WO1999026649A1 - Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity - Google Patents

Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity Download PDF

Info

Publication number
WO1999026649A1
WO1999026649A1 PCT/US1998/024905 US9824905W WO9926649A1 WO 1999026649 A1 WO1999026649 A1 WO 1999026649A1 US 9824905 W US9824905 W US 9824905W WO 9926649 A1 WO9926649 A1 WO 9926649A1
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
thrombospondin
tsp
tspl
analog
Prior art date
Application number
PCT/US1998/024905
Other languages
French (fr)
Other versions
WO1999026649A9 (en
Inventor
Andrea Rene Crombie
Ralph L. Nachman
Jeffrey C. Laurence
Original Assignee
Cornell Research Foundation, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cornell Research Foundation, Inc. filed Critical Cornell Research Foundation, Inc.
Priority to AU15971/99A priority Critical patent/AU1597199A/en
Priority to US09/555,090 priority patent/US6964763B1/en
Publication of WO1999026649A1 publication Critical patent/WO1999026649A1/en
Publication of WO1999026649A9 publication Critical patent/WO1999026649A9/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • the present invention relates to methods which inhibit HIV infectivity, block HIV binding to a cell, block chemokine binding to its receptor, and treat or prevent inflammatory states in a patient, as well as pharmaceutical compositions, contraceptives, and non-contraceptive prophylactic devices.
  • HIV Human immunodeficiency virus
  • a complete sequencing of the HIV genome indicates that it contains the same overall gag-pol-env organization as other retroviruses. Ratner et al., "Complete Nucleotide Sequence of the AIDS virus, HTLV-III,” Nature 313:277-84 (1985). HIV invades a host cell and uses the host cell's machinery to replicate itself.
  • HIV can be cultured from most tissues and body fluids of infected individuals. Saliva represents a significant exception.
  • HIV- 1 was isolated from only one of 71 saliva samples of HIV+ donors (Ho et al, "Infrequency of Isolation of HTLV-III Virus from Saliva in AIDS," New Engl. J. Med.. 313:1606 (1985)).
  • Recent work confirmed the paucity of infectious virus in saliva (Groopman et al, "HTLV-III in Saliva of People with AIDS-Related Complex and Healthy Homosexual Men at Risk for AIDS," Science.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Clinical support for the limited transmissibility of HIV by saliva includes: lack of infection following contamination of open wounds with saliva from HIV+ individuals (CDC, “Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and Other Blood-Borne Pathogens in Healthcare Settings," Morbid. Mortal. Wklv. Rep.. 37:377-388 (1988)); low occupational risk for HIV infection among dentists in practices with large numbers of patients at risk for HIV infection (Klein et al., “Low Occupational Risk of Human Immunodeficiency Virus Infection Among Dental Professionals," New Engl. J.
  • Such retarded transmission is not a general characteristic of viruses which can be shed orally.
  • the annual attack rate for hepatitis B virus among unvaccinated dentists is 2.6% (Remis et al., "Hepatitis B Infection in a Day School for Mentally Retarded Students: Transmission from Students to Staff," Am. J. Public Health, 77:1183-1186 (1987)), human T cell lymphotrophic virus type I is found in saliva (Achiron et al., "Detection of Proviralpuman T-Cell Lymphotropic Virus Type I DNA in Mouthwash Samples of HAM/TSP Patients and HTLV-I Carriers," Arch.
  • Particulate and filterable oral secretions capable of inhibiting HIV infection represent potential explanations for the paucity of HIV in saliva. Reports from several different groups imply that two processes are involved (Fultz,
  • Submandibular saliva contains sulfated polysaccharides of low (MG2) and high (MGI) molecular weights (Levine et al, "Structural Aspects of Salivary Glycoproteins,” J. Pent. Res.. 66:436-441 (1987)), with the latter forming an anionic charge barrier to binding of the high affinity HIV receptor, CP4, to the HIV envelope glycoprotein gpl20 (Amory et al., "The Large Molecular Weight Glycoprotein MGI, a Component of Human Saliva, Inhibits HIV-1 Infectivity," Clin. Res., 40:51A (1992)).
  • SLPI Secretory leukocyte protease inhibitor
  • Fibronectin a matrix adhesion molecule, binds directly to gpl20, but was shown to inhibit infectivity only at high concentrations (Su et al., "Interaction of the Envelope Glycoprotein of Human Immunodeficiency Virus with Clq and Fibronectin Under Conditions Present in Saliva," Mol. Immunol. 28:811-817 (1991)).
  • the present invention relates to a method for suppressing infectivity of HIV. This method is carried out by contacting the HIV or a cell targeted by HIV with an effective amount of a thrombospondin ("TSP") or a TSP analog. By contacting the HIV with an effective amount of a TSP or a TSP analog, the ability of HIV to bind to its cellular target is inhibited. Similarly, by contacting the cell targeted by HIV with an effective amount of a TSP or a TSP analog, the ability of HIV to bind to its cellular target is also inhibited. Both approaches are useful in suppressing HIV infectivity.
  • the present invention further relates to a method of inhibiting HIV infection in a patient which includes administering a TSP or a TSP analog to a patient under conditions effective to inhibit HIV infection.
  • the present invention also relates to a method of blocking HIV binding to a cell targeted by HIV.
  • This method includes contacting the HIV or the cell targeted by HIV with a TSP or a TSP analog under conditions effective to block binding of the HIV to the cell.
  • HIV is capable of spreading via sexual contact as well as non- sexual contact (e.g., surgical procedures)
  • pharmaceutical compositions, contraceptives, and non-contraceptive prophylactic devices capable of decreasing the likelihood of HIV infection are particularly desirable. Therefore, another embodiment of the present invention relates to a contraceptive that includes a contraceptive carrier and a TSP or a TSP analog.
  • a pharmaceutical composition that includes a pharmaceutically-acceptable carrier and a TSP or a TSP analog.
  • a non-contraceptive prophylactic device of the present invention includes a non-contraceptive carrier and a TSP or a TSP analog.
  • the present invention also relates to a method of blocking the binding of a chemokine to its receptor by contacting a chemokine with a TSP or a TSP analog under conditions effective to bind the chemokine. Because chemokines are generally implicated in inflammatory states, another aspect of the present invention relates to a method of treating or preventing inflammatory states in a patient by administering an effective amount of a TSP or a TSP analog to a patient.
  • TSP-1 purified thrombospondin- 1
  • Figure 1 is an amino acid sequence alignment of CO36/LIMPII TSP binding motifs with homologous sequences in HIV-1 g ⁇ l20. Results of a pattern- based Blast enhanced alignment utility search that matched a split motif in gpl20 domains C2 (top) and C3 (bottom). Asterisks (*) indicate disulfide-bonded cysteine residues of the V3 loop. The brackets above alignments show boundaries of CP36 Exon 5 coding region (CP36 aa 95-143). Amino acids identical between either CP36 or LIMPII and HIV-1 gpl20 are highlighted.
  • GenBankTM/EMBL accession numbers: huCP36, M24795; huLIMPII, P12676; HIV-1 partial sequence of isolate U37041 ; HIV-1 clade B consensus, MN and LAI isolate sequences were retrieved from the WHO HIV Sequence Oatabase.
  • FIG. 2B is a graph showing competitive inhibition of 125 I-TSP1 for HIV-1 g ⁇ l60, which demonstrates the specificity of the TSP 1 -HIV interaction.
  • Figure 3 is a graph showing how I-gpl20 interacts with the CP36 binding peptide of TSP 1.
  • Increasing concentrations of soluble I-labeled recombinant gpl20LAV (nM - 1 ⁇ M) were incubated with immobilized ligand for 2 hours at 37°C, and bound radioactivity measured as in Figures 2A and 2B.
  • Figure 4 A is a map showing the position of synthetic gpl20 peptides with respect to gpl20 C2-V3-C3 domains and TSP binding motifs.
  • a set of gpl20MN peptides ( ⁇ 20 aa) were immobilized either singly (numbered 1-7) or in pairs as indicated.
  • Figure 4B is a graph showing the binding of 125 I-TSP1 to gpl20
  • Figure 5 is a graph showing competitive inhibition of 125 I-CP4 binding
  • FIG. 6 is an image showing immunohistochemical detection of cell- associated TSPl in human gingival mucosa.
  • Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSPl and TSP2 (lower panel), or with pre-immune semm (upper panel), followed by biotinylated second antibody, and developed using avidin-conjugated peroxidase. Brown deposits indicate sites of TSP reactivity (magnified 200X).
  • Figure 7 is a graph showing the inhibitory effect of TSPl on HIV-1 infectivity.
  • HIV-1 isolate IIIB was pre-incubated in the absence of TSPl, or with various concentrations of purified TSPl for 1 hour at 37 °C.
  • Pre-incubated virus- TSP1 mixtures were added to target cells either directly (-), or first passed through 0.2 ⁇ m filters (+).
  • Phorbol myrisate acetate (“PMA”) activated primary peripheral blood mononuclear cells (“PBMC”), SK23 (T-cell line) or U937 (promonocytic line) were inoculated with the multiplicities of infection (“moi”) indicated.
  • PMA Phorbol myrisate acetate
  • PBMC primary peripheral blood mononuclear cells
  • SK23 T-cell line
  • U937 promonocytic line
  • infected cells were washed, cultured for 7 days, and HIV-1 p24 antigen measured by ELISA. Inhibition is expressed for a single data set as percent of maximum p24 detected in the absence of TSPl.
  • FIG 8 is a graph showing competitive inhibition of TSPl anti-HIV effect by LIMPII TSPl binding domain.
  • PMA-stimulated primary PBMC were infected with 0.02 moi HIV/IIIB that had been pre-incubated with TSPl alone, or with LIMPII fusion protein LFP75-155 alone or in the presence of TSPl, and filtered prior to incubation as in Figure 7.
  • TSP-mediated inhibition is expressed as percent decrease in p24 relative to maximum p24 detected in the absence of TSPl . Pata shown represent the average of two independent experiments (error as SP).
  • Figure 9 is a graph showing the binding of 125 I-RANTES to TSP 1.
  • RANTES measured by solid-binding radio-amino assay.
  • Figure 10 is a graph showing the Hanes analysis of 123 I-RANTES binding to immobilized TSPl .
  • the Hanes analysis provides a measure of assessing the possibility of binding one or more factors to a given molecule.
  • FIG. 11 is a diagram representing the structure of TSPl and TSP2.
  • Figure 12 is a graph showing the binding of 125 I-CP4 to TSPl and a
  • the present invention relates to a method for suppressing infectivity of HYV. This process includes contacting the HIV or a cell targeted by HIV with an effective amount of a TSP or a TSP analog.
  • TSPl is a trimeric sulfated glycoprotein which belongs to a family of high molecular weight extracellular matrix molecules reviewed in Bornstein, "Thrombospondins: Structure and Regulation of Expression,” FASEB, 6:3290-3299 (1992); Lahav, editor, “Thrombospondin,” CRC Press, (1993), which are hereby incorporated by reference.
  • TSPl is implicated in suppressing the infectivity of certain bacteria and protozoa, including Staphylococcus aureus, babesia, toxoplasma, leishmania, and the malaria sporozoite (Lahav, "The Functions of Thrombospondin and its Involvement in Physiology and Pathophysiology," Biochim. Biophys.
  • TSPl Lipoproteins Inhibit Malaria Sporozoite Invasion of Hepatocytes
  • J. Exp. Med., 184:945-954 (1996) , which are hereby incorporated by reference).
  • TSPl is found in very low quantities in plasma, but is stabilized during reversible binding to other matrix molecules, resulting in markedly elevated levels at certain cell surfaces (Taraboletti et al., "Platelet Thrombospondin Modulates Endothelial Cell Adhesion, Motility, and Growth: A Potential Angiogenesis Regulatory Factor," J.
  • TSP2 TSPl and thrombospondin-2
  • mice Bornstein, “ThiOmbospondins: Structure and Regulation of Expression,” FASEB, 6:3290-3299 (1992), which is hereby inco ⁇ orated by reference
  • humans LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region," Genomics 17:225-229 (1993), which is hereby incorporated by reference).
  • Human TSPl and human TSP2 are functionally and structurally similar and have an amino acid identity which is about 54 percent (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region,” Genomics 17:225-229 (1993), which is hereby inco ⁇ orated by reference).
  • human TSPl and mouse are functionally and structurally similar and have an amino acid identity which is about 54 percent (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region," Genomics 17:225-229 (1993), which is hereby inco ⁇ orated by reference).
  • TSP 1 have been shown to share a high degree of homology to one another (Bornstein, "Thrombospondins: Structure and Regulation of Expression,” FASEB, 6:3290-3299 (1992), which is hereby inco ⁇ orated by reference), as have human TSP2 and mouse TSP2 (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated
  • both TSPl and TSP2 contain similar functional domains or regions, including an N-terminal region 10, a procollagen-homologous region 20, type I TSP repeats 30, type II TSP repeats 40, type III calcium-binding repeats 50, and a carboxy-terminal region 60 (Bornstein, "Thrombospondins: Structure and Regulation of Expression,” FASEB, 6:3290-3299 (1992); Lawler et al., "The Structure of Human Thrombospondin, an Adhesive Glycoprotein with Multiple Calcium-binding Sites and Homologies with Several Different Proteins," J. Cell Biol.
  • TSPl and TSP2 each contain 21 exons. Exons 1-2 of each TSP gene encode the 5' untranslatable region ("UTR") and translation start sequences, while exon 22 of each TSP gene encodes a 3' UTR. The remaining exons all encode functional regions or domains of the mature TSPl or TSP2.
  • the N-terminal region 10 of TSPl and TSP2 includes a heparm-binding domain. This region includes two sequences of tandemly repeated basic residues that are believed to represent the specific sites which bind anionic heparin.
  • the N-terminal region 10 can be cleaved from TSPl or TSP2 by thermolysin or chymotrypsin.
  • a variable segment 70 of TSPl and TSP2, located between the N- terminal region 10 and the procollagen-homologous region 20, contains two cysteine residues which are believed to be involved in the formation of interchain di-sulfide bridges during trimerization.
  • the procollagen-homologous region 20 of TSPl and TSP2 is cysteine- rich and shares homology with the NH 2 -propeptide of the ⁇ (I) chain of type I procollagen.
  • TSPl and TSP2 each contain three type I TSP repeats 30 having between 50-54 amino acid residues.
  • the type I TSP repeat 30 has been found to be conserved in a number of different proteins including, among others, antistasin (Holt et al, "Properdin Binds to Sulfatide [Gal(350 4 ) beta 1-1 Cer] and Has a
  • the type I TSP repeat 30 contains a cys-ser-val-thr-cyg-gly (“CSVTCG”) motif (SEQ. ID. No. 1), which is found in both group 1 and group 2 repeats. This sequence has been implicated in the binding of various proteins to cells.
  • the amino acid sequence containing the CSVTCG motif (SEQ. ID. No. 1) is also conserved between TSPl and TSP2.
  • TSPl and TSP2 each contain three type II TSP repeats 40.
  • a role for the EGF-like domain in ligand-receptor interactions has been proposed (Appella et al., "Structure and Function of Epidermal Growth Factor-Like Regions in Proteins," FEBS Lett. 231 :1-4 (1988), which is hereby inco ⁇ orated by reference).
  • each of the type III repeats 50 contains two cysteine residues, presumably involved in a disulfide bond, and either seven or ten highly conserved asparagine, glutamine, and aspartic acid residues that could serve to complex with calcium.
  • the type III repeats 50 are functionally related to the E/F hand of calmodulin and also have features in common with calcium-binding structures in parvalbumin and fibrinogen (Lawler et al., "The Structure of Human Thrombospondin, an Adhesive Glycoprotein with Multiple Calcium-Binding Sites and Flomologies with Several Different Proteins," J. Cell. Biol.
  • the carboxy-terminal region 60 is highly conserved between TSPl and TSP2. This region has been implicated for involvement with platelets, melanoma cells, keratinocytes, and squamous cell carcinoma cells (reviewed in Prater et al., "The Properdin-like Type I Repeats of Human Thrombospondin Contain a Cell
  • CLESH-1 an evolutionary conserved TSPl binding domain, termed CLESH-1, was recently established as functional in at least two members of the CD36 gene family, cell surface adhesion receptor CD36 (Pearce et al.,
  • TSP can be isolated from tissue or fluid samples (e.g., plasma) by established techniques described in Silverstein et al., "Platelet Thrombospondin Forms a Trimolecular Complex with Plasminogen and Histadine-Rich Glycoprotein," J. Clin. Invest. 75:2065-2073 (1985), which is hereby inco ⁇ orated by reference.
  • the TSP is produced in purified form, i.e., preferably at least about 80%> pure, more preferably at least 90% pure, and most preferably at least about 95%> pure.
  • Purified TSPl derived from human platelets is also commercially available from Sigma Chemical Co. (St. Louis, Missouri). TSP can also be prepared using recombinant techniques.
  • TSPl DNA molecules encoding TSPl (Wolf et al., "Structure and Chromosomal Localization of the Human Thrombospondin Gene,” Genomics 6:685-691 (1990), which is hereby inco ⁇ orated by reference) and TSP2 (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region,” Genomics 17:225-229 (1993), which is hereby inco ⁇ orated by reference) have been isolated and described. Once obtained, the DNA molecule encoding TSP can be inco ⁇ orated in cells using conventional recombinant DNA technology.
  • the heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • U.S. Patent No. 4,237,224 to Cohen and Boyer which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. Transformed host cells capable of producing recombinant TSP can be grown in culture and the expressed TSP can be isolated from the growth medium using conventional protein separation techniques.
  • TSP analogs are fragments of TSP.
  • TSP analogs can be obtained following cleavage of TSP by proteases.
  • proteases include chymotrypsin, thermolysin, and thrombin (Dixit et al., "Isolation and Characterization of a Heparin-binding Domain from the Amino Terminus of Platelet Thrombosis," J. Biol. Chem. 259:10100-10105 (1984); Mumby et al, Interactions of Thrombospondin with Extracellular Matrix Proteins: Selective Binding to Type V Collagen," J. Cell Biol. 98:646-652 (1984), which are hereby inco ⁇ orated by reference).
  • TSP tissue-specific protein
  • fragments of TSP can be isolated using immunoselective techniques (Dixit et al., "Monoclonal Antibodies that Recognize Calcium-dependent Structures of Human Thrombospondin. Characterization of Mapping Their Epitopes," J. Biol. Chem. 261 :1962-1968 (1986); Galvin et al., “Mapping of Epitopes for Monoclonal Antibodies against Human Platelet Thrombospondin with Electron Microscopy and High Sensitivity Amino Acid Sequencing," J. Biol. Chem. 101 : 1434- 1441 (1985), which are hereby inco ⁇ orated by reference), HPLC, or gel electrophoresis.
  • TSP peptides (TSP analogs) according to the methods of Guo et al., "Heparin-binding Peptides from the Type I Repeats of Thrombospondin. Structural Requirements for Heparin Binding and Promotion of Melanoma Cell Adhesion and Chemotaxis," J. Biol. Chem. 267:19349-19355 (1992) and Guo et al, "Heparin- and Sulfatide-binding Peptides from the Type I Repeats of Human Thrombospondin Promote Melanoma Cell Adhesion.” Proc Natl. Acad. Sci.
  • TSP analogs can also be produced using conventional recombinant technologies as described above.
  • Exemplary TSP analogs include an N-terminal region of TSP (e.g.,
  • TSPl and TSP2 a procollagen-homologous region of TSP (e.g., TSPl and TSP2), a type I repeat of TSP (e.g., TSPl and TSP2), a type II repeat of TSP (e.g., TSPl and TSP2), a calcium-binding domain of TSP (e.g., TSPl and TSP2), a carboxy-terminal region of TSP (e.g., TSPl and TSP2), and TSP domains which bind to an HIV gpl20 envelope protein.
  • a preferred TSP analog is a polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1).
  • the TSP analogs can be defined as TSP domains which bind to an HIV g ⁇ l20 envelope protein, TSP domains which bind to a CD4 receptor on a cell, or TSP domains which bind to a chemokine (e.g., RANTES).
  • TSP analogs may include molecules, such as fusion proteins, containing TSP or any one of the above-identified TSP analogs.
  • the fusion protein can contain one or more domains/regions of TSP or the CSVTCG amino acid sequence (SEQ. ID. No. 1).
  • Fusion proteins can be prepared by ligating two DNA molecules together, one of which encodes TSP or the TSP analog and the other of which encodes a stable host protein.
  • Suitable host proteins include, among others, glutathione-S-transferase (Frangioni et al., "Solubilization and Purification of Enzymatically Active Glutathione-S-Transferase (pGEX) Fusion Proteins," Analyt.
  • the fusion protein is prepared with a protease cleavage site intermediate the host protein and the TSP or TSP analog, such that the TSP or TSP analog can be removed from the host protein by, for example, proteolytic cleavage following isolation of the fusion protein.
  • a linker or spacer peptide may also be included to promote proteolytic cleavage (Polyak et al., "Introduction of Spacer Peptides N-terminal to a Cleavage Recognition Motif in Recombinant Fusion Proteins Can Improve Site-specific Cleavage," Protein Eng. 10(6):615-619 (1997), which is hereby inco ⁇ orated by reference).
  • a number of suitable proteases e.g., factor Xa, chymosin, trypsin, etc.
  • their protease-specific cleavage sites are known in the art, and others continually being identified.
  • the RNA of the HIV-1 and HIV-2 viruses possess the following gene regions: so-called long terminal repeats at each end of the genome, gag, pol, env, and nef.
  • the gag gene encodes the core proteins p24 and pi 7.
  • the pol gene encodes the reverse transcriptase, RNAse H, and integrase.
  • the gene nef encodes a protein having a regulatory function.
  • the env gene encodes the glycoproteins of the viral coat, namely gp41 and gpl20.
  • the env gene has been associated particularly with antigenicity and infectivity. However, the env portion is also known to have regions which are highly divergent between different strains.
  • the HIV virus has been shown to bind to a cell surface receptor known as the CD4 or T4 region, which is present on the various cells susceptible to HIV infection, including T lymphocytes and macrophages (Shaw et al., "Molecular Characterization of Human T-Cell Leukemia (Lymphotrophic) Virus Type III in the Acquired Immune Deficiency Syndrome," Science 226:1165-1171 (1984), which is hereby inco ⁇ orated by reference).
  • HIV co-receptors have been implicated in HIV entry inside a cell, because the primary cell surface receptor, CD4, is often by itself insufficient to enable viral entry. HIV co-receptors are cell surface proteins that HIV uses to dock onto its cellular targets.
  • the method for suppressing HIV infectivity is carried out by contacting the HIV with an effective amount of TSP or the TSP analog.
  • the process of the present invention suppresses the ability of HIV to infect a host organism by blocking its ability to bind to its cellular target (e.g., T lymphocytes and macrophages).
  • the TSP or TSP analog are administered in a manner effective to contact the HIV gpl20 envelope protein. Because the gpl20 envelope protein is necessary for the binding of HIV to its cellular target, TSP or the TSP analog can inhibit or interfere with HIV binding to its cellular target.
  • the inhibition or interference with HIV binding to its cellular target results from the TSP or TSP analog binding to the gpl20 envelope protein in a manner which either prevents HIV from binding to the cell surface receptor (e.g., CD4) or co-receptor (e.g., chemokine receptor CCR5), or causes steric interference between the functional domains of the gpl20 envelope protein and the receptor/co- receptor.
  • the cell surface receptor e.g., CD4
  • co-receptor e.g., chemokine receptor CCR5
  • the method is carried out by contacting a cell targeted by HIV with an effective amount of a TSP or TSP analog.
  • the TSP or TSP analog is administered in a manner effective to contact the CD4 receptor on a cell. Because the CD4 receptor is necessary for the binding of HIV to its cellular target, TSP or the TSP analog can inhibit or interfere with HIV binding to its cellular target.
  • the method is carried out by both contacting the HIV with an effective amount of TSP or a TSP analog and contacting the cell targeted by HIV with an effective amount of TSP or a TSP analog.
  • the ability of HIV to bind and recognize its cellular target as well as the ability of the CD4 receptor (or the co-receptor) to recognize HIV can both be affected to further suppress HIV infectivity.
  • TSP or the TSP analog can be administered orally or intraorally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intravaginally or intrarectally (e.g., by suppository, ointment, etc.) intrasynovially, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes via inhalation or otherwise (e.g., using an aerosol or lavage).
  • the preferred route for administration is intraorally, intrarectally, or intravaginally.
  • TSP and the TSP analog can each be administered alone, with a spermicide, or with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions, which may also include microcapsules or liposomes.
  • a pharmaceutical composition which includes a pharmaceutically-acceptable carrier in combination with TSP or the TSP analog.
  • the pharmaceutical composition can also include additives commonly used for pharmaceutical preparations, if desired, such as excipients, stabilizers, antiseptics, solubilizers, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavors, antioxidants, and the like.
  • the solid unit dosage forms can be of the conventional type.
  • the solid form can be a capsule, such as an ordinary gelatin type which includes TSP or the TSP analog and a pharmaceutical carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch.
  • these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
  • conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin
  • disintegrating agents such as cornstarch, potato starch, or alginic acid
  • a lubricant like stearic acid or magnesium stearate.
  • TSP and the TSP analog can also be administered topically in the form of a solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier.
  • a pharmaceutical carrier include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carriers, including adjuvants, excipients or stabilizers.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • TSP and the TSP analog can be prepared in solution or suspension and packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • a preferred formulation for the pharmaceutical composition of the present invention contains TSPl or TSP2 in the pharmaceutically-acceptable carrier.
  • Another preferred formulation for the pharmaceutical composition of the present invention contains a TSP analog in the pharmaceutically-acceptable earlier.
  • the oral hygiene composition is a pharmaceutical composition which contains a carrier that is a pharmaceutically- acceptable topical oral carrier.
  • Suitable topical oral carriers include a toothpaste, a mouthwash or other oral rinse, a lozenge, and a gum. Details with regard to formulating oral hygiene compositions can be found in, for example, U.S. Patent No. 5,686,064 to Gaffar et al., U.S. Patent No. 5,672,351 to Chikindas, and U.S. Patent No. 5,578,295 to Francis et al., which are hereby incorporated by reference. Other processes for formulating oral hygiene compositions are well known in the art.
  • a contraceptive that includes a contraceptive carrier and a TSP or a TSP analog.
  • the contraceptive carrier can be a contraceptive composition or a contraceptive device.
  • Suitable contraceptive compositions include contraceptive foams, jellies, lubricants, and spermicides.
  • the contraceptive composition can be a pharmaceutical composition of the present invention. Details with regard to the formulation of contraceptive compositions, such as contraceptive foams, jellies, lubricants, spermicides, and the like, are generally known to those skilled in the art and are described in, for example, U.S. Patent No. 5,595,980 to Brode et al., which is hereby inco ⁇ orated by reference.
  • Suitable contraceptive devices include sponges, cervical diaphragms and caps, intravaginal devices, and condoms.
  • Contraceptive devices containing molecules which include TSP or TSP analogs can be prepared by methods described in, for example, U.S. Patent No. 5,571,567 to Shah et al., which is hereby inco ⁇ orated by reference. Further details regarding contraceptive formulation and manufacture can be found in U.S. Patent No. 5,545,615 to Maraganore and U.S. Patent No. 5,527,534 to Myhling, which are hereby inco ⁇ orated by reference. Other processes for preparing contraceptive formulations and contraceptive devices are well known in the art.
  • a contraceptive composition in accordance with the present invention can be used in combination with a contraceptive device of the present invention.
  • a condom or sponge of the present invention can contain a spermicide of the present invention.
  • a non-contraceptive prophylactic device capable of suppressing HIV infectivity.
  • another aspect of the present invention relates to a non-contraceptive prophylactic device that includes a non-contraceptive carrier and a TSP or a TSP analog.
  • the non-contraceptive carrier can be a carrier other than the contraceptive carriers described above. Specifically, it is intended that the non- contraceptive carrier is a type of physical barrier.
  • suitable non-contraceptive carriers can take any form or configuration, but, preferably, they are in the form of a surgical glove, a dental dam, or an oral dam.
  • Non-contraceptive carriers for this aspect of the present invention can be prepared by methods described in, for example, U.S. Patent No. 5,571,567 to Shah et al., which is hereby inco ⁇ orated by reference.
  • Suitable surgical gloves can be of any conventional design, including, but not limited to, surgical gloves of the type disclosed in U.S. Patent No. 5,317,759 to Pierce, U.S.
  • Suitable dental dams can be of any conventional design, including, but not limited to, dental dams of the type disclosed in U.S. Patent No. 4,721,465 to Barasz and U.S. Patent No. 3,662,466 to McConville.
  • Suitable oral dams also can be of any conventional design, including, but not limited to, oral dams of the type disclosed in U.S. Patent No. 4,949,731 to Harding and U.S. Patent No. 5,388,592 to Williams, which are hereby inco ⁇ orated by reference.
  • Another aspect of the present invention relates to a method of inhibiting HIV infection in a patient.
  • This method includes administering TSP or a TSP analog to the patient under conditions effective to inhibit HIV infection.
  • the TSP or TSP analog should be administered prior to and, optionally, immediately following exposure to the HIV.
  • a pharmaceutical composition of the present invention, a contraceptive of the present invention, a non-contraceptive prophylactic device of the present invention, or combinations thereof can be used.
  • the actual amount of the TSP or TSP analog to be administered according to the present invention will vary according to the particular compound, the particular composition, and the particular mode of administration. Many factors that may modify the action of the TSP or TSP analog can be taken into account by those skilled in the art; e.g., time of administration, route of administration, condition of the subject, drug combinations, etc Optimal administration rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage administration tests.
  • the TSP is TSPl or TSP2.
  • a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Because the threat of spreading HIV is greatest via oral, vaginal, or rectal routes, it is preferable that the administration of the TSP or TSP analog is intraorally, intravaginally, or intrarectally.
  • Still another aspect of the present invention relates to a method of blocking HIV binding to a cell targeted by HIV.
  • This method includes contacting HIV or the cell targeted by HIV with TSP or a TSP analog under conditions effective to block binding of the HIV to the cell.
  • the TSP is TSPl or TSP2.
  • a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Regardless of whether the TSP or TSP analog is used to contact the HIV, the TSP or TSP analog is administered in a manner effective to contact the HIV gpl20 envelope protein. By contacting the gpl20 envelope protein, the HIV is effectively blocked from binding to its cellular target as described above.
  • the TSP is TSPl or TSP2.
  • a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Regardless of whether the TSP or TSP analog is used to contact the cell, the TSP or TSP analog is administered in a manner effective to contact the CD4 receptor. By contacting the CD4 receptor, the HIV is effectively blocked from binding to its cellular target as described above.
  • TSP or TSP analogs can affect the activity of certain types of immunomodulatory proteins which are chemotactic cytokines, called "chemokines.”
  • Chemokines are small molecular weight immune ligands which are chemoattractants for leukocytes, such as neutrophils, basophils, monocytes, and T cells. There are two major classes of chemokines, an ⁇ -class and a ⁇ -class. Chemokines in each class contain four conserved cysteine residues that form disulfide bonds in the tertiary structure of the proteins.
  • the ⁇ -class is characterized by C-X-C motifs (where X is any amino acid) and includes IL-8, CTAP-III, gro/MGSA, and ENA-78.
  • the ⁇ -class is characterized by C-C motifs and includes MCP- 1 , MIP- 1 ⁇ , MIP- 1 ⁇ , and the regulated on activation normal T expressed and secreted protein ("RANTES").
  • RANTES regulated on activation normal T expressed and secreted protein
  • the designations of the classes are according to whether an intervening residue spaces the first two cysteines in the motif.
  • ⁇ -chemokines (C-X-C) are chemoattractants for neutrophils but not monocytes, whereas ⁇ -chemokines (C-C) appear to attract monocytes but not neutrophils.
  • chemokines the "C” group
  • lymphotactin a third group of chemokines
  • the chemokine family is believed to be critically important in the direction of lymphocytes and monocytes into sites of inflammation.
  • TSP and TSP analogs are capable of binding to chemokines, particularly to the chemokine RANTES.
  • RANTES is important for two reasons. First, RANTES is involved in inflammation (Barnes et al, "Polyclonal Antibody Directed Against Human RANTES Ameliorates Disease in the Lewis Rat Adjuvant-induced Arthritis Model," J. Clin. Invest. 101(2):2910-2919 (1998), which is hereby inco ⁇ orated by reference). Second, the natural receptor for RANTES is CCR5, the principal co-receptor that permits HIV to bind to and infect cells.
  • a further aspect of the present invention relates to a method of treating or preventing inflammatory states in a patient.
  • This method includes administering an effective amount of TSP or a TSP analog to the patient.
  • TSP is administered to the patient in accordance with this process of the present invention, it is preferred that the TSP is TSPl or TSP2.
  • a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1).
  • autoimmune diseases such as insulin-dependent diabetes, Goodpasture's syndrome, pemphigus and pemphigoid, primary biliary cirrhosis, ulcerative colitis, rheumatoid arthritis, scleroderma, mixed connective tissue disease and lupus erythematosus
  • graft versus host disease s
  • Patients which can be treated according to this aspect of the present invention include any mammal, such as a mouse, a rat, a dog, or a human.
  • administration of the TSP or TSP analog to humans suffering from various inflammatory states, such as those described above, is contemplated.
  • TSP or TSP analogs of the present invention can be administered by any suitable and accepted route of drug administration, including those described above.
  • TSP or TSP analogs can be administered as a component in a pharmaceutical composition of the present invention.
  • pharmaceutical compositions of the present invention can also contain other anti-inflammatory compounds and/or materials commonly used in pharmaceutical formulations.
  • Yet another aspect of the present invention relates to a method of blocking the binding of a chemokine to its receptor.
  • This method includes contacting a chemokine with a TSP or a TSP analog under conditions effective to bind the chemokine.
  • the binding of the chemokine is believed to interfere with its affinity for (i.e., its ability to bind) its receptor.
  • TSP When TSP is used to contact the chemokine in accordance with this process of the present invention, it is preferred that the TSP is TSPl or TSP2.
  • TSP analog When a TSP analog is used to contact the chemokine in accordance with this process of the present invention, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1).
  • This method of the present invention is particularly effective when the chemokine to be bound by the TSP or TSP analog is a ⁇ -chemokine and the corresponding receptor is a ⁇ -chemokine receptor.
  • a chemokine which is effectively bound by TSP or TSP analogs of the present invention is RANTES.
  • Other chemokines of the RANTES family are expected to be effectively blocked as well.
  • the chemokine receptor which is most effectively blocked against RANTES binding is a RANTES receptor, particularly a CCR5 receptor.
  • Human platelet-rich plasma was obtained from the NY Blood Center. Human thrombin was from Boeringer-Mannheim (Indianapolis, IN). Purified human calcium-replete TSPl was prepared from releasate of thrombin-activated washed platelets as described previously (Pearce et al., "Recombinant GST/CD36 Fusion
  • Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium- Dependent Binding Site on CD36," J. Biol. Chem., 270:2981-2986 (1995); Silverstein et al., "Platelet Thrombospondin Forms a Trimolecular Complex with Plasminogen and Histadine-Rich Glycoprotein,” J. Clin. Invest. 75:2065-2073 (1985), which are hereby inco ⁇ orated by reference).
  • Dot blot analysis with monoclonal antibodies (mAb) to fibronectin and vitronectin (Calbiochem, La Jolla, CA) showed no reactivity.
  • GST glutathione-S-transferase
  • CFP67-157 and LFP75-155 contain functionally homologous minimal TSPl binding domains (CP36 aa93-120) (Pearce et al, "Recombinant GST/CD36 Fusion Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium- Dependent Binding Site on CD36," J. Biol. Chem.. 270:2981-2986 (1995), which is hereby inco ⁇ orated by reference) while CFP298-439 and LFP156-243 represent downstream sequences.
  • a truncation mutant LFP75-78 was used as an additional GST1 control.
  • Recombinant baculovirus-expressed HIV envelope glycoprotein g ⁇ l60 was derived from HIV-1 isolate IIIB (IntraCel Co ⁇ ., Cambridge, MA). The following purified recombinant proteins were provided by the NIH AIDS Research and Reference Reagent Program: baculovirus-expressed HIV-1 gpl20 derived from LAV and MN isolates, CHO cell- expressed soluble CD4, and HIV-IMN env synthetic peptides.
  • TSPl (5-10 ⁇ g/ml) or fusion proteins and peptides (10-20 ⁇ g/ml) were immobilized on detachable 96-well microtiter plate strips (Immulon-4 Remov-a-well, Pynatech Laboratories, Inc.), by overnight incubation at 4°C in carbonate buffer (100 mM Na 2 CO 3 /l mM MgCl 2 /0.02% NaN 3 , pH 9.8). Washed wells were blocked with 0.5% bovine semm albumin ("BSA”), then incubated in triplicate with soluble radiolabeled ligand for 2.5 hours at 37°C. After extensive washing in 50 mM Tris pH 7.5, 150mM NaCI, 0.5%> Tween-20 (TBS-Tween), bound radioactivity was quantified by gamma counter.
  • BSA bovine semm albumin
  • Radiolabeling was performed with Na[ I] (Amersham Life Science Inc., Arlington Heights, IL) using immobilized chloramine T (Iodo-beadsTM; Pierce Chemical Co., Rockford, IL) (Silverstein et al, "Thrombospondin: A Versatile Multifunctional
  • Thin tissue sections of oral mucosa autopsy specimens were processed as described (Hajjar et al., "Lipoprotein (a) Modulation of Endothelial Cell Surface Fibrinolysis and its Potential Role in Atherosclerosis," Nature. 339:303-305 (1989), which is hereby inco ⁇ orated by reference).
  • Formalin-fixed paraffin-embedded sections were de- waxed, pronase treated, and permeabilized in Triton X-100. Endogenous peroxidase activity was blocked by treatment with a 3% solution of H O 2 for 30 minutes. Slides were pre-incubated with normal human semm for 1 hour at
  • ELISA sandwich enzyme-linked immunosorbent assay
  • PBMC peripheral blood mononuclear cells
  • PHA phytohemagglutinin
  • PBMC medium also contained 32 U/ml interleukin-2 (Sigma Chemical Co., St. Louis, MO).
  • TSP samples were pre-screened to assure that levels of TSP were ⁇ 10 ⁇ g/ml.
  • Acute HIV infection was performed using HIV-1 isolate IIIB stock vims as previously described (Laurence et al, "Human Immunodeficiency Vims Infection of Helper T-Cell Clones: Early Proliferative Oefects Pespite Intact Antigen-Specific Recognition and Interleukin 4 Secretion," J. Clin.
  • Saliva and TSPl inhibition assays 100 ⁇ l of HIV-1 /IIIB inocula were mixed with 100 ⁇ l of various concentrations of whole saliva, salivary fluid fractions, or purified platelet TSPl diluted in semm-free cell culture medium, and pre-incubated for 2 hours at 37°C. Then, 10-fold serial dilutions were added directly to target cell cultures for assay of infectivity. In some experiments, pre-incubated virus-saliva/TSP mixtures were filtered through 0.2 ⁇ m nitrocellulose syringe filters (Gelman Sciences, Ann Arbor, MI) prior to inoculation of target cells.
  • TSPl An affinity column for adso ⁇ tion of TSPl was prepared using fusion protein LFP75-155 coupled by N-hydroxysuccinimide to Sepharose (HiTRAPTM, Pharmacia). Two ml of clarified whole saliva was diluted 1 : 1 in PBS and divided into two aliquots, one of which was applied to the TSP-binding GST/LIMPII column, the other to an identical column containing N-hydroxysuccinimide-linked GST alone. Columns were incubated 45 minutes at 37°C, then each flushed with 1 ml PBS. The final products represented 1 :4 dilutions of saliva. TSP concentrations were determined before and after column adso ⁇ tion by sandwich ELISA, and total protein assessed using the micro-BCA reagent kit.
  • CP4 surface expression on HIV-1 infected or uninfected U937 cells was evaluated by indirect immunofluorescence flow cytometry using anti-CP4 mAb Leu3, as detailed elsewhere (Laurence et al, "Human Immunodeficiency Virus Infection of Helper T-Cell Clones: Early Proliferative Pefects Pespite Intact Antigen-Specific Recognition and Interleukin 4 Secretion," J. Clin. Invest.. 83:1843- 1848 (1989), which is hereby inco ⁇ orated by reference).
  • LIMPII homolog lysosomal integral membrane protein II
  • Vega et al "Cloning, Sequencing, and Expression of a cPNA Encoding the Rat LIMP II, a Novel 74-kPa Lysosomal Membrane Protein Related to the Surface Adhesion Protein CP36," J. Biol Chem.. 266:16818-16824 (1991); Calvo, P.
  • FIG. 1 shows the amino acid alignment of CP36/LIMPII CLESH-1 motifs with HIV-1 matched sequences. Strongest homology appeared as a split motif localized to domains C2 and C3 on either side of the V3 loop region of gpl20. Homology was highest to LIMPII, with 62.5%o identity, 87.5% similarity for the first half-site (C2 domain, 16 aa), and 55.6%> identity, 88.9% similarity for the second half-site (C3 domain, 18 aa).
  • FIG. 2 A shows concentration-dependent, saturatable binding of radiolabeled soluble TSPl to immobilized recombinant gpl60 (gpl20 non-covalently linked to transmembrane component gp41).
  • An apparent affinity of approximately 250 nM was comparable to that demonstrated for binding of TSPl to purified platelet CP36. Binding was effectively quenched in the presence of 10-fold molar excess unlabeled soluble TSPl ( Figure 2B, 94 ⁇ 6% inhibition), demonstrating specificity.
  • LIMPII fusion protein LFP75-155 containing the TSP binding domain partially blocked binding (47 ⁇ 14% inhibition), while control fusion protein LFP 156-243 representing downstream LIMPII sequences did not, supporting the existence of a functionally similar domain in HIV-1 env.
  • the CSVTCG (SEQ. IP. No. 1) peptides found in the Type 1 repeats of TSPl are binding sites for CP36 and LIMPII (Crombie et al, "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CP36 Pefines a conserveed Recognition Motif," J.
  • the gpl20 envelope protein was tested to determine whether it shared this same specificity. Saturation isotherms showed significant binding of radiolabeled gpl20 (LAI isolate) to immobilized CSVTCG peptide (SEQ. IP. No. 1), with an apparent affinity of 300 nM ( Figure 3). The activity was sequence-specific, as demonstrated by inefficient binding to scrambled control peptide VGSCCT (SEQ. IP. No. 2), or to an RGP-containing peptide similar to the GRGPA (SEQ. IP. No. 3) cell adhesion sequence of the last TSPl Type 3 calcium-binding repeat.
  • FIG. 4A shows the location of a series of 20 aa synthetic peptides with respect to gpl20 domain structure (MN isolate). Given the constraints of the solid phase assay, peptides were immobilized either singly or in pairs, and tested for ability to bind radiolabeled TSPl . As shown in Figure 4B, active peptides corresponded to regions of gpl20 containing homologous CLESH-1 motifs. Peptide pairs that extended the represented motif sequence showed augmented binding as compared to either peptide alone.
  • Figure 5 shows competitive inhibition of radiolabeled CP4 binding to gpl20 derived from two different viral isolates (MN and LAI).
  • MN and LAI different viral isolates
  • CSVTCG SEQ. IP. No. 1
  • peptide showed partial but significant inhibition of CP4-gpl20 complex formation (53 ⁇ 9%>), confirming a TSP- specific effect, while the RGP-containing peptide had little effect (4 ⁇ 4%), and the scrambled control actually enhanced binding.
  • TSPl -binding CP36- and LIMPII-derived fusion proteins proved strong competitors (both ⁇ 89% inhibition), whereas downstream LIMPII control protein had minimal effect (32 ⁇ 5%).
  • a 1 :2 dilution of whole saliva was a potent inhibitor in this assay system.
  • TSPl in whole saliva from either HIV+ or HIV- donors were at least 10-fold greater (1-12 ⁇ g/ml) compared to plasma.
  • Amounts of TSPl in parotid saliva fractions were equivalent to that of plasma, with the bulk of TSPl found in the submandibular secretions.
  • Table 1 Quantitation of TSPl in Human Plasma and Saliva Sample Source HIV Status TSP1/TSP2 cone, (range) n plasma negative 0.25 (0.1-0.34) ⁇ g/ml 8 whole saliva negative 4.1 (1.1-12.8) ⁇ g/ml 6 positive 3.3 ⁇ g/ml 2 parotid negative 0.1 ⁇ g/ml 1 submandibular negative 2.5 ⁇ g/ml 1
  • TSPl reduced HIV-1 infection of PHA-activated donor PBMC by >83% when pre-filtered, as measured by ELISA detection of p24 viral antigen (Figure 7). This was comparable to a 1 :2 dilution of whole saliva. In contrast, fibrinogen, another high molecular weight adhesive glycoprotein in saliva, had no effect in this system. Specificity was documented by abrogation of the inhibitory effect in the presence of a specific anti-TSP 1 polyclonal antibody (107%) of control p24), but not control IgG.
  • TSPl-virion mixture Prolonged incubation of the TSP-virion mixture was unnecessary, as exposures as brief as 5 minutes appeared sufficient to reduce infectivity by > 50% at 1 ⁇ g/ml TSPl.
  • the TSPl effect also was apparent for HIV-1 IIIB infection of CP4+ T- lymphoblastoid and monocytoid cell lines (SK23 > 90.7% and U937 > 83.0% inhibition, respectively).
  • two monocytotropic strains of HIV-1, pl3 and HA593 representing patient isolates obtained from the NIH AIPS Retroviral Repository were susceptible to inhibition by purified TSPl . At viral moi's of 0.8, 100 ⁇ g/ml TSPl inhibited HIV infectivity by > 98 ⁇ 1%.
  • TSP depletion correlated with >70% reduction in anti-viral activity, in contrast to saliva adsorbed using a control fusion protein affinity column (GST-1). The data suggest that TSP may account for a major proportion of HlV-specific inhibitory activity in saliva.
  • LIMPII TSP-binding peptide L75-155 (10 kPa product purified after removal of GST moiety by proteolytic cleavage) was included in HIV-TSP1 pre-incubation mixtures as a competitor.
  • Figure 8 shows that 1 ⁇ M LIMPII peptide abrogated the inhibitory effect of even high concentrations of TSPl (50-100 ⁇ g/ml) by 83-90%.
  • Incubation of virus in the presence of LIMPII peptide alone resulted in minimal decrease of HIV-1 infectivity ( ⁇ 9%), suggesting that amounts of peptide able to block the TSPl anti-viral effect were not sufficient to compete for HIV-1 env binding sites on PBMC target cells.
  • the ability of the LIMPII peptide to restore infectivity supports a direct role for a CD36/LIMPII-related TSPl binding domain on HIV-1 gpl20, and provides further evidence of a common binding site on TSPl.
  • TSP-mediated blockade to HIV infection would be a direct effect on target cells, whereby alterations in cell function would decreased the capacity to support productive infection.
  • CD4+ Jurkat and SK23 T-cell lines, as well as PHA-activated PBMC were monitored for differences in CD4 surface expression after culture for 3 days in the absence or presence of 100 ⁇ g/ml TSP 1.
  • flow cytometric analysis of cells stained with fluorescein-conjugated anti-CD4 IgG no change in relative fluorescence intensity or percent CD4+ cell was detected.
  • TSPl likely does not reduce cell susceptibility.
  • TSPl secretion was monitored in three groups of cells: an uninfected line of U937 promonocytic cells, Ul.l cells representing chronically infected U937 containing 2 stably integrated copies of HIVOl/LAI, and U937 acutely infected with HIV-1 to high copy number (> 1000 proviral copies/cell, (Laurence et al, "Human
  • PMA phorbol myrisate acetate
  • the present invention concerns, in part, the identification of a TSPl binding site in the C2 and C3 regions of gpl20, conserved areas of the HIV envelope which are important in binding to CD4, and the demonstration of direct interaction between gpl20 and a specific cell adhesion sequence found in the TSPl type I repeat. Characterization by in vitro binding and competition studies substantiates that these CP36/LIMPII-related CLESH-1 motifs in gpl20 represent authentic TSPl binding domains.
  • TSPl-gpl20 complex formation The physiological significance of TSPl-gpl20 complex formation is supported by observations that: salivary inhibition of HIV-1 infectivity was markedly reduced by affinity depletion of TSPl; saliva samples that block infection following filtration contained levels of TSPl that correlate with inhibitory concentrations of purified TSPl; and higher amounts of TSPl required to block HIV-1 infectivity in vitro are comparable to the greater quantities of saliva required to obtain an anti -viral effect (Archibald et al, "In Vitro Inhibition of HIV-1 Infectivity by Human Salivas," AIPS Res. Hum. Retrovimses. 6:1425-1432 (1990); Bergey et al, "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic Syndr., 7:995-1002 (1994), which are hereby inco ⁇ orated by reference).
  • Our findings establish a distinct mechanism to explain HlV-specific blockade of transmission via saliva.
  • TSPl is synthesized in low amounts by monocytes/macrophages, epithelial cells, fibroblasts, smooth muscle cells, pneumocytes, and endothelial cells, and in larger quantities by platelets (Lahav, editor, "Thrombospondin,” CRC Press, (1993), which is hereby inco ⁇ orated by reference).
  • platelets Lahav, editor, "Thrombospondin," CRC Press, (1993), which is hereby inco ⁇ orated by reference.
  • HIV may be exposed to levels of TSPl over 2-log higher on surfaces in the oral cavity.
  • the fluid distribution of TSPl with very low concentrations in plasma, sweat, tears, and urine, reflects the relative frequency with which HIV can be isolated from these secretions, but not from saliva.
  • Breast secretions present another issue. HIV can be cultured from some samples of breast milk, which has been implicated in HIV transmission.
  • Colostrum often contains high concentrations of TSPl (upwards of 145 ⁇ g/ml), while lower, more variable amounts (to ⁇ 1 ⁇ g/ml) have been measured in other breast secretions (Pawes et al, "Thrombospondin in Milk, Other Breast Secretions and Breast Tissue," Sem. Thromb. Heost, 13:378-384 (1987), which is hereby inco ⁇ orated by reference).
  • HIV has not been recovered from breast milk devoid of cells (Guay et al, "Petection of Human Immunodeficiency Vims Type 1 (HIV-1) PNA and p24 Antigen in Breast Milk of HIV-1 -Infected Kenyan Women and Verticle Transmission," Pediatrics, 98:438-444 (1996), which is hereby inco ⁇ orated by reference), and breast milk contains factors which inhibit HIV infection (VandePerre et al, "Infective and Anti-Infective Properties of Breast Milk from HIV-1 Infected Women," Lancet, 341 :914-918 (1993), which is hereby inco ⁇ orated by reference), one of which may be TSP.
  • fibronectin binds free g ⁇ l20 (Su et al, "Interaction of the Envelope Glycoprotein of Human Immunodeficiency Virus with Clq and Fibronectin Under Conditions Present in Saliva," Moi Immunol, 28:811-817 (1991), which is hereby inco ⁇ orated by reference), and thereby might sequester HIV virions.
  • FN actually may facilitate HIV-mediated syncycium formation (Ushijima et al, "Effect of Semm Components on Syncytium Formation and Virus Production by Cells Infected with Human Immunodeficiency Viruses In Vitro," AIPS Res. Hum.
  • Retroviruses 8:513-520 (1992), which is hereby inco ⁇ orated by reference), and promote the growth of AIPS-KS (Kaposi sarcoma) cells constitutively expressing high levels of FN receptor (Barillari et al, "The RGP Motif and the Integrin Receptors are Involved in the Vascular Cell Growth and Adhesive Properties of Extracellular HIV-1 Tat Protein," J. AIPS. 6:688A (1993), which is hereby inco ⁇ orated by reference), bringing further into question the physiological significance of FN-gpl20 binding.
  • AIPS-KS Koaposi sarcoma
  • TSPl proteolytic fragments and peptides show opposite effects, inhibiting KS/endothelial cell proliferation (Taraboletti et al, "Platelet Thrombospondin Modulates Endothelial Cell Adhesion, Motility, and Growth: A Potential Angiogenesis Regulatory Factor,” J. Cell Biol. 1 11 :765-772 (1990); Roberts et al, “Modulation of Tumor Growth In Vitro and In Vivo by Stable Analogs of Thrombospondin Peptides," AIPS Res. Hum. Retrovimses, 21 :S73 (1995), which are hereby inco ⁇ orated by reference).
  • the requirement for pre-filtration may have its in vivo counte ⁇ art in the continued cleansing of oral surfaces by salivary flow, with elimination of enmeshed viral particles from potential attachment sites.
  • Experiments testing direct inhibition by whole saliva are complicated by the fact that additional salivary components may contribute to the anti-HIV effect. Indeed, TSPl affinity depletion removed only ⁇ 70% of the HIV-inhibitory activity.
  • nonspecific antiviral phenomena may occur with saliva dilutions of 1:1 to 1:4 (Liuzzi et al, "Analysis of HIV-1 Load in Blood, Semen and Saliva: Evidence for Pifferent Viral Compartments in a Cross- Sectional and Longitudinal Study," AIPS.
  • Piscontinuity created by intervening residues between the first and second motif half- site could induce conformational strain to distort or physically disrupt V3 loop integrity, with profound negative effects on gpl20-CP4 association.
  • Binding of TSP to the first highly homologous half-site in the C2 domain might lead to subsequent binding to weakly homologous downstream residues extending into the V3 loop, freeing the second full-length TSP binding site in the C3 domain.
  • the second motif encompasses a serine pair, KQSS (SEQ. IP. No.
  • TSPl could compete directly with CP4, occupying an identical or overlapping site.
  • the bulky TSPl trimer may sterically block access to the CP4 binding site, as well as V3 loop determinants. This model could explain why only partial inhibition is seen using CSVTCG peptides (SEQ. IP. No. 1). Elucidation of the molecular basis for TSPl interference of CP4-gpl20 association awaits additional structural studies. Anti-HIV activities vary quantitatively among individual saliva donors.
  • TSPl binding motifs in highly conserved HIV domains makes these sites attractive targets for blocking agents that would be broadly reactive to HIV-1 and HIV-2 substrains.
  • the ability of TSPl to block CD4-gpl20 complex formation suggests the potential utility of this matrix molecule in the development of non-toxic natural inhibitors of local transmission of HIV-1, perhaps as a candidate topical adjuvant that could serve as a preventive physical barrier for rectogenital and GI tract mucosa.
  • I-CP4 binded to immobilized TSPl in a concentration dependent saturatable manner, with biphasic kinetics.
  • the results of this study are shown in Figure 12. Additionally, 125 I-CP4 binded directly to the TSP analog having an amino acid sequence CSVTCG (SEQ. IP. No. 1), implicating the presence of a CP36-related binding site (i.e., a CLESH-1 motif) in CP4.
  • CSVTCG SEQ. IP. No. 1
  • a CP36-related binding site i.e., a CLESH-1 motif

Abstract

The present invention relates to a method for suppressing infectivity of HIV which includes contacting the HIV or a cell targeted by HIV with an effective amount of a thrombospondin or a thrombospondin analog. Other aspects of the invention relate to contraceptives, pharmaceutical compositions, and non-contraceptive prophylactic devices that include a carrier and a thrombospondin or a thrombospondin analog. Methods of inhibiting HIV infection in a patient, blocking HIV binding to a cell, blocking chemokine binding to its receptor, and treating or preventing inflammatory states in a patient are also disclosed.

Description

METHODS AND COMPOSITIONS FOR INHIBITING HIV INFECTIVITY AND BLOCKING CHEMOKINE ACTIVITY
This application claims benefit of U.S. Provisional Patent Application Serial No. 60/066,294, filed November 25, 1997, and U.S. Provisional Patent Application Serial No. 60/078,873, filed March 20, 1998. This invention was made with support under National Institutes of Health Grant Nos. DE-11348, HL-55646, AI-41327, HL422540, and T32HL07029. The U.S. Government may have certain rights to this invention.
FIELD OF THE INVENTION
The present invention relates to methods which inhibit HIV infectivity, block HIV binding to a cell, block chemokine binding to its receptor, and treat or prevent inflammatory states in a patient, as well as pharmaceutical compositions, contraceptives, and non-contraceptive prophylactic devices.
BACKGROUND OF THE INVENTION
Acquired immunodeficiency syndrome ("AIDS") has been described as the first great pandemic of the second half of the twentieth century. Gallo, Sci. Am., 256:39 (1987). Human immunodeficiency virus ("HIV") is the etiological agent of AIDS. A complete sequencing of the HIV genome indicates that it contains the same overall gag-pol-env organization as other retroviruses. Ratner et al., "Complete Nucleotide Sequence of the AIDS virus, HTLV-III," Nature 313:277-84 (1985). HIV invades a host cell and uses the host cell's machinery to replicate itself.
HIV can be cultured from most tissues and body fluids of infected individuals. Saliva represents a significant exception. In an early report, HIV- 1 was isolated from only one of 71 saliva samples of HIV+ donors (Ho et al, "Infrequency of Isolation of HTLV-III Virus from Saliva in AIDS," New Engl. J. Med.. 313:1606 (1985)). Recent work confirmed the paucity of infectious virus in saliva (Groopman et al, "HTLV-III in Saliva of People with AIDS-Related Complex and Healthy Homosexual Men at Risk for AIDS," Science. 226:447-449 (1994)), with a mean viral load in 25 samples of 162 genome equivalent/ml, at the limits of detection by reverse transcription-polymerase chain reaction ("RT-PCR") (Liuzzi et al., "Analysis of HIV- 1 Load in Blood, Semen and Saliva: Evidence for Different Viral Compartments in a Cross-Sectional and Longitudinal Study," AIDS. 10:F10-F56 (1996)). Clinical support for the limited transmissibility of HIV by saliva includes: lack of infection following contamination of open wounds with saliva from HIV+ individuals (CDC, "Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and Other Blood-Borne Pathogens in Healthcare Settings," Morbid. Mortal. Wklv. Rep.. 37:377-388 (1988)); low occupational risk for HIV infection among dentists in practices with large numbers of patients at risk for HIV infection (Klein et al., "Low Occupational Risk of Human Immunodeficiency Virus Infection Among Dental Professionals," New Engl. J. Med., 318:86-90 (1988)); and the inability to infect adult chimpanzees by direct application of HIV to intact oral mucosa (Fultz, "Components of Saliva Inactivate Human Immunodeficiency Virus," Lancet. ii:1215 (1986)).
Such retarded transmission is not a general characteristic of viruses which can be shed orally. The annual attack rate for hepatitis B virus among unvaccinated dentists is 2.6% (Remis et al., "Hepatitis B Infection in a Day School for Mentally Retarded Students: Transmission from Students to Staff," Am. J. Public Health, 77:1183-1186 (1987)), human T cell lymphotrophic virus type I is found in saliva (Achiron et al., "Detection of Proviralpuman T-Cell Lymphotropic Virus Type I DNA in Mouthwash Samples of HAM/TSP Patients and HTLV-I Carriers," Arch. Virol., 141 :147-153 (1996)), and the type D retrovirus etiologic in a simian immune-deficiency syndrome can be readily isolated from macaque saliva and spread by this fluid (Lercke et al., "Inapparent Carrier of Simian Acquired Immune Deficiency Type D Retrovirus and Disease Transmission with Saliva," J. Natl. Cancer Inst, 77:489-495 (1986)). The ability of saliva to suppress HIV-1 also is relatively specific. It does not alter the infectivity of Herpes simplex virus (Malamud et al., "Human Submandibular Saliva Aggregates HIV," AIDS Res. Hum. Retroviruses.
9:633-637 (1993)), and both cytomegalovirus and Epstein-Barr virus are readily shed in oral secretions of HIV seronegative (Fox et al., "Saliva Inhibits HIV-1 Infectivity," J. Am. Dent. Assoc, 116:635-637 (1988)) and seropositive persons (Alsip et al., "Increased Epstein-Barr Virus DNA in Oropharygeal Secretions from Patients with AIDS, AIDS-Related Complex, or Asymptomatic Human Immunodeficiency Virus Infections," J. Infect. Pis., 157:1072-1076 (1988); Scholes et al, "Oral Shedding of CMV and HSV in Relation to HIV Disease," IXth Intl. Conf. AIDS. Berlin, June 6- l l :Abst. PO-B18-1801 (1993)).
In contrast, other body fluids from HIV+ individuals contain HIV in relatively high titers, including tears (CDC, "Recommendations for Preventing Possible Transmission of Human T-Lymphotropic Virus Type III/Lymphadenopathy- Associated Vims from Tears," Morbid. Mortal. Wkly. Rep.. 34:533-534 (1986)), genital secretions (Mostad et al., "Shedding of HIV in the Genital Tract," AIDS, 10:1305-1315 (1996)), feces (Yolken et al., "Persistent Diarrhea and Fecal Shedding of Retroviral Nucleic Acids in Children Infected with Human Immunodeficiency Virus," J. Infect. Pis., 164:61-66 (1991)), and breast milk (VandePerre et al., "Infective and Anti-Infective Properties of Breast Milk from HIV-1 Infected
Women," Lancet, 341 :914-918 (1993)). Genital secretions, feces, and breast milk have all been implicated in HIV transmission.
Particulate and filterable oral secretions capable of inhibiting HIV infection represent potential explanations for the paucity of HIV in saliva. Reports from several different groups imply that two processes are involved (Fultz,
"Components of Saliva Inactivate Human Immunodeficiency Virus," Lancet, ii:1215 (1986); Malamud et al., "Human Submandibular Saliva Aggregates HIV," AIDS Res. Hum. Retrovimses. 9:633-637 (1993); Fox et al, "Saliva Inhibits HIV-1 Infectivity," J. Am. Dent. Assoc, 116:635-637 (1988); Fox et al, "Salivary Inhibition of HIV-1 Infectivity: Functional Properties and Distribution in Men, Women and Children," J. Am. Dent. Assoc, 118:709-711 (1989); Archibald et al, "In Vitro Inhibition of HIV- 1 Infectivity by Human Salivas," AIPS Res. Hum. Retroviruses, 6:1425-1432 (1990); McNeely et al., "Secretory Leukocyte Protease Inhibitor: A Human Saliva Protein Exhibiting Anti-Human Immunodeficiency Vims I Activity In Vitro," J. Clin. Invest., 96:456-464 (1995); Malamud et al, "HIV in the Oral Cavity: Virus, Viral Inhibitory Activity, and Antibodies: A Review," Crit. Rev. Oral. Biol. Med.. 4:461-466 (1993); Amory et al., "The Large Molecular Weight Glycoprotein MGI, a Component of Human Saliva, Inhibits HIV-1 Infectivity," Clin. Res.. 40:51A (1992); Yeh et al., "Further Studies of Salivary Inhibition of HIV-1 Infectivity," J. Acquired Immune Pefi Svndr.. 5:898-903 (1992); Bergey et al, "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic Syndr., 7:995-1002 (1994); Phillips et al., "Low Level of Cell-Free Virus Petected at High Frequency in Saliva from HIV-1 Infected Individuals," AIPS, 8:1011-1012 (1994)). Some studies found that whole saliva and submandibular secretions, but not parotid fluid, could sequester HIV virions (Malamud et al, "Human Submandibular Saliva Aggregates HIV," AIPS Res. Hum. Retrovimses, 9:633-637 (1993); Fox et al, "Saliva Inhibits HIV-1 Infectivity," J. Am. Pent. Assoc. 116:635-637 (1988); Fox et al, "Salivary Inhibition of HIV-1 Infectivity: Functional Properties and Pistribution in Men, Women and Children," J. Am. Pent. Assoc, 118:709-711 (1989); Archibald et al., "In Vitro Inhibition of HIV- 1 Infectivity by Human Salivas," AIPS Res. Hum. Retrovimses. 6:1425-1432 (1990); Bergey et al., "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic Syndr., 7:995-1002 (1994)), while others identified soluble inhibitory factors capable of direct inhibition in secretions from all salivary glands, but only at very high concentrations (Yeh et al., "Further Studies of Salivary Inhibition of HIV-1 Infectivity," J. Acquired Immune Pefic Svndr.. 5:898-903 (1992); Phillips et al., "Low Level of Cell-Free Vims Petected at High Frequency in Saliva from HIV-1 Infected Individuals," AIPS. 8:1011-1012 (1994)). Submandibular saliva contains sulfated polysaccharides of low (MG2) and high (MGI) molecular weights (Levine et al, "Structural Aspects of Salivary Glycoproteins," J. Pent. Res.. 66:436-441 (1987)), with the latter forming an anionic charge barrier to binding of the high affinity HIV receptor, CP4, to the HIV envelope glycoprotein gpl20 (Amory et al., "The Large Molecular Weight Glycoprotein MGI, a Component of Human Saliva, Inhibits HIV-1 Infectivity," Clin. Res., 40:51A (1992)). Secretory leukocyte protease inhibitor (SLPI), a 12kPa protein found in whole saliva, has an effect independent of HIV binding to CP4 (McNeely et al., "Secretory Leukocyte Protease Inhibitor: A Human Saliva Protein Exhibiting Anti-Human Immunodeficiency Virus I Activity In Vitro," J. Clin. Invest., 96:456-464 (1995)), albeit its significance in vivo has been questioned (Bu et al., "Secretory Leukocyte Protease Inhibitor (SLPI) does not Effectively Inhibit HIV-1 Replication," 35th ICAAC. San Francisco, CA, Sept. 17-20:Abst. 1142 (1995)). Fibronectin, a matrix adhesion molecule, binds directly to gpl20, but was shown to inhibit infectivity only at high concentrations (Su et al., "Interaction of the Envelope Glycoprotein of Human Immunodeficiency Virus with Clq and Fibronectin Under Conditions Present in Saliva," Mol. Immunol. 28:811-817 (1991)).
None of the above-cited references has positively identified the factor in saliva which is capable of inhibiting HIV infectivity. The present invention is directed to overcoming this and other deficiencies in the art.
SUMMARY OF THE INVENTION
The present invention relates to a method for suppressing infectivity of HIV. This method is carried out by contacting the HIV or a cell targeted by HIV with an effective amount of a thrombospondin ("TSP") or a TSP analog. By contacting the HIV with an effective amount of a TSP or a TSP analog, the ability of HIV to bind to its cellular target is inhibited. Similarly, by contacting the cell targeted by HIV with an effective amount of a TSP or a TSP analog, the ability of HIV to bind to its cellular target is also inhibited. Both approaches are useful in suppressing HIV infectivity. The present invention further relates to a method of inhibiting HIV infection in a patient which includes administering a TSP or a TSP analog to a patient under conditions effective to inhibit HIV infection.
The present invention also relates to a method of blocking HIV binding to a cell targeted by HIV. This method includes contacting the HIV or the cell targeted by HIV with a TSP or a TSP analog under conditions effective to block binding of the HIV to the cell. Because HIV is capable of spreading via sexual contact as well as non- sexual contact (e.g., surgical procedures), pharmaceutical compositions, contraceptives, and non-contraceptive prophylactic devices capable of decreasing the likelihood of HIV infection are particularly desirable. Therefore, another embodiment of the present invention relates to a contraceptive that includes a contraceptive carrier and a TSP or a TSP analog. Also encompassed by the present invention is a pharmaceutical composition that includes a pharmaceutically-acceptable carrier and a TSP or a TSP analog. A non-contraceptive prophylactic device of the present invention includes a non-contraceptive carrier and a TSP or a TSP analog.
The present invention also relates to a method of blocking the binding of a chemokine to its receptor by contacting a chemokine with a TSP or a TSP analog under conditions effective to bind the chemokine. Because chemokines are generally implicated in inflammatory states, another aspect of the present invention relates to a method of treating or preventing inflammatory states in a patient by administering an effective amount of a TSP or a TSP analog to a patient.
By demonstrating the ability of purified thrombospondin- 1 ("TSP-1") to block HIV-1 infection of primary and transformed target cell lines through the binding of TSP 1 to C2 and C3 conserved regions adjoining the V3 loop of HIV-1 envelope glycoprotein gpl20 or through the binding of TSP 1 to the CO4 cell surface receptor, potential targets for therapeutic intervention in HIV infectivity has been identified. Previously, it has been shown that the V3 loop region of gpl20 is a key determinant for the binding of this glycoprotein to its high affinity CP4 cellular receptor (Ivanhoff, L.A. et al., "V3 Loop Region of the HIN-1 Envelope Protein is Essential for Vims Infectivity," Virology, 187:423-432 (1992), which is hereby incoφorated by reference). The effect of TSP 1, particularly the polypeptide sequence cys-ser-val-thr-cys-gly, on CP4-gpl20 complex formation provides a target for therapeutic intervention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an amino acid sequence alignment of CO36/LIMPII TSP binding motifs with homologous sequences in HIV-1 gρl20. Results of a pattern- based Blast enhanced alignment utility search that matched a split motif in gpl20 domains C2 (top) and C3 (bottom). Asterisks (*) indicate disulfide-bonded cysteine residues of the V3 loop. The brackets above alignments show boundaries of CP36 Exon 5 coding region (CP36 aa 95-143). Amino acids identical between either CP36 or LIMPII and HIV-1 gpl20 are highlighted. Bold residues represent conservative substitutions according to the following groups: basic (KRH), acidic (PE), charged (KRH, PE), aromatic (YRW), hydroxy (STY), aliphatic (AG), nonpolar/branched (IVL), hydrophobic (AGP, IVL, FM), polar/hydrophilic (ST, KRH, PNEQ, CWY), X = any aa, period = gap. GenBank™/EMBL accession numbers: huCP36, M24795; huLIMPII, P12676; HIV-1 partial sequence of isolate U37041 ; HIV-1 clade B consensus, MN and LAI isolate sequences were retrieved from the WHO HIV Sequence Oatabase.
Figure 2A is a graph showing concentration-dependent binding of I- TSP1 to LIMPII fusion protein. Increasing concentrations of soluble I-labeled TSP1 (1 nM - μM) were added to immobilized recombinant HIV-1 gpl60 for 3 hours at 22°C, and bound TSP1 measured after extensive washing. Non-linear curve fit was generated with Excel™ version 5.0. Apparent affinity was estimated from Scatchard analysis, (n = 2; error calculated as standard deviation, SP).
Figure 2B is a graph showing competitive inhibition of 125I-TSP1 for HIV-1 gρl60, which demonstrates the specificity of the TSP 1 -HIV interaction. A fixed concentration of 125I-labeled TSP1 (50 nM) was added to immobilized rgplόO in the absence or presence of 10-fold molar excess (0.5 μM) unlabeled TSP1, fusion protein LFP75-155 containing the LIMPII TSP binding domain (aa 75-155), or downstream fusion protein LFP 156-243. Samples were incubated, and bound TSP1 was measured as in Figure 2A. Plots represent single data sets of triplicate samples, (n = 3, error as SP). Figure 3 is a graph showing how I-gpl20 interacts with the CP36 binding peptide of TSP 1. Increasing concentrations of soluble I-labeled recombinant gpl20LAV (nM - 1 μM) were incubated with immobilized ligand for 2 hours at 37°C, and bound radioactivity measured as in Figures 2A and 2B. Shown are 125I-gpl20 binding to CSVTCG (SEQ. IP. No. 1) peptide derived from TSP1 properdin-like Type 1 repeat, scrambled control peptide VGSCCT (SEQ. IP. No. 2), and GRGPS (SEQ. IP. No. 3) derived from TSP1 calcium binding Type 3 repeat. This plot represents a single data set of triplicate samples (η = 2, errors as SP).
Figure 4 A is a map showing the position of synthetic gpl20 peptides with respect to gpl20 C2-V3-C3 domains and TSP binding motifs. A set of gpl20MN peptides (~ 20 aa) were immobilized either singly (numbered 1-7) or in pairs as indicated. Corresponding amino acid positions are: 1 = aa 271-290; 2 = aa 281-300; 3 = aa 291-310; 4 = aa 311-330; 5 = aa 331-351 ; 6 = aa 351-370; 7 = aa 3361-380, 1+2 and 6+7 overlap by 10 aa; gpl20 = full length rgpl20MN; V3 loop = aa 305-332; motif 1 = aa 272-321; motif 2 = aa 331-384.
Figure 4B is a graph showing the binding of 125I-TSP1 to gpl20
1 peptides. Increasing concentrations of soluble I-labeled TSPl (25 nM - 1 μM) were added to immobilized gpl20MN peptides. Samples were incubated, and bound TSPl was measured as in Figure 2. A single data set is shown for 25 nM TSPl binding to ~75 - 130 μmol peptide or ~2 μmol gpl20.
Figure 5 is a graph showing competitive inhibition of 125I-CP4 binding
1 S to gpl20. A fixed concentration of I-labeled soluble recombinant CP4 (50 nM) was added to immobilized rgpl60 (solid MN or hatched LAV isolate-derived) in the absence or presence of 10-fold molar excess (0.5 μM) unlabeled TSPl, TSPl-derived peptides CSVTCG (SEQ. IP. No. 1) or GRGPS (SEQ. IP. No. 3), scrambled control peptide VGSCCT (SEQ. IP. No. 2), fusion proteins containing the TSPl binding domain of CP36 (CFP, aa 67-157) or LIMPII (aa 75-155), downstream fusion protein LFP156-243, or an equal volume of saliva (final 2-fold dilution). Samples were incubated, and bound TSPl was measured as in Figure 2. A single data set of triplicate samples (η = 3, error as SP) is shown.
Figure 6 is an image showing immunohistochemical detection of cell- associated TSPl in human gingival mucosa. Fixed oral epithelial tissue thin section was incubated with polyclonal antiserum reactive against both TSPl and TSP2 (lower panel), or with pre-immune semm (upper panel), followed by biotinylated second antibody, and developed using avidin-conjugated peroxidase. Brown deposits indicate sites of TSP reactivity (magnified 200X).
Figure 7 is a graph showing the inhibitory effect of TSPl on HIV-1 infectivity. HIV-1 isolate IIIB was pre-incubated in the absence of TSPl, or with various concentrations of purified TSPl for 1 hour at 37 °C. Pre-incubated virus- TSP1 mixtures were added to target cells either directly (-), or first passed through 0.2 μm filters (+). Phorbol myrisate acetate ("PMA") activated primary peripheral blood mononuclear cells ("PBMC"), SK23 (T-cell line) or U937 (promonocytic line) were inoculated with the multiplicities of infection ("moi") indicated. After an additional 1 hour at 37 °C, infected cells were washed, cultured for 7 days, and HIV-1 p24 antigen measured by ELISA. Inhibition is expressed for a single data set as percent of maximum p24 detected in the absence of TSPl.
Figure 8 is a graph showing competitive inhibition of TSPl anti-HIV effect by LIMPII TSPl binding domain. PMA-stimulated primary PBMC were infected with 0.02 moi HIV/IIIB that had been pre-incubated with TSPl alone, or with LIMPII fusion protein LFP75-155 alone or in the presence of TSPl, and filtered prior to incubation as in Figure 7. TSP-mediated inhibition is expressed as percent decrease in p24 relative to maximum p24 detected in the absence of TSPl . Pata shown represent the average of two independent experiments (error as SP). Figure 9 is a graph showing the binding of 125I-RANTES to TSP 1.
Increasing concentrations of soluble 125I-labeled RANTES (0.01 μM - 10.0 μM) were
1 added to immobilized TSPl . Samples were incubated and bound I-labeled
RANTES measured by solid-binding radio-amino assay.
Figure 10 is a graph showing the Hanes analysis of 123I-RANTES binding to immobilized TSPl . The Hanes analysis provides a measure of assessing the possibility of binding one or more factors to a given molecule.
Figure 11 is a diagram representing the structure of TSPl and TSP2.
Below each domain or region are listed some of the major interactions that have been established previously for human platelet TSPl. Figure 12 is a graph showing the binding of 125I-CP4 to TSPl and a
TSP analog. Increasing concentrations of soluble I-labeled CP4 (0.1 μM -
1.0 μM) were added to immobilized TSPl or immobilized CVSTCG polypeptide
1 ( (SSEEQQ.. IIPP.. NNoo.. 11)).. SSaammpplleess wweerree incubated and bound I-labeled CP4 measured by solid-binding radio-amino assay.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for suppressing infectivity of HYV. This process includes contacting the HIV or a cell targeted by HIV with an effective amount of a TSP or a TSP analog.
TSPl is a trimeric sulfated glycoprotein which belongs to a family of high molecular weight extracellular matrix molecules reviewed in Bornstein, "Thrombospondins: Structure and Regulation of Expression," FASEB, 6:3290-3299 (1992); Lahav, editor, "Thrombospondin," CRC Press, (1993), which are hereby incorporated by reference. TSPl is implicated in suppressing the infectivity of certain bacteria and protozoa, including Staphylococcus aureus, babesia, toxoplasma, leishmania, and the malaria sporozoite (Lahav, "The Functions of Thrombospondin and its Involvement in Physiology and Pathophysiology," Biochim. Biophys. Acta, 1182:1-14 (1993); Sinnis et al., "Remnant Lipoproteins Inhibit Malaria Sporozoite Invasion of Hepatocytes," J. Exp. Med., 184:945-954 (1996) , which are hereby incorporated by reference). Unlike other multifunctional glycoproteins, TSPl is found in very low quantities in plasma, but is stabilized during reversible binding to other matrix molecules, resulting in markedly elevated levels at certain cell surfaces (Taraboletti et al., "Platelet Thrombospondin Modulates Endothelial Cell Adhesion, Motility, and Growth: A Potential Angiogenesis Regulatory Factor," J. Cell BioL, 111 -.765-772 (1990), which is hereby incorporated by reference). TSPl and thrombospondin-2 ("TSP2") have been shown to have a similar structure in mice (Bornstein, "ThiOmbospondins: Structure and Regulation of Expression," FASEB, 6:3290-3299 (1992), which is hereby incoφorated by reference) and in humans (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region," Genomics 17:225-229 (1993), which is hereby incorporated by reference). Human TSPl and human TSP2 are functionally and structurally similar and have an amino acid identity which is about 54 percent (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region," Genomics 17:225-229 (1993), which is hereby incoφorated by reference). In addition, human TSPl and mouse
TSP 1 have been shown to share a high degree of homology to one another (Bornstein, "Thrombospondins: Structure and Regulation of Expression," FASEB, 6:3290-3299 (1992), which is hereby incoφorated by reference), as have human TSP2 and mouse TSP2 (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated
Region," Genomics 17:225-229 (1993), which is hereby incoφorated by reference). Referring to Figure 11, both TSPl and TSP2 contain similar functional domains or regions, including an N-terminal region 10, a procollagen-homologous region 20, type I TSP repeats 30, type II TSP repeats 40, type III calcium-binding repeats 50, and a carboxy-terminal region 60 (Bornstein, "Thrombospondins: Structure and Regulation of Expression," FASEB, 6:3290-3299 (1992); Lawler et al., "The Structure of Human Thrombospondin, an Adhesive Glycoprotein with Multiple Calcium-binding Sites and Homologies with Several Different Proteins," J. Cell Biol. 103:1635-1648 (1986), which are hereby incoφorated by reference). Each of these regions is addressed more fully below. The genes encoding TSPl and TSP2 each contain 21 exons. Exons 1-2 of each TSP gene encode the 5' untranslatable region ("UTR") and translation start sequences, while exon 22 of each TSP gene encodes a 3' UTR. The remaining exons all encode functional regions or domains of the mature TSPl or TSP2.
Referring again to Figure 11, the N-terminal region 10 of TSPl and TSP2 includes a heparm-binding domain. This region includes two sequences of tandemly repeated basic residues that are believed to represent the specific sites which bind anionic heparin. The N-terminal region 10 can be cleaved from TSPl or TSP2 by thermolysin or chymotrypsin.
A variable segment 70 of TSPl and TSP2, located between the N- terminal region 10 and the procollagen-homologous region 20, contains two cysteine residues which are believed to be involved in the formation of interchain di-sulfide bridges during trimerization.
The procollagen-homologous region 20 of TSPl and TSP2 is cysteine- rich and shares homology with the NH2-propeptide of the α(I) chain of type I procollagen.
Following the procollagen-homologous region is the region containing the type I TSP repeats 30. TSPl and TSP2 each contain three type I TSP repeats 30 having between 50-54 amino acid residues. The type I TSP repeat 30 has been found to be conserved in a number of different proteins including, among others, antistasin (Holt et al, "Properdin Binds to Sulfatide [Gal(3504) beta 1-1 Cer] and Has a
Sequence Homology with Other Proteins That Bind Sulfated Glycoconjugates," J. Biol. Chem. 265:2852-2855 (1990), which is hereby incoφorated by reference), proteins involved in the complement cascade such as properdin (Smith et al., "Neutron and X-ray Scattering Studies on the Human Complement Protein Properdin Provide an Analysis of the Thrombospondin Repeat," Biochem. 30:8000-8008 (1991), which is hereby incoφorated by reference), and f-spondin (Klar et al., "F-spondin: A Gene Expressed at High Levels in the Floor Plate Encodes a Secreted Protein that Promotes Neural Cell Adhesion and Neurite Extension," Cell 69:95-110 (1992), which is hereby incoφorated by reference). The type I repeats of TSP fall into group 1, as characterized by Smith et al., "Neutron and X-ray Scattering Studies on the Human Complement Protein Properdin Provide an Analysis of the Thrombospondin Repeat," Biochem. 30:8000-8008 (1991), which is hereby incoφorated by reference, whereas the type I repeats of f-spondin fall into group 2. The type I TSP repeat 30 contains a cys-ser-val-thr-cyg-gly ("CSVTCG") motif (SEQ. ID. No. 1), which is found in both group 1 and group 2 repeats. This sequence has been implicated in the binding of various proteins to cells. The amino acid sequence containing the CSVTCG motif (SEQ. ID. No. 1) is also conserved between TSPl and TSP2.
Following the type I TSP repeats is the region containing the type II TSP repeats 40, also known as EGF-like repeats. TSPl and TSP2 each contain three type II TSP repeats 40. A role for the EGF-like domain in ligand-receptor interactions has been proposed (Appella et al., "Structure and Function of Epidermal Growth Factor-Like Regions in Proteins," FEBS Lett. 231 :1-4 (1988), which is hereby incoφorated by reference).
Following the type II TSP repeats is the region containing the type III calcium-binding repeats 50. Each of the type III repeats 50 contains two cysteine residues, presumably involved in a disulfide bond, and either seven or ten highly conserved asparagine, glutamine, and aspartic acid residues that could serve to complex with calcium. The type III repeats 50 are functionally related to the E/F hand of calmodulin and also have features in common with calcium-binding structures in parvalbumin and fibrinogen (Lawler et al., "The Structure of Human Thrombospondin, an Adhesive Glycoprotein with Multiple Calcium-Binding Sites and Flomologies with Several Different Proteins," J. Cell. Biol. 103:1635-1648 (1986), which is hereby incoφorated by reference). Because the stmcture of this region of the molecule could be sensitive to calcium ion concentration, it is possible that the interaction of TSP 1 with cell-surface integrins could also be modulated by calcium (Frazier, "Thrombospondin: A Modular Adhesive Glycoprotein of Platelets and Nucleated Cells," J. Cell Biol. 105:625-632 (1987); Lawler et al., "Cell Attachment to Thrombospondin: The Role of ARG-GLY-ASP, Calcium, and Integrin Receptors," J. Cell Biol. 107:2351-2361 (1988), which are hereby incoφorated by reference).
The carboxy-terminal region 60 is highly conserved between TSPl and TSP2. This region has been implicated for involvement with platelets, melanoma cells, keratinocytes, and squamous cell carcinoma cells (reviewed in Prater et al., "The Properdin-like Type I Repeats of Human Thrombospondin Contain a Cell
Attachment Site," J. Cell Biol. 112:1031-1040 (1991), which is hereby incorporated by reference).
The presence of an evolutionary conserved TSPl binding domain, termed CLESH-1, was recently established as functional in at least two members of the CD36 gene family, cell surface adhesion receptor CD36 (Pearce et al.,
"Recombinant GST/CD36 Fusion Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium-Pependent Binding Site on CD36," J. Biol. Chem.. 270:2981-2986 (1995), which is hereby incoφorated by reference) and lysosomal integral membrane protein II ("LIMPII") (Crombie et al., "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Stmcture-Function Homology with the Cell Adhesion Molecule CD36 Defines a Conserved Recognition Motif," J. Biol. Chem., 273(9):4855-63 (1998), which is hereby incoφorated by reference). Referring to Figure 1, the CLESH-1 motif appears within the bracketed portions of each sequence alignment. TSP can be isolated from tissue or fluid samples (e.g., plasma) by established techniques described in Silverstein et al., "Platelet Thrombospondin Forms a Trimolecular Complex with Plasminogen and Histadine-Rich Glycoprotein," J. Clin. Invest. 75:2065-2073 (1985), which is hereby incoφorated by reference. Preferably the TSP is produced in purified form, i.e., preferably at least about 80%> pure, more preferably at least 90% pure, and most preferably at least about 95%> pure. Purified TSPl derived from human platelets is also commercially available from Sigma Chemical Co. (St. Louis, Missouri). TSP can also be prepared using recombinant techniques. DNA molecules encoding TSPl (Wolf et al., "Structure and Chromosomal Localization of the Human Thrombospondin Gene," Genomics 6:685-691 (1990), which is hereby incoφorated by reference) and TSP2 (LaBell et al., "Sequence and Characterization of the Complete Human Thrombospondin 2 cDNA: Potential Regulatory Role for the 3' Untranslated Region," Genomics 17:225-229 (1993), which is hereby incoφorated by reference) have been isolated and described. Once obtained, the DNA molecule encoding TSP can be incoφorated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. Transformed host cells capable of producing recombinant TSP can be grown in culture and the expressed TSP can be isolated from the growth medium using conventional protein separation techniques.
Suitable TSP analogs are fragments of TSP. TSP analogs can be obtained following cleavage of TSP by proteases. Exemplary proteases include chymotrypsin, thermolysin, and thrombin (Dixit et al., "Isolation and Characterization of a Heparin-binding Domain from the Amino Terminus of Platelet Thrombosis," J. Biol. Chem. 259:10100-10105 (1984); Mumby et al, Interactions of Thrombospondin with Extracellular Matrix Proteins: Selective Binding to Type V Collagen," J. Cell Biol. 98:646-652 (1984), which are hereby incoφorated by reference). Once fragments of TSP are produced, they can be isolated using immunoselective techniques (Dixit et al., "Monoclonal Antibodies that Recognize Calcium-dependent Structures of Human Thrombospondin. Characterization of Mapping Their Epitopes," J. Biol. Chem. 261 :1962-1968 (1986); Galvin et al., "Mapping of Epitopes for Monoclonal Antibodies Against Human Platelet Thrombospondin with Electron Microscopy and High Sensitivity Amino Acid Sequencing," J. Biol. Chem. 101 : 1434- 1441 (1985), which are hereby incoφorated by reference), HPLC, or gel electrophoresis.
Alternatively, chemical synthesis can be used to prepare TSP peptides (TSP analogs) according to the methods of Guo et al., "Heparin-binding Peptides from the Type I Repeats of Thrombospondin. Structural Requirements for Heparin Binding and Promotion of Melanoma Cell Adhesion and Chemotaxis," J. Biol. Chem. 267:19349-19355 (1992) and Guo et al, "Heparin- and Sulfatide-binding Peptides from the Type I Repeats of Human Thrombospondin Promote Melanoma Cell Adhesion." Proc Natl. Acad. Sci. USA 89:3040-3044 (1992), both of which are hereby incoφorated by reference. Basically, this approach involves using standard Merrifield solid-phase synthesis protocols and t-butoxycarbonyl chemistry. Resulting peptides can be analyzed by reverse-phase HPLC and/or mass spectrometry.
Other conventional peptide synthesis schemes, such as liquid-phase protocols, can also be employed.
TSP analogs can also be produced using conventional recombinant technologies as described above. Exemplary TSP analogs include an N-terminal region of TSP (e.g.,
TSPl and TSP2), a procollagen-homologous region of TSP (e.g., TSPl and TSP2), a type I repeat of TSP (e.g., TSPl and TSP2), a type II repeat of TSP (e.g., TSPl and TSP2), a calcium-binding domain of TSP (e.g., TSPl and TSP2), a carboxy-terminal region of TSP (e.g., TSPl and TSP2), and TSP domains which bind to an HIV gpl20 envelope protein. A preferred TSP analog is a polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Depending upon the intended use of the TSP analogs, the TSP analogs can be defined as TSP domains which bind to an HIV gρl20 envelope protein, TSP domains which bind to a CD4 receptor on a cell, or TSP domains which bind to a chemokine (e.g., RANTES). Other suitable TSP analogs may include molecules, such as fusion proteins, containing TSP or any one of the above-identified TSP analogs. Thus, the fusion protein can contain one or more domains/regions of TSP or the CSVTCG amino acid sequence (SEQ. ID. No. 1). Fusion proteins can be prepared by ligating two DNA molecules together, one of which encodes TSP or the TSP analog and the other of which encodes a stable host protein. Suitable host proteins include, among others, glutathione-S-transferase (Frangioni et al., "Solubilization and Purification of Enzymatically Active Glutathione-S-Transferase (pGEX) Fusion Proteins," Analyt. Biochem., 210:179-187 (1993), which is hereby incoφorated by reference) and maltose-binding protein (Rodriguez et al., "Improved Factor Xa Cleavage of Fusion Proteins Containing Maltose Binding Protein," Biotechniques 18(2):238, 241-243 (1995), which is hereby incoφorated by reference). The two DNA molecules must be ligated in a manner which allows their proper expression. A number of efficient expression schemes for preparing fusion proteins have been developed and are well known in the art. According to one approach the fusion protein is prepared with a protease cleavage site intermediate the host protein and the TSP or TSP analog, such that the TSP or TSP analog can be removed from the host protein by, for example, proteolytic cleavage following isolation of the fusion protein. A linker or spacer peptide may also be included to promote proteolytic cleavage (Polyak et al., "Introduction of Spacer Peptides N-terminal to a Cleavage Recognition Motif in Recombinant Fusion Proteins Can Improve Site-specific Cleavage," Protein Eng. 10(6):615-619 (1997), which is hereby incoφorated by reference). A number of suitable proteases (e.g., factor Xa, chymosin, trypsin, etc.) and their protease-specific cleavage sites are known in the art, and others continually being identified.
The complete nucleotide sequence of the HIV viruses have been reported by several investigators (Ratner et al., "Complete Nucleotide Sequence of the AIDS virus, HTLV-III," Nature 313:277-84 (1985); Muesing et al., "Nucleic Acid Structure and Expression of the Human AIDS/lymphadenopathy Retrovirus," Nature 313:450-458 (1985); Wain-Hobson et al., "Nucleotide Sequence of the AIDS Virus, LAV," Cell 40:9-17 (1985), which are hereby incoφorated by reference). The RNA of the HIV-1 and HIV-2 viruses possess the following gene regions: so-called long terminal repeats at each end of the genome, gag, pol, env, and nef. The gag gene encodes the core proteins p24 and pi 7. The pol gene encodes the reverse transcriptase, RNAse H, and integrase. The gene nef encodes a protein having a regulatory function. The env gene encodes the glycoproteins of the viral coat, namely gp41 and gpl20. The env gene has been associated particularly with antigenicity and infectivity. However, the env portion is also known to have regions which are highly divergent between different strains.
The HIV virus has been shown to bind to a cell surface receptor known as the CD4 or T4 region, which is present on the various cells susceptible to HIV infection, including T lymphocytes and macrophages (Shaw et al., "Molecular Characterization of Human T-Cell Leukemia (Lymphotrophic) Virus Type III in the Acquired Immune Deficiency Syndrome," Science 226:1165-1171 (1984), which is hereby incoφorated by reference). In addition, HIV co-receptors have been implicated in HIV entry inside a cell, because the primary cell surface receptor, CD4, is often by itself insufficient to enable viral entry. HIV co-receptors are cell surface proteins that HIV uses to dock onto its cellular targets. Thus, a cooperative interaction between CD4, a co-receptor, and HIV gpl20 envelope protein must occur. There are presently thirteen known HIV co-receptors, including CCR5, CXCR4, and CCR2B (Baiter, "AIDS Researchers Negotiate Tricky Slopes of Science." Science 280:825-826 (1998), which is hereby incoφorated by reference).
According to one embodiment, the method for suppressing HIV infectivity is carried out by contacting the HIV with an effective amount of TSP or the TSP analog. By doing so, the process of the present invention suppresses the ability of HIV to infect a host organism by blocking its ability to bind to its cellular target (e.g., T lymphocytes and macrophages). Specifically, the TSP or TSP analog are administered in a manner effective to contact the HIV gpl20 envelope protein. Because the gpl20 envelope protein is necessary for the binding of HIV to its cellular target, TSP or the TSP analog can inhibit or interfere with HIV binding to its cellular target. It is believed that the inhibition or interference with HIV binding to its cellular target results from the TSP or TSP analog binding to the gpl20 envelope protein in a manner which either prevents HIV from binding to the cell surface receptor (e.g., CD4) or co-receptor (e.g., chemokine receptor CCR5), or causes steric interference between the functional domains of the gpl20 envelope protein and the receptor/co- receptor.
According to another embodiment, the method is carried out by contacting a cell targeted by HIV with an effective amount of a TSP or TSP analog. Specifically, the TSP or TSP analog is administered in a manner effective to contact the CD4 receptor on a cell. Because the CD4 receptor is necessary for the binding of HIV to its cellular target, TSP or the TSP analog can inhibit or interfere with HIV binding to its cellular target. According to another embodiment, the method is carried out by both contacting the HIV with an effective amount of TSP or a TSP analog and contacting the cell targeted by HIV with an effective amount of TSP or a TSP analog. Thus, the ability of HIV to bind and recognize its cellular target as well as the ability of the CD4 receptor (or the co-receptor) to recognize HIV can both be affected to further suppress HIV infectivity.
TSP or the TSP analog can be administered orally or intraorally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intravaginally or intrarectally (e.g., by suppository, ointment, etc.) intrasynovially, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes via inhalation or otherwise (e.g., using an aerosol or lavage). When TSP or the TSP analog are used in accordance with the methods of the present invention concerning HIV infectivity or binding as described herein, the preferred route for administration is intraorally, intrarectally, or intravaginally.
TSP and the TSP analog can each be administered alone, with a spermicide, or with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions, which may also include microcapsules or liposomes.
Thus, another aspect of the present invention relates to a pharmaceutical composition which includes a pharmaceutically-acceptable carrier in combination with TSP or the TSP analog. The pharmaceutical composition can also include additives commonly used for pharmaceutical preparations, if desired, such as excipients, stabilizers, antiseptics, solubilizers, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavors, antioxidants, and the like. The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type which includes TSP or the TSP analog and a pharmaceutical carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
TSP and the TSP analog can also be administered topically in the form of a solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carriers, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
For use as aerosols, TSP and the TSP analog can be prepared in solution or suspension and packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer. A preferred formulation for the pharmaceutical composition of the present invention contains TSPl or TSP2 in the pharmaceutically-acceptable carrier. Another preferred formulation for the pharmaceutical composition of the present invention contains a TSP analog in the pharmaceutically-acceptable earlier.
Because of the possibility for transmission of HIV from an infected person to another via oral exchange, it is preferable for the pharmaceutical composition to be an oral hygiene composition. Thus, the oral hygiene composition is a pharmaceutical composition which contains a carrier that is a pharmaceutically- acceptable topical oral carrier. Suitable topical oral carriers include a toothpaste, a mouthwash or other oral rinse, a lozenge, and a gum. Details with regard to formulating oral hygiene compositions can be found in, for example, U.S. Patent No. 5,686,064 to Gaffar et al., U.S. Patent No. 5,672,351 to Chikindas, and U.S. Patent No. 5,578,295 to Francis et al., which are hereby incorporated by reference. Other processes for formulating oral hygiene compositions are well known in the art.
Because of the possibility for transmission of HIV from an infected person to another via sexual contact, it is also desirable to provide a contraceptive capable of suppressing HIV infectivity. Thus, another aspect of the present invention relates to a contraceptive that includes a contraceptive carrier and a TSP or a TSP analog.
The contraceptive carrier can be a contraceptive composition or a contraceptive device.
Suitable contraceptive compositions include contraceptive foams, jellies, lubricants, and spermicides. The contraceptive composition can be a pharmaceutical composition of the present invention. Details with regard to the formulation of contraceptive compositions, such as contraceptive foams, jellies, lubricants, spermicides, and the like, are generally known to those skilled in the art and are described in, for example, U.S. Patent No. 5,595,980 to Brode et al., which is hereby incoφorated by reference. Suitable contraceptive devices include sponges, cervical diaphragms and caps, intravaginal devices, and condoms. Contraceptive devices containing molecules which include TSP or TSP analogs (e.g., the CSVTCG amino acid sequence of SEQ. ID. No. 1) can be prepared by methods described in, for example, U.S. Patent No. 5,571,567 to Shah et al., which is hereby incoφorated by reference. Further details regarding contraceptive formulation and manufacture can be found in U.S. Patent No. 5,545,615 to Maraganore and U.S. Patent No. 5,527,534 to Myhling, which are hereby incoφorated by reference. Other processes for preparing contraceptive formulations and contraceptive devices are well known in the art. In addition, in some instances, a contraceptive composition in accordance with the present invention can be used in combination with a contraceptive device of the present invention. For example, a condom or sponge of the present invention can contain a spermicide of the present invention. Because of the possibility for transmission of HIV from an infected person to another via non-sexual contact (e.g., surgical procedures) as well as sexual contact, it is also desirable to provide a non-contraceptive prophylactic device capable of suppressing HIV infectivity. Thus, another aspect of the present invention relates to a non-contraceptive prophylactic device that includes a non-contraceptive carrier and a TSP or a TSP analog.
The non-contraceptive carrier can be a carrier other than the contraceptive carriers described above. Specifically, it is intended that the non- contraceptive carrier is a type of physical barrier. Thus, suitable non-contraceptive carriers can take any form or configuration, but, preferably, they are in the form of a surgical glove, a dental dam, or an oral dam. Non-contraceptive carriers for this aspect of the present invention can be prepared by methods described in, for example, U.S. Patent No. 5,571,567 to Shah et al., which is hereby incoφorated by reference. Suitable surgical gloves can be of any conventional design, including, but not limited to, surgical gloves of the type disclosed in U.S. Patent No. 5,317,759 to Pierce, U.S. Patent No. 5,817,433 to Darras, and U.S. Patent No. 4,901,372 to Pierce, which are hereby incoφorated by reference. Suitable dental dams can be of any conventional design, including, but not limited to, dental dams of the type disclosed in U.S. Patent No. 4,721,465 to Barasz and U.S. Patent No. 3,662,466 to McConville. Suitable oral dams also can be of any conventional design, including, but not limited to, oral dams of the type disclosed in U.S. Patent No. 4,949,731 to Harding and U.S. Patent No. 5,388,592 to Williams, which are hereby incoφorated by reference.
In some instances, it may be possible to utilize a pharmaceutical composition of the present invention together with a non-contraceptive prophylactic device of the present invention.
Another aspect of the present invention relates to a method of inhibiting HIV infection in a patient. This method includes administering TSP or a TSP analog to the patient under conditions effective to inhibit HIV infection. To inhibit HIV infection, the TSP or TSP analog should be administered prior to and, optionally, immediately following exposure to the HIV. To administer the TSP or TSP analog, a pharmaceutical composition of the present invention, a contraceptive of the present invention, a non-contraceptive prophylactic device of the present invention, or combinations thereof can be used.
It will be appreciated that the actual amount of the TSP or TSP analog to be administered according to the present invention will vary according to the particular compound, the particular composition, and the particular mode of administration. Many factors that may modify the action of the TSP or TSP analog can be taken into account by those skilled in the art; e.g., time of administration, route of administration, condition of the subject, drug combinations, etc Optimal administration rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage administration tests.
When this method of the present invention is carried out by administering TSP to the patient, it is preferable that the TSP is TSPl or TSP2. When this method of the present invention is carried out by administering a TSP analog to the patient, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Because the threat of spreading HIV is greatest via oral, vaginal, or rectal routes, it is preferable that the administration of the TSP or TSP analog is intraorally, intravaginally, or intrarectally.
Still another aspect of the present invention relates to a method of blocking HIV binding to a cell targeted by HIV. This method includes contacting HIV or the cell targeted by HIV with TSP or a TSP analog under conditions effective to block binding of the HIV to the cell.
When this method of the present invention is carried out by contacting HIV with TSP, it is preferable that the TSP is TSPl or TSP2. When this method of the present invention is carried out by contacting HIV with a TSP analog, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Regardless of whether the TSP or TSP analog is used to contact the HIV, the TSP or TSP analog is administered in a manner effective to contact the HIV gpl20 envelope protein. By contacting the gpl20 envelope protein, the HIV is effectively blocked from binding to its cellular target as described above. When this method of the present invention is carried out by contacting the cell with TSP, it is preferable that the TSP is TSPl or TSP2. When this method of the present invention is carried out by contacting the cell with a TSP analog, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1). Regardless of whether the TSP or TSP analog is used to contact the cell, the TSP or TSP analog is administered in a manner effective to contact the CD4 receptor. By contacting the CD4 receptor, the HIV is effectively blocked from binding to its cellular target as described above.
It has also been discovered that TSP or TSP analogs can affect the activity of certain types of immunomodulatory proteins which are chemotactic cytokines, called "chemokines." Chemokines are small molecular weight immune ligands which are chemoattractants for leukocytes, such as neutrophils, basophils, monocytes, and T cells. There are two major classes of chemokines, an α-class and a β-class. Chemokines in each class contain four conserved cysteine residues that form disulfide bonds in the tertiary structure of the proteins. The α-class is characterized by C-X-C motifs (where X is any amino acid) and includes IL-8, CTAP-III, gro/MGSA, and ENA-78. The β-class is characterized by C-C motifs and includes MCP- 1 , MIP- 1 α, MIP- 1 β, and the regulated on activation normal T expressed and secreted protein ("RANTES"). Thus, the designations of the classes are according to whether an intervening residue spaces the first two cysteines in the motif. In general, α-chemokines (C-X-C) are chemoattractants for neutrophils but not monocytes, whereas β-chemokines (C-C) appear to attract monocytes but not neutrophils. Recently, a third group of chemokines, the "C" group, was designated by the discovery of a new protein called lymphotactin (Kelner et al., "Lymphotactin: A Cytokine That Represents a New Class of Chemokine," Science 266:1395-1399 (1994), which is hereby incoφorated by reference). The chemokine family is believed to be critically important in the direction of lymphocytes and monocytes into sites of inflammation.
In particular, it has been discovered that TSP and TSP analogs are capable of binding to chemokines, particularly to the chemokine RANTES. RANTES is important for two reasons. First, RANTES is involved in inflammation (Barnes et al, "Polyclonal Antibody Directed Against Human RANTES Ameliorates Disease in the Lewis Rat Adjuvant-induced Arthritis Model," J. Clin. Invest. 101(2):2910-2919 (1998), which is hereby incoφorated by reference). Second, the natural receptor for RANTES is CCR5, the principal co-receptor that permits HIV to bind to and infect cells.
Therefore, a further aspect of the present invention relates to a method of treating or preventing inflammatory states in a patient. This method includes administering an effective amount of TSP or a TSP analog to the patient. When TSP is administered to the patient in accordance with this process of the present invention, it is preferred that the TSP is TSPl or TSP2. When a TSP analog is administered to the patient in accordance with this process of the present invention, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1).
Specific conditions in which TSP or TSP analogs of the present invention may have therapeutic value would include situations in which an undesirable immune response has occurred, including, but not limited to, autoimmune diseases such as insulin-dependent diabetes, Goodpasture's syndrome, pemphigus and pemphigoid, primary biliary cirrhosis, ulcerative colitis, rheumatoid arthritis, scleroderma, mixed connective tissue disease and lupus erythematosus; graft versus host disease; septic shock; reperfusion injury (including injury subsequent to myocardial or cerebral infarction); atherosclerosis; asthma; and inflammatory lung disease. Patients which can be treated according to this aspect of the present invention include any mammal, such as a mouse, a rat, a dog, or a human. In particular, administration of the TSP or TSP analog to humans suffering from various inflammatory states, such as those described above, is contemplated. TSP or TSP analogs of the present invention can be administered by any suitable and accepted route of drug administration, including those described above. Moreover, TSP or TSP analogs can be administered as a component in a pharmaceutical composition of the present invention. When prepared for such use, pharmaceutical compositions of the present invention can also contain other anti-inflammatory compounds and/or materials commonly used in pharmaceutical formulations. Yet another aspect of the present invention relates to a method of blocking the binding of a chemokine to its receptor. This method includes contacting a chemokine with a TSP or a TSP analog under conditions effective to bind the chemokine. The binding of the chemokine is believed to interfere with its affinity for (i.e., its ability to bind) its receptor.
When TSP is used to contact the chemokine in accordance with this process of the present invention, it is preferred that the TSP is TSPl or TSP2. When a TSP analog is used to contact the chemokine in accordance with this process of the present invention, a preferred TSP analog is the polypeptide containing a CSVTCG amino acid sequence (SEQ. ID. No. 1).
This method of the present invention is particularly effective when the chemokine to be bound by the TSP or TSP analog is a β-chemokine and the corresponding receptor is a β-chemokine receptor. A chemokine which is effectively bound by TSP or TSP analogs of the present invention is RANTES. Other chemokines of the RANTES family are expected to be effectively blocked as well. Thus, the chemokine receptor which is most effectively blocked against RANTES binding is a RANTES receptor, particularly a CCR5 receptor.
EXAMPLES
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Example 1 - Methodology
Protein sequence analysis and homology search
Deduced amino acid sequences encoded by Exon 5 of CD36 or homolog LIMPII (Figure 1) that contain previously characterized TSPl binding motifs (Crombie et al., "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CD36 Defines a Conserved Recognition Motif," J. Biol. Chem., 273(9):4855-63 (1998), which is hereby incoφorated by reference) were used as query for a BLAST Enhanced Alignment Utility search ("BEAUTY") (Worley et al, "BEAUTY: An
Enhanced BLAST-Based Search Tool that Integrates Multiple Biological Information Resources Into Sequence Similarity Search Results," Genome Res.. 5:173-184 (1995), which is hereby incoφorated by reference), which incoφorates Pattern-Induced Multiple Alignment ("PIMA") (Smith et al, "Pattern-Induced Multi-Sequence Alignment (PIMA) Algorithm Employing Secondary Structure-Dependent Gap Penalties for Comparative Protein Modelling," Protein Eng., 5:35-41 (1992), which is hereby incoφorated by reference) sequence family clusters, conserved cluster domains and PROSITE annotated libraries (Bairoch, "The PROSITE Dictionary of Sites and Patterns, its Current Status," Nucl. Acids Res., 21 :3097-3103 (1993), which is hereby incoφorated by reference). HIV-1 consensus and subtype sequence alignments were retrieved from the Los Alamos Sequence Database.
Preparation of TSPl
Human platelet-rich plasma was obtained from the NY Blood Center. Human thrombin was from Boeringer-Mannheim (Indianapolis, IN). Purified human calcium-replete TSPl was prepared from releasate of thrombin-activated washed platelets as described previously (Pearce et al., "Recombinant GST/CD36 Fusion
Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium- Dependent Binding Site on CD36," J. Biol. Chem., 270:2981-2986 (1995); Silverstein et al., "Platelet Thrombospondin Forms a Trimolecular Complex with Plasminogen and Histadine-Rich Glycoprotein," J. Clin. Invest. 75:2065-2073 (1985), which are hereby incoφorated by reference). Dot blot analysis with monoclonal antibodies (mAb) to fibronectin and vitronectin (Calbiochem, La Jolla, CA) showed no reactivity. Endotoxin content was monitored by the Limulus amoebocyte lysate test, and was <1 U/μg protein. Polyclonal rabbit anti-TSP antisera and murine anti-TSP +2 mAbs 11.4 and 45.2 were prepared and characterized as reported (Silverstein et al, "Thrombospondin Binds to Monocytes-Macrophages and Mediates Platelet-Monocyte Adhesion," J. Clin. Invest., 79:867-874 (1987); Pearce et al., "A Carboxy Terminal Truncation Mutant of CD36 is Secreted and Binds Thrombospondin: Evidence for a Single Transmembrane Domain," Blood. 84:384-389 (1994), which are hereby incoφorated by reference). Purified fusion proteins and peptides
Characterization of glutathione-S-transferase ("GST") fusion proteins derived from CP36 or CP36 homo log LIMPII are reported elsewhere (Crombie et al., "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CP36 Pefines a Conserved Recognition Motif," J. Biol. Chem.. 273(9):4855-63 (1998), which is hereby incoφorated by reference), and are numbered to indicate encoded amino acids. CFP67-157 and LFP75-155 contain functionally homologous minimal TSPl binding domains (CP36 aa93-120) (Pearce et al, "Recombinant GST/CD36 Fusion Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium- Dependent Binding Site on CD36," J. Biol. Chem.. 270:2981-2986 (1995), which is hereby incoφorated by reference) while CFP298-439 and LFP156-243 represent downstream sequences. A truncation mutant LFP75-78 was used as an additional GST1 control. Large-scale production and purification of soluble fusion protein followed the method of Frangioni and Neel (Frangioni et al, "Solubilization and Purification of Enzymatically Active Glutathione-S-Transferase (pGEX) Fusion Proteins," Analyt. Biochem., 210:179-187 (1993), which is hereby incoφorated by reference). For some experiments, LIMPII peptide (L75-155) was cleaved from the GST moiety with coagulation Factor Xa (Boehringer Mannheim, Indianapolis, IN), and purified by size exclusion chromatography (Centricon 10; Amicon, Beverly, MA). TSPl synthetic peptides were from Chiron Mimotopes (Australia). Recombinant baculovirus-expressed HIV envelope glycoprotein gρl60 (gpl20 non- covalently linked to transmembrane component gp41) was derived from HIV-1 isolate IIIB (IntraCel Coφ., Cambridge, MA). The following purified recombinant proteins were provided by the NIH AIDS Research and Reference Reagent Program: baculovirus-expressed HIV-1 gpl20 derived from LAV and MN isolates, CHO cell- expressed soluble CD4, and HIV-IMN env synthetic peptides.
Solid phase binding assays In vitro binding experiments were performed as described previously for TSPl binding to CD36 and LIMPII (Pearce et al., "Recombinant GST/CD36 Fusion Proteins Define a Thrombospondin Binding Domain: Evidence for a Single Calcium-Pependent Binding Site on CP36," J. Biol. Chem.. 270:2981-2986 (1995); Crombie et al., "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CP36 Pefines a Conserved Recognition Motif," J. Biol. Chem., 273(9):4855-63 (1998), which are hereby incorporated by reference). Briefly, TSPl (5-10 μg/ml) or fusion proteins and peptides (10-20 μg/ml) were immobilized on detachable 96-well microtiter plate strips (Immulon-4 Remov-a-well, Pynatech Laboratories, Inc.), by overnight incubation at 4°C in carbonate buffer (100 mM Na2CO3/l mM MgCl2/0.02% NaN3, pH 9.8). Washed wells were blocked with 0.5% bovine semm albumin ("BSA"), then incubated in triplicate with soluble radiolabeled ligand for 2.5 hours at 37°C. After extensive washing in 50 mM Tris pH 7.5, 150mM NaCI, 0.5%> Tween-20 (TBS-Tween), bound radioactivity was quantified by gamma counter.
19*ϊ
Radiolabeling was performed with Na[ I] (Amersham Life Science Inc., Arlington Heights, IL) using immobilized chloramine T (Iodo-beads™; Pierce Chemical Co., Rockford, IL) (Silverstein et al, "Thrombospondin: A Versatile Multifunctional
Glycoprotein," Atherosclerosis. 6:245 (1986); MacGregor et al., "Rapid Purification and Partial Characterization of Human Platelet Glycoprotein Illb," J. Biol. Chem.. 264:501-506 (1989), which are hereby incoφorated by reference). Specific activity of Na[125I] was determined for each experiment, ranging from 0.06-1.0 μCi/μg. Specific binding was determined as quenchable in the presence of excess unlabeled ligand, above background binding to BSA-coated wells.
Immunohistochemistry
Thin tissue sections of oral mucosa autopsy specimens were processed as described (Hajjar et al., "Lipoprotein (a) Modulation of Endothelial Cell Surface Fibrinolysis and its Potential Role in Atherosclerosis," Nature. 339:303-305 (1989), which is hereby incoφorated by reference). Formalin-fixed paraffin-embedded sections were de- waxed, pronase treated, and permeabilized in Triton X-100. Endogenous peroxidase activity was blocked by treatment with a 3% solution of H O2 for 30 minutes. Slides were pre-incubated with normal human semm for 1 hour at
22°C, then incubated overnight at 4°C with 1 :1000 dilutions of either polyclonal rabbit anti-TSP 1/TSP2, or pre-immune rabbit serum. After a brief blocking step with normal goat semm, successive rinses in PBS were performed between incubations with a 1:250 dilution of biotinylated goat anti-rabbit IgG (Oako, Caφenteria, CA), followed by avidin-biotin-peroxidase complex (Pako). Peroxidase deposition was visualized with 3,3'-diaminobenzidine tetrachloride. Samples were rinsed in distilled water, counterstained with hematoylin, and viewed by light microscopy.
Quantitative immunodetection of TSP
A sandwich enzyme-linked immunosorbent assay ("ELISA") was used to measure levels of TSP in saliva and cell culture supernatants. Polystyrene 96-well microtiter plates (Falcon, Oxnard, CA) were coated overnight at 4°C with 50 μg/well of 5 μg/ml anti-TSP 1 mAb 45.2 in carbonate buffer, pH 9.6. Plates were extensively washed with carbonate buffer, then blocked with 1% BSA in TBS-Tween. This was followed by incubating 50 μl of sample diluted 1 :16 in TBS-Tween for 2 hours at 37°C, followed by alkaline-phosphatase-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Labs ("KPL"), Gaithersberg, MO). Washed plates were developed with 100 μi/well of 1 mg/ml p-nitrophenyl phosphate in 50 mM carbonate buffer, pH 9.6 (KPL), incubated for 30 minutes at 37°C, and absorbance at 410 nm measured using a Microtek plate reader. Quantitation was estimated relative to a standard curve constmcted using purified TSPl diluted in TBS-Tween and compared with saliva samples to which known quantities of TSPl were added.
Saliva collection
Unstimulated whole saliva was collected by expectoration into chilled centrifuge tubes and placed on ice. All donors, whether HIV seropositive or negative, had no active periodontal disease or oral lesions. Whole saliva was clarified by microcentrifugation (12,000 x g for 10 minutes at 4°C) and used immediately, while aliquots were stored at <20°C. Parotid saliva was collected from one gland using a modified Curby cup (Smith et al., "Immunological Features of Minor Salivary Gland Saliva," J. Clin. Immunol, 7:449-454 (1987), which is hereby incoφorated by reference). Submandibular salivary fluid was collected using surgical sponges
(Weck-Cel) cut into approximately 0.25 x 0.5 cm rectangles and touched to blebs of fluid which formed at the duct orifi. Saliva was removed from sponges by centrifugation in polyethylene tubes, as described in Smith et al, "Immunological Features of Minor Salivary Gland Saliva," J. Clin. Immunol. 7:449-454 (1987), which is hereby incoφorated by reference. Protein content was estimated using a micro-BCA reagent kit (Pierce).
HIV-1 infectivity assays
CP4+ T lymphoblastoid (SK23, Jurkat) and promonocytic (U937) cell lines, or peripheral blood mononuclear cells ("PBMC") obtained from HIV seronegative donors and activated with phytohemagglutinin ("PHA", 2 μg/ml for 72 hours), were cultured in RPMI 1640, 5% fetal bovine serum ("FBS", frozen/thawed and complement-inactivated 30 minutes at 56°C), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO/BRL, Grand Island, NY). PBMC medium also contained 32 U/ml interleukin-2 (Sigma Chemical Co., St. Louis, MO). FBS samples were pre-screened to assure that levels of TSP were <10 μg/ml. Acute HIV infection was performed using HIV-1 isolate IIIB stock vims as previously described (Laurence et al, "Human Immunodeficiency Vims Infection of Helper T-Cell Clones: Early Proliferative Oefects Pespite Intact Antigen-Specific Recognition and Interleukin 4 Secretion," J. Clin. Invest., 83:1843-1848 (1989); Laurence et al, "Human Immunodeficiency Vims Infection of Monocytes: Relationship to Fc-Gamma Receptors and Antibody-Pependent Viral Enhancement," Immunology, 70:338-343 (1990), which are hereby incoφorated by reference). Briefly, 2.5 x 105 target cells were exposed to stock vims at a moi of either 0.02 or 0.15 for 2 hours at 37°C, washed once with PBS, and re-plated in tissue culture-treated microwells with 0.3 ml fresh culture medium. At 3-4 days post-inoculation, one half of culture supernatant from each well was replaced with fresh medium. HIV activity was determined after 7 days using an ELISA antigen capture assay for HIV-1 p24 (gag) core protein (Pupont Medical Products, Boston, MA) of Triton X-100 solubilized culture supernatants.
Saliva and TSPl inhibition assays 100 μl of HIV-1 /IIIB inocula were mixed with 100 μl of various concentrations of whole saliva, salivary fluid fractions, or purified platelet TSPl diluted in semm-free cell culture medium, and pre-incubated for 2 hours at 37°C. Then, 10-fold serial dilutions were added directly to target cell cultures for assay of infectivity. In some experiments, pre-incubated virus-saliva/TSP mixtures were filtered through 0.2 μm nitrocellulose syringe filters (Gelman Sciences, Ann Arbor, MI) prior to inoculation of target cells.
Salivary TSPl depletion experiments
An affinity column for adsoφtion of TSPl was prepared using fusion protein LFP75-155 coupled by N-hydroxysuccinimide to Sepharose (HiTRAP™, Pharmacia). Two ml of clarified whole saliva was diluted 1 : 1 in PBS and divided into two aliquots, one of which was applied to the TSP-binding GST/LIMPII column, the other to an identical column containing N-hydroxysuccinimide-linked GST alone. Columns were incubated 45 minutes at 37°C, then each flushed with 1 ml PBS. The final products represented 1 :4 dilutions of saliva. TSP concentrations were determined before and after column adsoφtion by sandwich ELISA, and total protein assessed using the micro-BCA reagent kit.
Cell surface expression of CP4
CP4 surface expression on HIV-1 infected or uninfected U937 cells was evaluated by indirect immunofluorescence flow cytometry using anti-CP4 mAb Leu3, as detailed elsewhere (Laurence et al, "Human Immunodeficiency Virus Infection of Helper T-Cell Clones: Early Proliferative Pefects Pespite Intact Antigen-Specific Recognition and Interleukin 4 Secretion," J. Clin. Invest.. 83:1843- 1848 (1989), which is hereby incoφorated by reference).
Example 2 - Characterization of a Putative TSPl Binding Domain in HIV-1 env
Stmctural mapping studies have elucidated a TSPl binding domain within the region encoded by CP36 exon 5 (Pearce et al, "Recombinant GST/CP36 Fusion Proteins Pefine a Thrombospondin Binding Pomain: Evidence for a Single Calcium-Pependent Binding Site on CP36," J. Biol Chem.. 270:2981-2986 (1995), which is hereby incoφorated by reference). It has also been shown that the corresponding sequence of the CP36 homolog lysosomal integral membrane protein II ("LIMPII") (Vega et al, "Cloning, Sequencing, and Expression of a cPNA Encoding the Rat LIMP II, a Novel 74-kPa Lysosomal Membrane Protein Related to the Surface Adhesion Protein CP36," J. Biol Chem.. 266:16818-16824 (1991); Calvo, P. et al, "The CP36, CLA-1 (CP36L1), and LIMP II (CP36L2) Gene Family: Cellular Pistribution, Chromosomal Location, and Genetic Evolution," Genomics, 25:100-106 (1995), which are hereby incoφorated by reference) bound TSPl with high affinity, confirming a functionally identical domain (Crombie et al, "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CP36 Pefines a Conserved Recognition Motif," J. Biol. Chem., 273(9):4855-63 (1998), which is hereby incoφorated by reference). Computer assisted motif analysis of 14 CP36 gene family homologs revealed highly conserved blocks implicating an evolutionarily maintained functional module designated CLESH-1 (Crombie et al, "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Stmcture-Function Homology with the Cell Adhesion Molecule CP36 Pefines a Conserved Recognition Motif," J. Biol Chem., 273(9):4855-63 (1998), which is hereby incoφorated by reference). Using this sequence as the query in a pattern-based homology search to identify putative TSP binding sequences in proteins outside the CP36 gene family, CLESH-1 related sequences in HIV-1, HIV-2, and SIV-1 were identified. Figure 1 shows the amino acid alignment of CP36/LIMPII CLESH-1 motifs with HIV-1 matched sequences. Strongest homology appeared as a split motif localized to domains C2 and C3 on either side of the V3 loop region of gpl20. Homology was highest to LIMPII, with 62.5%o identity, 87.5% similarity for the first half-site (C2 domain, 16 aa), and 55.6%> identity, 88.9% similarity for the second half-site (C3 domain, 18 aa). Comparison with the clade B subtype consensus and two laboratory isolates used in this study (MN and LAI) shows that homology extends throughout both full-length motifs. The overall range of 30-36% identity, 79-84% similarity between LIMPII and HIV-1 isolates is comparable to 37% identity, 75% similarity observed between gene family members CP36 and LIMPII. This discovery prompted a biochemical approach to determine whether TSPl could interact directly with gpl20.
Solid phase binding assays were employed to assess direct binding of TSPl to the HIV-1 envelope complex. Figure 2 A shows concentration-dependent, saturatable binding of radiolabeled soluble TSPl to immobilized recombinant gpl60 (gpl20 non-covalently linked to transmembrane component gp41). An apparent affinity of approximately 250 nM was comparable to that demonstrated for binding of TSPl to purified platelet CP36. Binding was effectively quenched in the presence of 10-fold molar excess unlabeled soluble TSPl (Figure 2B, 94 ± 6% inhibition), demonstrating specificity. In addition, a LIMPII fusion protein LFP75-155 containing the TSP binding domain partially blocked binding (47 ± 14% inhibition), while control fusion protein LFP 156-243 representing downstream LIMPII sequences did not, supporting the existence of a functionally similar domain in HIV-1 env. The CSVTCG (SEQ. IP. No. 1) peptides found in the Type 1 repeats of TSPl are binding sites for CP36 and LIMPII (Crombie et al, "Lysosomal Integral Membrane Protein LIMP II Binds Thrombospondin- 1 : Structure-Function Homology with the Cell Adhesion Molecule CP36 Pefines a Conserved Recognition Motif," J. Biol Chem., 273(9):4855-63 (1998); Asch et al, "Cellular Attachment to Thrombospondin: Cooperative Interactions Between Receptor Systems," J. Biol Chem., 266:1740-1745 (1991); Catimel et al, "Human Platelet Glycoprotein Illb Binds to Thrombospondin Fragments Bearing the C-Terminal Region, and/or the Type I Repeats (CSVTCG Motif), but not to the N-Terminal Heparin-Binding Region," Biochem. J., 284:231-236 (1992); Li et al, "Identification of SVTCG in Thrombospondin as the Conformation-Pependent, High Affinity Binding Site for its Receptor, CP36," J. Biol Chem., 268:16179-16184 (1993), which are hereby incoφorated by reference).
The gpl20 envelope protein was tested to determine whether it shared this same specificity. Saturation isotherms showed significant binding of radiolabeled gpl20 (LAI isolate) to immobilized CSVTCG peptide (SEQ. IP. No. 1), with an apparent affinity of 300 nM (Figure 3). The activity was sequence-specific, as demonstrated by inefficient binding to scrambled control peptide VGSCCT (SEQ. IP. No. 2), or to an RGP-containing peptide similar to the GRGPA (SEQ. IP. No. 3) cell adhesion sequence of the last TSPl Type 3 calcium-binding repeat. This is further evidence that the TSPl-gpl20 interaction is mediated through a CP36/LIMPII -related structural domain. Figure 4A shows the location of a series of 20 aa synthetic peptides with respect to gpl20 domain structure (MN isolate). Given the constraints of the solid phase assay, peptides were immobilized either singly or in pairs, and tested for ability to bind radiolabeled TSPl . As shown in Figure 4B, active peptides corresponded to regions of gpl20 containing homologous CLESH-1 motifs. Peptide pairs that extended the represented motif sequence showed augmented binding as compared to either peptide alone. In addition, single peptides representing sequences outside strongly homologous split motif half-sites also showed significant TSPl binding. Interestingly, a V3 containing peptide (#4, aa311-330) with TSPl binding activity also displayed augmented binding in combination with an inactive C2 peptide within the first CLESH-1 motif (#3, aa291-310). These data suggest the presence of two component TSPl binding elements in the predicted regions of gpl20, including V3 sequences in the first motif, with the potential for interchangeable combinations of site usage, and possible TSP-mediated structural alterations that might disrupt conformation-dependent binding of gpl20 to CP4 receptor.
Figure 5 shows competitive inhibition of radiolabeled CP4 binding to gpl20 derived from two different viral isolates (MN and LAI). In the presence of a 10- fold molar excess of TSPl, complete inhibition of 12:,I-CP4 binding to immobilized gpl20 was seen. The CSVTCG (SEQ. IP. No. 1) peptide showed partial but significant inhibition of CP4-gpl20 complex formation (53 ± 9%>), confirming a TSP- specific effect, while the RGP-containing peptide had little effect (4 ± 4%), and the scrambled control actually enhanced binding. Consistent with structural homology data, TSPl -binding CP36- and LIMPII-derived fusion proteins proved strong competitors (both ~ 89% inhibition), whereas downstream LIMPII control protein had minimal effect (32 ± 5%). For comparison, a 1 :2 dilution of whole saliva was a potent inhibitor in this assay system. These observations support a role for salivary TSPl as a direct inhibitor of HIV infectivity.
Example 3 - Analysis of TSPl-Mediated Inhibition of HIV-1 Infectivity
As shown in Table 1 below, levels of TSPl in whole saliva from either HIV+ or HIV- donors were at least 10-fold greater (1-12 μg/ml) compared to plasma. Amounts of TSPl in parotid saliva fractions were equivalent to that of plasma, with the bulk of TSPl found in the submandibular secretions. These values are consistent with previous reports of anti-HIV activity predominately in submandibular and not parotid gland fluids (Fox et al, "Saliva Inhibits HIV-1 Infectivity," J. Am. Pent. Assoc, 116:635-637 (1988); Archibald et al, "In Vitro Inhibition of HIV-1
Infectivity by Human Salivas," AIPS Res. Hum. Retrovimses. 6:1425-1432 (1990), which are hereby incorporated by reference). To document that elevated levels of TSP 1 in saliva may be secondary to local production, rather than leakage and concentration from plasma, immunohistochemical staining of fixed tissue from oral mucosa was performed. Moderate to intense levels of TSPl -directed immuno- reactivity were evident on gingival epithelium (Figure 6), confirming that cell- associated TSPl may correspond to significantly high local concentrations in the oral cavity.
Table 1 : Quantitation of TSPl in Human Plasma and Saliva Sample Source HIV Status TSP1/TSP2 cone, (range) n plasma negative 0.25 (0.1-0.34) μg/ml 8 whole saliva negative 4.1 (1.1-12.8) μg/ml 6 positive 3.3 μg/ml 2 parotid negative 0.1 μg/ml 1 submandibular negative 2.5 μg/ml 1
To investigate whether purified TSPl could inhibit HIV-1 infection similar to saliva, viral isolate IIIB was added to target cells after pre-incubation with TSPl . Many studies have shown that filtration of vims-saliva mixtures is required for maximum inhibition (Malamud et al, "Human Submandibular Saliva Aggregates HIV," AIPS Res. Hum. Retrovimses, 9:633-637 (1993); Fox et al, "Saliva Inhibits HIV-1 Infectivity," J. Am. Pent. Assoc. 116:635-637 (1988); Fox et al, "Salivary Inhibition of HIV-1 Infectivity: Functional Properties and Pistribution in Men, Women and Children," J. Am. Pent. Assoc, 118:709-711 (1989); Bergey et al, "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic Syndr., 7:995-1002 (1994), which are hereby incoφorated by reference). The physiologic equivalent of such filtration is thought to be the constant cleansing of the oral cavity by salivary flow. Therefore, in some experiments, pre-incubated virus- TSP1 mixtures were passed through 0.2 μm filters. Pifferent moi vims per target cell were tested in both systems. At concentrations found in saliva (2-10 μg/ml), TSPl reduced HIV-1 infection of PHA-activated donor PBMC by >83% when pre-filtered, as measured by ELISA detection of p24 viral antigen (Figure 7). This was comparable to a 1 :2 dilution of whole saliva. In contrast, fibrinogen, another high molecular weight adhesive glycoprotein in saliva, had no effect in this system. Specificity was documented by abrogation of the inhibitory effect in the presence of a specific anti-TSP 1 polyclonal antibody (107%) of control p24), but not control IgG. Prolonged incubation of the TSP-virion mixture was unnecessary, as exposures as brief as 5 minutes appeared sufficient to reduce infectivity by > 50% at 1 μg/ml TSPl. The TSPl effect also was apparent for HIV-1 IIIB infection of CP4+ T- lymphoblastoid and monocytoid cell lines (SK23 > 90.7% and U937 > 83.0% inhibition, respectively). In addition, two monocytotropic strains of HIV-1, pl3 and HA593 representing patient isolates obtained from the NIH AIPS Retroviral Repository, were susceptible to inhibition by purified TSPl . At viral moi's of 0.8, 100 μg/ml TSPl inhibited HIV infectivity by > 98±1%. In contrast to pre-filtration experiments, direct addition of TSPl was able to inhibit HIV-1 by 50-75%) only when a high concentration of TSPl and a low moi inoculum was used (Figure 7). In parallel assays, saliva could inhibit HIV-1 as a direct addition only when added in dilutions of less than 1 :4. However, pre-incubation of vims with saliva permitted dilutions greater than 1 :10. The results again are consistent with levels of TSPl found in these dilutions of saliva.
To determine the extent of TSPl contribution to saliva inhibition of HIV infectivity, clarified saliva samples were passed over affinity columns of immobilized TSP-binding LIMPII fusion protein LFP75-155 prior to virus pre- incubation. HIV-lni-B (0.15 moi) was admixed with clarified whole saliva that was untreated or first adsorbed with immobilized fusion proteins for 1 hour at 37°C (final 1 :4 dilute). Pre-incubated and filtered virus-saliva mixtures then were used to infect PHA-activated PBMC, and HIV-1 activity determined on day 7 post- inoculation as in Figure 7. Percentages, shown in parentheses in Table 2 below, are relative to pre- adsorbed concentrations. Anti-HIV-1 inhibitory effect is expressed as percent decrease of maximum HIV-1 p24 antigen in the absence of saliva. Table 2: Effect of TSPl Depletion on Salivary Inhibition of HIV-1 Saliva Protein cone. Percent
Adsorption (pre-adsorbed cone.) TSP 1/TSP2 cone. Inhibition
None 8.16 1.20 99.8
TSP-affinity 6.20 (76.0 %) 0.08 (6.3%) 31.1
GST Control 7.52 (92.2%) 1.18 (98.3%) 99.7
Adsoφtion removed ~ 95% of TSP, as assessed by sandwich ELISA, while decreases in total protein was substantially less (-15%). TSP depletion correlated with >70% reduction in anti-viral activity, in contrast to saliva adsorbed using a control fusion protein affinity column (GST-1). The data suggest that TSP may account for a major proportion of HlV-specific inhibitory activity in saliva.
To delineate further a TSP-specific effect, LIMPII TSP-binding peptide L75-155 (10 kPa product purified after removal of GST moiety by proteolytic cleavage) was included in HIV-TSP1 pre-incubation mixtures as a competitor.
Figure 8 shows that 1 μM LIMPII peptide abrogated the inhibitory effect of even high concentrations of TSPl (50-100 μg/ml) by 83-90%. Incubation of virus in the presence of LIMPII peptide alone resulted in minimal decrease of HIV-1 infectivity (~ 9%), suggesting that amounts of peptide able to block the TSPl anti-viral effect were not sufficient to compete for HIV-1 env binding sites on PBMC target cells. The ability of the LIMPII peptide to restore infectivity supports a direct role for a CD36/LIMPII-related TSPl binding domain on HIV-1 gpl20, and provides further evidence of a common binding site on TSPl.
Another mechanism to explain TSP-mediated blockade to HIV infection would be a direct effect on target cells, whereby alterations in cell function would decreased the capacity to support productive infection. To address whether exogenous purified TSPl induced down-modulation of the high affinity HIV receptor, CD4+ Jurkat and SK23 T-cell lines, as well as PHA-activated PBMC, were monitored for differences in CD4 surface expression after culture for 3 days in the absence or presence of 100 μg/ml TSP 1. By flow cytometric analysis of cells stained with fluorescein-conjugated anti-CD4 IgG, no change in relative fluorescence intensity or percent CD4+ cell was detected. Thus, TSPl likely does not reduce cell susceptibility. To examine whether HIV-1 infection modulates TSPl expression, TSPl secretion was monitored in three groups of cells: an uninfected line of U937 promonocytic cells, Ul.l cells representing chronically infected U937 containing 2 stably integrated copies of HIVOl/LAI, and U937 acutely infected with HIV-1 to high copy number (> 1000 proviral copies/cell, (Laurence et al, "Human
Immunodeficiency Virus Infection of Monocytes: Relationship to Fc-Gamma Receptors and Antibody-Dependent Viral Enhancement," Immunology, 70:338-343 (1990), which is hereby incoφorated by reference). Cells were pre-incubated 1 hour in the presence or absence of phorbol myrisate acetate ("PMA"), a mitogenic inducer of HIV from chronically infected cells (Laurence et al, "Human Immunodeficiency Virus Infection of Monocytes: Relationship to Fc-Gamma Receptors and Antibody- Dependent Viral Enhancement," Immunology, 70:338-343 (1990), which is hereby incoφorated by reference), also shown to stimulate TSPl expression in cell lines (Varani et al, "Thrombospondin Production and Thrombospondin-Mediated Adhesion in U937 Cells," Exp. Cell Res.. 195:177-182 (1991), which is hereby incorporated by reference). After 24 hours in culture medium (RPMI 1640 with 5% FCS), cells were switched to semm-free medium for an additional 18 hours, then supernatants were collected for TSPl quantitation by ELISA. As shown in Table 3 below, HIV-1 infection did not diminish TSPl production, although acute infection blunted the response to PMA induction of TSP 1.
Table 3: Production of TSP by Monocytoid Cells in Presence/ Absence of HIV-1 Infection
Cell Line Infection Status PMA (5 ng/ml) TSP1/TSP2 cone, (ng/ml)
U937 parental, uninfected - < 5
U937 promonocytic line + 51
Ul .l U937, chronic - 50
Ul .l 2 stable copies LAI + 125
U937/HIV acute, high copy - 180
U937/HIV IIIB (0.02 moi) + 135
The present invention concerns, in part, the identification of a TSPl binding site in the C2 and C3 regions of gpl20, conserved areas of the HIV envelope which are important in binding to CD4, and the demonstration of direct interaction between gpl20 and a specific cell adhesion sequence found in the TSPl type I repeat. Characterization by in vitro binding and competition studies substantiates that these CP36/LIMPII-related CLESH-1 motifs in gpl20 represent authentic TSPl binding domains. The physiological significance of TSPl-gpl20 complex formation is supported by observations that: salivary inhibition of HIV-1 infectivity was markedly reduced by affinity depletion of TSPl; saliva samples that block infection following filtration contained levels of TSPl that correlate with inhibitory concentrations of purified TSPl; and higher amounts of TSPl required to block HIV-1 infectivity in vitro are comparable to the greater quantities of saliva required to obtain an anti -viral effect (Archibald et al, "In Vitro Inhibition of HIV-1 Infectivity by Human Salivas," AIPS Res. Hum. Retrovimses. 6:1425-1432 (1990); Bergey et al, "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic Syndr., 7:995-1002 (1994), which are hereby incoφorated by reference). Our findings establish a distinct mechanism to explain HlV-specific blockade of transmission via saliva.
The likelihood that HIV inhibitors in saliva identified in vitro are active in vivo is bolstered by two lines of evidence. First, in a study of 48 HIV+ patients, 88% of PBMC samples, but no saliva samples, were positive for replication- competent HIV (Qureshi et al, "Prevalence of HIV-1 Proviral PNA an Virion- Associated RNA in Saliva," J. Pent. Res.. 73:2564A (1994), which is hereby incoφorated by reference). Second, recovery rates for HIV in saliva do not differ before and after dental procedures accompanied by bleeding into the oral cavity (Moore et al, "HIV Recovery from Saliva Before an After Pental Treatment: Inhibitors May Have a Crucial Role in Viral Inactivation," J. Am. Pent. Assoc, 124:67-74 (1993), which is hereby incoφorated by reference), indicating that free virus from blood was removed or inactivated. The fact that HIV is not found within salivary ancinar and ductal elements (Fox, "Saliva and Salivary Gland Alterations in HIV Infection," J. Am. Pent. Assoc. 122:46-48 (1991) , which is hereby incoφorated by reference) implies that while virions and infected cells may traffic into the salivary glands, they cannot establish a productive infection.
TSPl is synthesized in low amounts by monocytes/macrophages, epithelial cells, fibroblasts, smooth muscle cells, pneumocytes, and endothelial cells, and in larger quantities by platelets (Lahav, editor, "Thrombospondin," CRC Press, (1993), which is hereby incoφorated by reference). However, HIV may be exposed to levels of TSPl over 2-log higher on surfaces in the oral cavity. The fluid distribution of TSPl, with very low concentrations in plasma, sweat, tears, and urine, reflects the relative frequency with which HIV can be isolated from these secretions, but not from saliva. Breast secretions present another issue. HIV can be cultured from some samples of breast milk, which has been implicated in HIV transmission. Colostrum often contains high concentrations of TSPl (upwards of 145 μg/ml), while lower, more variable amounts (to < 1 μg/ml) have been measured in other breast secretions (Pawes et al, "Thrombospondin in Milk, Other Breast Secretions and Breast Tissue," Sem. Thromb. Heost, 13:378-384 (1987), which is hereby incoφorated by reference). However, HIV has not been recovered from breast milk devoid of cells (Guay et al, "Petection of Human Immunodeficiency Vims Type 1 (HIV-1) PNA and p24 Antigen in Breast Milk of HIV-1 -Infected Ugandan Women and Verticle Transmission," Pediatrics, 98:438-444 (1996), which is hereby incoφorated by reference), and breast milk contains factors which inhibit HIV infection (VandePerre et al, "Infective and Anti-Infective Properties of Breast Milk from HIV-1 Infected Women," Lancet, 341 :914-918 (1993), which is hereby incoφorated by reference), one of which may be TSP.
The concept that an extracellular matrix molecule may serve as an inhibitor of microbial pathogens is not new. For example, fibronectin ("FN") binds free gρl20 (Su et al, "Interaction of the Envelope Glycoprotein of Human Immunodeficiency Virus with Clq and Fibronectin Under Conditions Present in Saliva," Moi Immunol, 28:811-817 (1991), which is hereby incoφorated by reference), and thereby might sequester HIV virions. The compartmentalization of FN in gingival crevicular fluid, whole and submandibular saliva, but not parotid fluid (Tynelius-Bratthall et al, "Fibronectin in Saliva and Gingival Crevices," J. Period. Res., 21 :563-568 (1986), which is hereby incoφorated by reference), parallels our findings with TSP. However, concentrations required to inhibit HIV infectivity in vitro exceed those found in saliva by 10-fold. While high levels of FN are present in plasma (~ 300 μg/ml) (Torre et al, "Plasma Fibronectin Concentrations in Patients with Human Immunodeficiency Virus Infection," J. Clin. Pathol, 43:560-562 (1990), which is hereby incoφorated by reference), so is recoverable infectious HIV. In fact, FN actually may facilitate HIV-mediated syncycium formation (Ushijima et al, "Effect of Semm Components on Syncytium Formation and Virus Production by Cells Infected with Human Immunodeficiency Viruses In Vitro," AIPS Res. Hum. Retroviruses, 8:513-520 (1992), which is hereby incoφorated by reference), and promote the growth of AIPS-KS (Kaposi sarcoma) cells constitutively expressing high levels of FN receptor (Barillari et al, "The RGP Motif and the Integrin Receptors are Involved in the Vascular Cell Growth and Adhesive Properties of Extracellular HIV-1 Tat Protein," J. AIPS. 6:688A (1993), which is hereby incoφorated by reference), bringing further into question the physiological significance of FN-gpl20 binding. In contrast, TSPl proteolytic fragments and peptides show opposite effects, inhibiting KS/endothelial cell proliferation (Taraboletti et al, "Platelet Thrombospondin Modulates Endothelial Cell Adhesion, Motility, and Growth: A Potential Angiogenesis Regulatory Factor," J. Cell Biol. 1 11 :765-772 (1990); Roberts et al, "Modulation of Tumor Growth In Vitro and In Vivo by Stable Analogs of Thrombospondin Peptides," AIPS Res. Hum. Retrovimses, 21 :S73 (1995), which are hereby incoφorated by reference). Pefming the relationship between salivary inhibitors of HIV for in vivo versus in vitro model systems is important. Concentrations of TSPl required to inhibit HIV infectivity by < 50% following direct addition were equivalent to that found in dilutions of saliva used in HIV inhibition experiments by other investigators (Malamud et al, "Human Submandibular Saliva Aggregates HIV," AIPS Res. Hum. Retroviruses, 9:633-637 (1993); Fox et al, "Saliva Inhibits HIV-1 Infectivity," J. Am. Pent. Assoc, 116:635-637 (1988); Fox et al, "Salivary Inhibition of HIV-1 Infectivity: Functional Properties and Pistribution in Men, Women and Children," L Am. Pent. Assoc, 118:709-711 (1989); Bergey et al, "Interaction of HIV-1 and Human Salivary Mucins," J. Acquired Immune Pefic. Svndr., 7:995-1002 (1994), which are hereby incoφorated by reference). Much lower doses of TSPl were required to abrogate HIV infectivity when HIV-TSP mixtures were pre-filtered, suggesting aggregation of virion-TSPl complexes as a potential mechanism of inhibition. The requirement for pre-filtration may have its in vivo counteφart in the continued cleansing of oral surfaces by salivary flow, with elimination of enmeshed viral particles from potential attachment sites. Experiments testing direct inhibition by whole saliva are complicated by the fact that additional salivary components may contribute to the anti-HIV effect. Indeed, TSPl affinity depletion removed only ~ 70% of the HIV-inhibitory activity. In addition, nonspecific antiviral phenomena may occur with saliva dilutions of 1:1 to 1:4 (Liuzzi et al, "Analysis of HIV-1 Load in Blood, Semen and Saliva: Evidence for Pifferent Viral Compartments in a Cross- Sectional and Longitudinal Study," AIPS. 10:F10-F56 (1996), which is hereby incoφorated by reference). For example, highly charged sulfated polysaccharides, such as dextran sulfate and salivary mucins, present nonspecific anionic charge barriers to CD4-gpl20 interactions at high concentrations (Amory et al, "The Large Molecular Weight Glycoprotein MGI, a Component of Human Saliva, Inhibits HIV-1 Infectivity," Clin. Res.. 40:51A (1992); Baba et al, "Novel Sulfated Polysaccharides: Dissociation of Anti-Human Immunodeficiency Virus Activity from Anti-Thrombin Activity," J. Infect. Pis., 161:208-213 (1990), which are hereby incorporated by reference).
The presence of properdin-related sequences and properdin binding activity described for gpl20 and gp41 (Stoiber et al, "Human Complement Proteins C3b, C4b, Factor H and Properdin React with Specific Sites in gpl20 and gp41, the Envelope Proteins of HIV-1," Immunobiol. 193/1 :98-113 (1995), which is hereby incoφorated by reference) support the functional significance of our sequence search results. The CSVTCG (SEQ. IP. No. 1) motif is found in two of the three Type 1 properdin/malaria-like repeats of TSPl . Consistent with homologies detected between CP36/LIMPII TSPl binding domains and conserved sequences surrounding the V3 region of gpl20, in vitro binding and peptide inhibition data indicate a highly specific gpl20 interaction mediated through this CLESH-1 site. A high affinity TSPl binding site on gpl20 traversing the cysteine bridge of the V3 loop was suspected. However, envelope peptide mapping data suggested the presence of two full-length functional sites, with potential for direct involvement of the V3 loop, and presented a more complex model in which multiple or sequential site utilization is possible. Piscontinuity created by intervening residues between the first and second motif half- site could induce conformational strain to distort or physically disrupt V3 loop integrity, with profound negative effects on gpl20-CP4 association. Binding of TSP to the first highly homologous half-site in the C2 domain might lead to subsequent binding to weakly homologous downstream residues extending into the V3 loop, freeing the second full-length TSP binding site in the C3 domain. The second motif encompasses a serine pair, KQSS (SEQ. IP. No. 4), shown to be critical for CP4 binding (Kowalski et al, "Functional Regions of the Envelope Glycoprotein of Human Immunodeficiency Virus Type 1," Science, 237:1351-1355 (1987), which is hereby incoφorated by reference). Mechanistically, TSPl could compete directly with CP4, occupying an identical or overlapping site. Alternatively, the bulky TSPl trimer may sterically block access to the CP4 binding site, as well as V3 loop determinants. This model could explain why only partial inhibition is seen using CSVTCG peptides (SEQ. IP. No. 1). Elucidation of the molecular basis for TSPl interference of CP4-gpl20 association awaits additional structural studies. Anti-HIV activities vary quantitatively among individual saliva donors.
There also are differences in whether filtration is required to detect an inhibitory effect (Nagashunmugam et al, "Human Submandibular Saliva Specifically Inhibits HIV Type 1," AIDS Res. Hum. Retroviruses, 13:371-376 (1997), which is hereby incoφorated by reference). In general, however, titers of salivary inhibitory factors decline with disease progression, in parallel with decreased total protein concentrations (Lai et al, "Pilot Study Comparing the Salivary Cationic Protein Concentrations in Healthy Adults and AIDS Patients: Correlation with Antifungal Activity," J. AIDS, 5:904-914 (1992), which is hereby incoφorated by reference). There is a greater chance of HIV recovery from saliva with advancing clinical stage, albeit the rate is still low in comparison with other body fluids (Moore et al, "HIV Recovery from Saliva Before an After Dental Treatment: Inhibitors May Have a Crucial Role in Viral Inactivation," J. Am. Dent. Assoc, 124:67-74 (1993), which is hereby incoφorated by reference). This raises the possibility that HIV may directly suppress production of saliva inhibitory factors, or elicit blocking molecules. TSPl production is down-regulated by DNA vimses (Mosher, "Physiology of
Thrombospondin," Ann. Rev. Med.. 41:85-97 (1990), which is hereby incoφorated by reference), and production of FN is depressed by retro viral infection (Adams et al, "Modulation of Fibronectin Gene Expression in Chondrocytes by Viral Transformation and Substrate Attachment," J. Cell Biol.. 105:483-488 (1987), which is hereby incoφorated by reference). However, production of TSPl by PMA- activated monocytes was not affected by HIV infection in our system, and acute or chronic HIV infection actually upregulated TSPl production by these cells, although to levels lower than shown to affect HIV infectivity (< 2ng/ml). Regardless of normal levels of TSPl production in the oral cavity, mechanical alterations also may contribute to decreased saliva inhibitory activity in vivo, as decreased salivary flow rates and buffering capacity correlate with advanced HIV infection (Madigan et al, "Caries and Criogenic Flora in HIV-Positive Children Versus Uninfected Children," J. Dent. Res., 73T898A (1994), which is hereby incoφorated by reference).
The location of TSPl binding motifs in highly conserved HIV domains makes these sites attractive targets for blocking agents that would be broadly reactive to HIV-1 and HIV-2 substrains. The ability of TSPl to block CD4-gpl20 complex formation suggests the potential utility of this matrix molecule in the development of non-toxic natural inhibitors of local transmission of HIV-1, perhaps as a candidate topical adjuvant that could serve as a preventive physical barrier for rectogenital and GI tract mucosa.
Example 4 - RANTES Binding to TSPl
Further analysis of the CLESH-1 structure was performed by searching a crystal stmcture database that combines primary sequence homology and predicted secondary stmcture. The CLESH-1 sequence of LIMPII was used to perform this search using BEAUTY as described above. The highest scoring match for LIMPII was to RANTES, a β-chemokine that competes with gρl20 for binding to the CCR5 co-receptor for M-tropic HIV-1 strains.
In solid phase assays, 125I-RANTES binded to immobilized TSP. Saturatable binding showed biphasic higher order kinetics, half maximal at ~330nM (Figure 9). Estimated stoichiometry at equilibrium was low (~0.5 RANTES molecule for every 10 TSP monomer), although the steep slope of the second phase indicated a cooperative interaction (Hanes plot, Figure 10). One inteφretation of the data is that RANTES homodimerization is favored over binding to TSP. At a concentration that shifts equilibrium toward formation of the heterologous complex, positive cooperativity likely reflects a higher apparent affinity of RANTES monomers for binding to TSP, with an increase in monomers being a function of the odd number of sites presented by TSP homotrimers. The ability of RANTES to bind directly to TSP suggests that RANTES may compete for TSP binding sites in gpl20, and supports further the potential for CLESH-1 -containing conserved domains of gpl20 to share structural homology with the β-turn configuration of RANTES.
EXAMPLE 5 - CD4 Binding to TSPl and the CSVTCG TSP Analog
1
In solid phase assays, I-CP4 binded to immobilized TSPl in a concentration dependent saturatable manner, with biphasic kinetics. The results of this study are shown in Figure 12. Additionally, 125I-CP4 binded directly to the TSP analog having an amino acid sequence CSVTCG (SEQ. IP. No. 1), implicating the presence of a CP36-related binding site (i.e., a CLESH-1 motif) in CP4. These results are consistent with TSP saturating CLESH-1 sites in gpl20, which is believed to free properdin sequences in gpl20 to form a tri-molecular complex with CP4.
Although the invention has been described in detail for the puφose of illustration, it is understood that such detail is solely for that puφose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:
1. A method for suppressing infectivity of HIV comprising: contacting the HIV or a cell targeted by HIV with an effective amount of a thrombospondin or a thrombospondin analog.
2. The method according to claim 1 , wherein said method is carried out with an effective amount of a thrombospondin and wherein the thrombospondin is thrombospondin- 1 or thrombospondin-2.
3. The method according to claim 1, wherein said method is carried out with an effective amount of a thrombospondin analog and wherein the tlirombospondin analog is selected from a group consisting of an N-terminal region of a thrombospondin, a procollagen-homologous region of a thrombospondin, a type I repeat of a thrombospondin, a type II repeat of a thrombospondin, a calcium-binding domain of a thrombospondin, and a carboxy-terminal region of a thrombospondin, thrombospondin domains which bind to an HIV gpl20 envelope protein, and thrombospondin domains which bind to a CP4 receptor on the cell.
4. The method according to claim 1, wherein said method is carried out with an effective amount of a thrombospondin analog and wherein the thrombospondin analog is a polypeptide comprising an amino acid sequence of SEQ. IP. No. 1.
5. The method according to claim 1, wherein said method is carried out by contacting the HIV with a thrombospondin or a thrombospondin analog which binds to an HIV g l20 envelope protein.
6. The method according to claim 1, wherein said method is carried out by contacting the cell targeted by HIV with a thrombospondin or a thrombospondin analog which binds to a CP4 receptor on the cell.
7. The method according to claim 1, wherein said contacting the HIV or the cell is carried out intraorally, intrarectally, or intravaginally.
8. A contraceptive comprising: a contraceptive carrier and a thrombospondin or a thrombospondin analog.
9. The contraceptive according to claim 8, wherein said contraceptive carrier is a contraceptive composition selected from the group consisting of a contraceptive foam, jelly, lubricant, and spermicide.
10. The contraceptive according to claim 8, wherein said contraceptive carrier is a contraceptive device selected from the group consisting of a sponge, intravaginal device, cervical diaphragm, and condom.
11. The contraceptive according to claim 8, wherein said contraceptive contains thrombospondin- 1 or thrombospondin-2.
12. The contraceptive according to claim 8, wherein said contraceptive contains a thrombospondin analog selected from a group consisting of an N-terminal region of a thrombospondin, a procollagen-homologous region of a thrombospondin, a type I repeat of a thrombospondin, a type II repeat of a thrombospondin, a calcium-binding domain of a thrombospondin, and a carboxy- terminal region of a thrombospondin, thrombospondin domains which bind to an HIV gρl20 envelope protein, and thrombospondin domains which bind to a CP4 receptor on a cell.
13. The contraceptive according to claim 8, wherein said contraceptive contains a thrombospondin analog and wherein said thrombospondin analog is a polypeptide comprising an amino acid sequence of SEQ. IP. No. 1.
PCT/US1998/024905 1997-11-25 1998-11-24 Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity WO1999026649A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU15971/99A AU1597199A (en) 1997-11-25 1998-11-24 Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity
US09/555,090 US6964763B1 (en) 1997-11-25 1998-11-24 Methods and compositions for inhibiting HIV infectivity and blocking chemokine activity

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US6629497P 1997-11-25 1997-11-25
US60/066,294 1997-11-25
US7887398P 1998-03-20 1998-03-20
US60/078,873 1998-03-20

Publications (2)

Publication Number Publication Date
WO1999026649A1 true WO1999026649A1 (en) 1999-06-03
WO1999026649A9 WO1999026649A9 (en) 2000-07-06

Family

ID=26746580

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/024905 WO1999026649A1 (en) 1997-11-25 1998-11-24 Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity

Country Status (2)

Country Link
AU (1) AU1597199A (en)
WO (1) WO1999026649A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1377253A4 (en) * 2001-04-13 2008-09-03 Gen Hospital Corp Methods of preventing uvb-induced skin damage

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992017499A1 (en) * 1991-04-08 1992-10-15 Cornell Research Foundation, Inc. A unique hexapeptide derived from thrombospondin and its uses
WO1996038480A1 (en) * 1995-06-02 1996-12-05 Allelix Biopharmaceuticals, Inc. Anti-inflammatory thrombospondin-derived peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992017499A1 (en) * 1991-04-08 1992-10-15 Cornell Research Foundation, Inc. A unique hexapeptide derived from thrombospondin and its uses
WO1996038480A1 (en) * 1995-06-02 1996-12-05 Allelix Biopharmaceuticals, Inc. Anti-inflammatory thrombospondin-derived peptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CROMBIE R ET AL: "Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.", JOURNAL OF EXPERIMENTAL MEDICINE, (1998 JAN 5) 187 (1) 25-35, XP002101396 *
CROMBIE R ET AL: "Lysosomal integral membrane protein II binds thrombospondin-1. Structure-function homology with the cell adhesion molecule CD36 defines a conserved recognition motif.", JOURNAL OF BIOLOGICAL CHEMISTRY, (1998 FEB 27) 273 (9) 4855-63, XP002101397 *
CROMBIE, R. ET AL: "Blockade of human immunodeficiency virus infectivity by salivary thrombospondin.", JOURNAL OF INVESTIGATIVE MEDICINE, (1997) VOL. 45, NO. 3, PP. 201A. ANNUAL MEETING OF THE ASSOCIATION OF AMERICAN PHYSICIANS, XP002101395 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1377253A4 (en) * 2001-04-13 2008-09-03 Gen Hospital Corp Methods of preventing uvb-induced skin damage

Also Published As

Publication number Publication date
WO1999026649A9 (en) 2000-07-06
AU1597199A (en) 1999-06-15

Similar Documents

Publication Publication Date Title
Crombie et al. Identification of a CD36-related thrombospondin 1–binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1–specific inhibitory factors in human saliva
McNeely et al. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.
Shugars Endogenous mucosal antiviral factors of the oral cavity
Sun et al. Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120
Ho et al. Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization
US6190871B1 (en) Immunoreagents reactive with a conserved epitope of human immunodeficiency virus type I (HIV-1) gp120 and methods of use
EP0269712A1 (en) Peptides involved in the pathogenesis of hiv infection
US5854400A (en) Monoclonal antibodies which neutralize HIV-1 infection
KR920008744B1 (en) Monoclonal antibodies to hiv and related peptides
MXPA01010985A (en) Method and compositions for preventing or reducing hiv infection by use of inhibitors for leucine aminopeptidase.
O'Brien et al. Macrophage-tropic and T-cell line-adapted chimeric strains of human immunodeficiency virus type 1 differ in their susceptibilities to neutralization by soluble CD4 at different temperatures
Shugars et al. Endogenous salivary inhibitors of human immunodeficiency virus
US6132721A (en) Non-Toxic immunogens derived from a retroviral regulatory protein, antibodies, preparation method therefor, and pharmaceutical compositions containing same
Lin et al. Characterization of siamycin I, a human immunodeficiency virus fusion inhibitor
US20120213790A1 (en) Methods of treatment using il-16 antagonist peptides
US20130189264A1 (en) A novel chimeric protein for prevention and treatment of hiv infection
CA2190613A1 (en) Methods for the identification of compounds capable of abrogating hiv-1 vpr-rip-1 binding interactions
US6242564B1 (en) Treatment of tropical spastic paresis with peptide T
US5017688A (en) Peptides involved in the pathogenesis of HIV infection
US5981278A (en) Chimeric monoclonal antibodies which neutralize HIV-1 infection and their applications in therapy and prevention for AIDS
JPH05506031A (en) Suppression of diseases associated with immunodeficiency virus infection
US6964763B1 (en) Methods and compositions for inhibiting HIV infectivity and blocking chemokine activity
AU3889600A (en) A novel chimeric protein for prevention and treatment of hiv infection
WO1999026649A1 (en) Methods and compositions for inhibiting hiv infectivity and blocking chemokine activity
Würzner et al. Human Immunodeficiency Virus Type 1 gp41 Binds to Candida albicans via Complement C3-like Region

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

AK Designated states

Kind code of ref document: C2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 48-53, CLAIMS, ADDED

WWE Wipo information: entry into national phase

Ref document number: 09555090

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA