc-His tag from the plasmid pcDNA3.1/myc-His (Invitrogen, Carlsbad, CA).
- Plasmid pBG64 has the ectodomain of HSV-1 (KOS) gD (amino acids 1-343) fused in frame to the Fc region of a rabbit IgG heavy chain, and cloned into pcDNA3.
- Plasmids described elsewhere include those expressing m3-OST-l (Shworak et al., 1997), h3-OST-3A and h3-OST-3B (Liu et al., 1999), pBG38 expressing HveC (Geraghty et al., 1998), and pRE4 expressing HSV-1 gD (Cohen et al., 1988). Degradation analysis of 3-OST-3-modified HS
- the [ S] disaccharides were isolated by Biogel P-2 (Indianapolis, IN) and analyzed on reverse phase ion pairing HPLC. The identities of the [ 35 S] disaccharides were determined by co- elution with [ 3 H] disaccharide standards. The disaccharides (about 60% of total loaded 35 S counts) were subfractionated on reversed-phase ion pairing HPLC with appropriate disaccharide standards. Viral Entry Assays
- Entry assays were based on quantitation of ⁇ -galactosidase expressed from the viral genome or by CHO-IE8 cells in which ⁇ -galactosidase expression is inducible by HSV infection (Montgomery et al., 1996). Cells were transiently transfected in 6-well dishes, using
- Lipofectamine (Gibco-BRL, Gaithersburg, MD) with plasmids expressing one of the entry mediators or control plasmid, pcDNA3, at 1.5 to 2.0 ⁇ g per well in 1 ml. At 24 hours after
- the cells were replated in 96 well tissue culture dishes (2-4 X 10 cells/well) at least
- Results are presented as the means of triplicate or quadruplicate determinations. Assays for inhibition of infection by soluble gD were similar, except that dilutions of virus were mixed with gD-l( ⁇ 290-299t) or BSA and incubated for 30 minutes at 37°C prior to addition of the mixtures (50 ⁇ l/well) to washed cell monolayers. gD Interference Assay
- Wild-type CHO cells were co-transfected, using Lipofectamine (Gibco-BRL, Gaithersburg, MD), with m3-OST-3B expression plasmid (pDS43/myc-His) and gD expression plasmid (pRE4 for HSV-1 wild-type gD) or control plasmid (pcDNA3), in 1 :4 (m3-OST-3B:gD or control) ratio, in 6-well dishes. The total amount of plasmid DNA added per well was 2.0 ⁇ g. After 24 firs the transfected cells were replated in 96 well plates and, 24 hrs later, were exposed to various doses of ⁇ -galactosidase-expressing HSV-1. Six hours later, viral entry was quantitated as described herein. Binding of gD-1 to 3-O-sulfated HS
- the average number of 3-O-sulfate groups introduced per HS molecule from wild-type CHO cells was 8 for 3-OST-l and 14 for h3-OST-3A, based on the 35 S count transferred to the HS, given the specific activities of [ 35 S]PAPS (18 Ci/mmole) and [ 3 H]HS (727 Ci/mmole), and assuming a molecular weight of 81 ,000 for HS.
- Each preparation of enzyme- modified or unmodified HS (1-2 x 10 dpm of H) was mixed with gD-l(306t) (10 ⁇ g) in 50 ⁇ l of binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.01% Triton X-100) for 30 min at room temperature.
- the anti-gD antiserum R7 (or the anti-gD monoclonal antibody DL6) (5 ⁇ l) was added for incubation at 4°C for 1 hr, followed by addition of Protein A-agarose gel (80 ⁇ l of 1 : 1 slurry) and agitation at 4°C for an additional hour.
- Metabolically-labeled [ S]HS was modified by incubation with purified h3-OST-3 A as above except that unlabeled PAPS (10 ⁇ M) was the sulfate donor.
- the molecules of modified [ 35 S]HS capable of binding gD- 1 ([ 35 S]HS gD ' Act ) were collected by co-precipitation with gD- l(306t) as described above.
- the molecules that failed to co-precipitate were designated [ 35 S]HS gD*Inact although this fraction probably contained some gD-1 -binding material.
- gD-l(306t) was cast in low melting point 1% agarose gel (Gibco-BRL, Gaithersburg, MD) in a buffer containing 125 mM sodium acetate, 50 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-l-propane-sulfonate, pH 7.0, at 9 different concentrations for [ 35 S]HS gD*Act , ranging from 0 to 1 1 ⁇ M, and at 4 different concentrations for [ 35 S]HS gD " Inact , ranging from 0 to 83 ⁇ M, in each separation zone.
- the [ 35 S]- intensity was plotted against the migration distance through the separation zone, and the migration point with the highest [ S] intensity was defined as the migration of [ 35 S]HS gD*Act .
- the retardation coefficient R (M 0 -M)/M 0 , where M 0 is the migration of free [ 35 S]HS gD,Act and M is the observed migration of [ 35 S]HS gD ' Act .
- [ 35 S]HS gD*Act and gD-1 forms 1 : 1 complex and the amount of [ 35 S]HS gD#Act is far less than gD-1 , a plot of R/[gD- ljtotai vs.
- gD binds to 3-OST-3-modified HS, but not to 3-OST-3 enzyme.
- HS extracted from wild-type CHO cells was unmodified or was sulfated by incubation in vitro with 3-OST-l or h3-OST-3A, and was tested for binding to gD in co-immunoprecipitation experiments.
- Table 1 shows that the fraction of labeled HS bound to immunoprecipitated gD- l(306t) was enhanced almost 10-fold for HS by incubation with 3-OST-3A. Modification by 3- OST-1 did not significantly enhance the binding to gD-l(306t).
- the HS from pgsF-17 cells After incubation with 3-OST- 3 A, the HS from pgsF-17 cells, a cell line that fails to modify the hexuronic acid residues of HS by the addition of 2-O-sulfate groups, bound to gD-l(306t) with about the same efficiency as unmodified HS from CHO cells.
- the sulfated structure generated is -GlcA-GlcNS ⁇ 6S3S-.
- the sulfated structure generated is -Ido A2 S - GlcN+6 S 3 S - .
- 3-OST-3B-mediated viral entry depends on HS production and 2-O-sulfation of HS
- mutant cell lines defective in various aspects of GAG biosynthesis have been isolated from CHO cells (Bai and Esko, 1996; Esko, 1991). Wild-type CHO cells and several mutant cell lines were transfected with plasmids expressing myc-tagged version of m3-OST-3B or HveC, or with the empty vector, pcDNA3. The cells were then exposed to HSV-1 (KOS)gL86 for quantitation of viral entry. Various cell lines transfected with the m3-OST-3B-expressing plasmid produced equivalent amounts of the protein. Mutations affecting HS biosynthesis had different consequences for HSV-1 entry, depending on whether the entry mediator was m3-OST- 3B or HveC.
- HSV-1 entry None of the cells transfected with control plasmid was susceptible to HSV-1 entry.
- Expression of m3-OST-3B resulted in HSV-1 entry only in cells capable of producing HS with the modifications characteristic of CHO cells.
- HSV-1 entry occurred in wild-type CHO cells and mutant cells pgsE-606, which produce HS that is under-sulfated due to the absence of one of the N-sulfotransferase isoforms (Aikawa and Esko, 1999; Bame and Esko, 1989), provided 3-OST-3 was expressed.
- HSV-1 entry did not occur in mutant lines that fail to produce HS.
- HSV-1 entry did not occur in the following HS defective cell lines: pgsA-745, a cell line that does not produce glycosaminoglycans (Esko et al., 1985); pgsD-677, a cell line that produces only chondroitin sulfate (Lidholt et al., 1992); and pgsF-17, a cell line that fails to modify the hexuronic acid residues of HS by the addition of 2-O-sulfate groups (Bai and Esko, 1996).
- 3-OST-3s is a glucosamine residue adjacent to 2-O- sulfated IdoA
- incubation of HS from pgsF-17 cells with purified 3-OST-3A should not yield the characteristic disaccharides resulting from 3-OST-3 activity, as was confirmed experimentally.
- HveC rendered all the cell lines susceptible to HSV-1 entry, although the wild-type CHO cells became significantly more susceptible than any of the mutant cell lines. Binding of virus to the wild-type cells should be enhanced by interactions of gB or gC with HS as well as of gD with HveC.
- HveC can confer greater susceptibility to HSV-1 entry than does m3-OST-3B.
- new gD-binding receptors are over-expressed from the transfected plasmid whereas, in the case of m3-OST-3B, the enzyme is thought to modify endogenous molecules to generate receptors for virus. Accordingly, the levels of endogenous receptor are believed to control the degree of susceptibility to infection resulting from 3-OST-3 activity.
- Heparinases removed gD-binding receptors and reduced viral entry in 3-OST-3 cells
- Heparinases were shown to remove the receptors that bind gD and mediate viral entry from 3-OST-3-expressing cells. Wild-type CHO cells or pgsA-745 cells were transfected with plasmids expressing m3-OST-3B or h3-OST-3A, or with control plasmid. The transfected cells were either incubated with a mixture of heparinases I and III or in buffer without the heparinases. The cells were next incubated with a gD hybrid protein consisting of the ectodomain of gD fused to the Fc of rabbit IgG (gD-l:Fc).
- Heparinase I heparin lyase I; heparinase; EC 4.2.2.8
- heparinase III heparitin sulfate lyase III; heparitinase, EC 4.2.2.8
- Transfected cells plated in 96-well dishes were washed twice with PBS lacking magnesium and calcium ions, overlaid with several dilutions of heparinase I and heparinase III or PBS alone, and incubated at 30°C for 2 h. The cells were then washed with PBS and used for viral entry assays or for quantitation of gD-l:Fc binding to the cells.
- the cells assayed for gD-binding were processed as follows. The cells were next incubated with a gD hybrid protein consisting of the ectodomain of gD fused to the Fc of rabbit IgG (gD-1 :Fc). Expression of 3-OST-3A or m3-OST-3B caused enhanced binding of gD-1 :Fc. In contrast, expression of 3-OST-3A or m3-OST-3B in pgs-745 cells did not enhance binding of gD-1 :Fc. The enhanced binding in CHO cells was eliminated by pre-treatment of the cells with heparinases I/III.
- the cells assayed for viral entry were processed as follows. The cells were exposed to HSV-l(KOS)gL86 at 7.5 x 10 5 PFU/well and viral entry was quantitated 6 hrs later. Entry into the heparinase I/III-treated cells was compared to percentage of the entry into mock-treated cells. For CHO cells expressing m3-OST-3B, treatment with heparinases I/III almost completely eliminated susceptibility to HSV-1 entry whereas, for cells expressing HveC, this treatment reduced susceptibility to HSV-1 infection by 50%). Both membrane bound and soluble gD competes with virus for receptors generated by m3-OST- 3B
- HSV-1 entry was inhibited in CHO cells transformed with HSV-1 gD and m3-OST-3B.
- CHO cells were transfected with a m3-OST-3B-expressing plasmid mixed in 1 :4 ratio with a gD- 1 -expressing plasmid or the empty vector pcDNA3.
- the transfected cells were then exposed to HSV-1 (KOS)gL86 and viral entry quantitated after 6 hrs. Viral entry was assayed using the entry assay described herein.
- HSV-1 entry was inhibited in m3-OST-3B-expressing CHO cells that were pre-incubated with soluble gD.
- CHO cells transfected with a plasmid expressing m3-OST-3B were exposed to HSV-l(KOS)gL86 (7.5 x 10 5 PFU/well) that had been pre-incubated with gD-l( ⁇ 290-299t), a soluble form of gD-1.
- Viral entry was assayed using the entry assay described herein.
- h3-OST-3A K162 mutant does not express 3-OST-3 activity
- h3-OST-3A A mutated form of h3-OST-3A, in which the Lys at position 162 was changed to Ala, was shown by Western blot analysis to be expressed at a level comparable to that of wild-type enzyme but to have little or no enzymatic activity and no viral entry activity, as described below.
- the 3-OST-3A mutant was transfected into COS-7 cells and the cell extract was assayed for 3- OST-3 activity.
- enzyme-modified HS to viral glycoproteins was determined by immunoprecipitation using Protein A-agarose (Pierce, Rockford, IL). Briefly, enzyme-modified [ 3 H/ 35 S]HS (1 x 10 5 cpm of 3 H counts) was incubated with recombinant purified HSV glycoprotein gD-1 (306t, 10 ⁇ g) (Tal-Singer et al., 1995) in 50 ⁇ l of a buffer containing 50 mM Tris, 150 mM NaCl and 0.01% Triton X-100, pH 7.5 (binding buffer) at room temperature for 30 min.
- CHO cells and pgsA-745 cells were transfected with pDS43 or pcDNA3 and plated in 96- well plates. Some cultures were treated with heparinases as described below prior to the assay. After washing the cells and blocking with PBS containing 3% BSA for 30 min, the cells were incubated with purified gD-1 :Fc (1 ⁇ g/ml) in 1% BSA-PBS for 45 min at room temperature. The cells were then washed and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS.
- the human PRR2 gene related to the human poliovirus receptor gene (PVR), is the true homolog of the murine MPH gene. Gene 159, 267-272. Esko, J. D. (1991). Genetic analysis of proteoglycan structure, function and metabolism. Curr.Opin.Cell Biol. 3, 805-816.
- Glycoprotein C-independent binding of herpes simplex virus to cells requires of cell surface heparan sulphate and glycoprotein B. J.Gen. Virol. 75, 1211-1222.
- Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J.Virol. 65, 1090-1098.
- Herpes simplex virus glycoprotein D can bind to poliovirus receptor- related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry. J.Virol. 72, 7064-7074.
- the first Ig-like domain of HveC is sufficient to bind HSV gD with full affinity while the third domain is involved in oligomerization of HveC. J.Virol, in press.
- LIGHT a new member of the TNF superfamily, and lymphotoxin a are ligands for herpesvirus entry mediator.
- Cellular receptor for poliovirus molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56, 855-865.
- glycoprotein D loses importance for replication of bovine herpesvirus 1 in cell culture. Journal of Virology 71, 25-33. Shieh, M.-T., WuDunn, D., Montgomery, R. I., Esko, J. D., and Spear, P. G. (1992). Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J.Cell Biol. 116, 1273- 1281.
- aVb5 integrin a co-receptor for adeno-associated virus type 2 infection. Nature Med. 5, 78-82. Summerford, C, and Samulski, R. J. (1998).
- Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J.Virol. 72, 1438-1445.
- Nectin PRR an immunoglobulin- like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with afadin, a PDZ domain-containing protein. J. Cell Biol. 145, 539-549.
- a cell surface protein with herpesvirus entry activity confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus.
- HveB herpesvirus entry activity
- HBV herpes simplex virus
- HVEM HveA herpesvirus entry mediator
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EP3572079A1 (en) * | 2018-05-22 | 2019-11-27 | Dobecure, S.L. | Etamsylate for treatment of herpes virus infections |
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WO1998005341A1 (en) * | 1996-08-06 | 1998-02-12 | Medicarb Ab | The use of heparin or heparan sulphate in combination with chitosan for the prevention or treatment of infections caused by herpes virus |
WO1999022005A2 (en) * | 1997-10-24 | 1999-05-06 | Massachusetts Institute Of Technology | Heparan sulfate d-glucosaminyl 3-o-sulfotransferases, an uses therefor |
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WO1998005341A1 (en) * | 1996-08-06 | 1998-02-12 | Medicarb Ab | The use of heparin or heparan sulphate in combination with chitosan for the prevention or treatment of infections caused by herpes virus |
WO1999022005A2 (en) * | 1997-10-24 | 1999-05-06 | Massachusetts Institute Of Technology | Heparan sulfate d-glucosaminyl 3-o-sulfotransferases, an uses therefor |
Non-Patent Citations (4)
Title |
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LIU JIAN ET AL: "Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 8, 19 February 1999 (1999-02-19), pages 5185-5192, XP002166605 ISSN: 0021-9258 * |
LIU JIAN ET AL: "Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A sulfates N-unsubstituted glucosamine residues." JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 274, no. 53, 31 December 1999 (1999-12-31), pages 38155-38162, XP002166604 ISSN: 0021-9258 * |
SHIEH M -T ET AL: "CELL SURFACE RECEPTORS FOR HERPES SIMPLEX VIRUS ARE HEPARAN SULFATE PROTEOGLYCANS" JOURNAL OF CELL BIOLOGY,ROCKEFELLER UNIVERSITY PRESS, NEW YORK, US,US, vol. 116, no. 5, March 1992 (1992-03), pages 1273-1281, XP000941011 ISSN: 0021-9525 * |
SHUKLA D ET AL: "A NOVEL ROLE FOR 3-O-SULFATED HEPARAN SULFATE IN HERPES SIMPLEX VIRUS 1 ENTRY" CELL,CELL PRESS, CAMBRIDGE, NA,US, vol. 99, 1 October 1999 (1999-10-01), pages 13-22, XP000982864 ISSN: 0092-8674 * |
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EP3572079A1 (en) * | 2018-05-22 | 2019-11-27 | Dobecure, S.L. | Etamsylate for treatment of herpes virus infections |
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