AU5603999A - Creation of Inventions and Patents with Artificial Intelligence

AU5603999A – Antiviral proteins, DNA coding sequences therefor, and uses thereof
– Google Patents

AU5603999A – Antiviral proteins, DNA coding sequences therefor, and uses thereof
– Google Patents
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Download PDF
Info

Publication number
AU5603999A

AU5603999A
AU56039/99A
AU5603999A
AU5603999A
AU 5603999 A
AU5603999 A
AU 5603999A
AU 56039/99 A
AU56039/99 A
AU 56039/99A
AU 5603999 A
AU5603999 A
AU 5603999A
AU 5603999 A
AU5603999 A
AU 5603999A
Authority
AU
Australia
Prior art keywords
antiviral
peptide
conjugate
protein
isolated
Prior art date
1995-04-27
Legal status (The legal status 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 status listed.)

Granted

Application number
AU56039/99A
Other versions

AU746809B2
(en

Inventor
Michael R. Boyd
Kirk R Gustafson
James B. Mcmahon
Robert H. Shoemaker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

National Institutes of Health NIH

Original Assignee
United States, Represented By Secretary
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.)
1995-04-27
Filing date
1999-10-22
Publication date
2000-01-06

1999-10-22
Application filed by United States, Represented By Secretary
filed
Critical
United States, Represented By Secretary

1999-10-22
Priority to AU56039/99A
priority
Critical
patent/AU746809B2/en

2000-01-06
Publication of AU5603999A
publication
Critical
patent/AU5603999A/en

2002-05-02
Application granted
granted
Critical

2002-05-02
Publication of AU746809B2
publication
Critical
patent/AU746809B2/en

2016-04-26
Anticipated expiration
legal-status
Critical

Status
Ceased
legal-status
Critical
Current

Links

Espacenet

Global Dossier

Discuss

Description

-1-
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
a Name of Applicant/s: Actual Inventor/s: The United States of America, represented by the Secretary, Department of Health and Human Services Michael R. Boyd and Kirk R. Gustafson and Robert H. Shoemaker and James B. McMahon BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 ‘ANTIVIRAL PROTEINS, DNA CODING SEQUENCES THEREFOR, AND USES THEREOF’ Address for Service: Invention Title: Details of Original Application No. 56691/96 dated 26 APR 1996 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 25765AUP01 ANTIVIRAL PROTEINS, DNA CODING SEQUENCES THEREFOR, AND USES THEREOF TECHNICAL FIELD OF THE INVENTION This invention relates to antiviral proteins 10 (collectively referred to as cyanovirins), as well as conjugates thereof, antibodies thereto, DNA sequences encoding same, compositions comprising same, host cells transformed to produce same, compositions comprising same, and methods of using and obtaining same, especially in clinical applications, such as in antiviral therapy and prophylaxis.
BACKGROUND OF THE INVENTION Acquired immune deficiency syndrome (AIDS) is a fatal disease, reported cases of which have increased dramatically within the past two decades. The virus that causes AIDS was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphotropic virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. This particular retrovirus has also been known as lymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and, presently, as human immunodeficiency virus (HIV). Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2.
The acronym HIV is used herein to refer generically to human immunodeficiency viruses.
HIV exerts profound cytopathic effects on the CD4′ helper/inducer T-cells, thereby severely compromising the immune system. HIV infection also results in neurological deterioration and, ultimately, in death of infected individuals. Tens of millions of people are infected with HIV worldwide, and, without effective 10 therapy, most of these are doomed to die. During the long latency, the period of time from initial infection to ‘the appearance of symptoms, or death, due to AIDS, infected individuals spread the infection further, by sexual contacts, exchanges of contaminated needles during i.v. drug abuse, transfusions of blood or blood products, or maternal transfer of HIV to a fetus or newborn. Thus, there is not only an urgent need for effective therapeutic agents to inhibit the progression of HIV ‘disease in individuals already infected, but also for methods of prevention of the spread of HIV infection from infected individuals to noninfected individuals. Indeed, the World Health Organization (WHO) has assigned an urgent international priority to the search for an effective anti-HIV prophylactic virucide to help curb the further expansion of the AIDS pandemic (Balter, Science 266, 1312-1313, 1994; Merson, Science 260, 1266-1268, 1993; Taylor, J. NIH Res. 6, 26-27, 1994; Rosenberg et al., Sex. Transm. Dis. 20, 41-44, 1993; Rosenberg, Am. J.
Public Health 82, 1473-1478, 1992).
The field of viral therapeutics has developed in response to the need for agents effective against retroviruses, especially HIV. There are many ways in which an agent can exhibit anti-retroviral activity (see, DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J. Accuir. Immun. Def. Svnd. 4, 207-218, 1991; Mitsuya et al., Science 249, 1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit the viral reverse transcriptase, are among the few clinically active agents that are currently available commercially for anti-HIV therapy. Although very useful in some patients, the 10 utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy. Also, given more recent revelations of the dynamics of HIV infection (Coffin, Science 267, 483- 489, 1995; Cohen, Science 267, 179, 1995; Perelson et al., Science 271, 1582-1586, 1996), it is now increasingly apparent that agents acting as early as Spossible in the viral replicative cycle are needed to inhibit infection of newly produced, uninfected immune cells generated in the body in response to the virusinduced killing of infected cells. Also, it is essential to neutralize or inhibit new infectious virus produced by infected cells.
Infection of CD4’ cells by HIV-1 and related primate immunodeficiency viruses begins with interaction of the respective viral envelope glycoproteins (generically termed “gpl20”) with the cell-surface receptor CD4, followed by fusion and entry (Sattentau, AIDS 2, 101-105, 1988; Koenig et al., PNAS USA 86, 2443-2447, 1989).
Productively infected, virus-producing cells express gpl20 at the cell surface; interaction of gpl20 of infected cells with CD4 on uninfected cells results in formation of dysfunctional multicellular syncytia and further spread of viral infection (Freed et al., Bull.
Inst. Pasteur 88, 73, 1990). Thus, the gpl20/CD4 interaction is a particularly attractive target for interruption of HIV infection and cytopachogenesis, either by prevention of initial virus-to-cell binding or by blockage of cell-to-cell fusion (Capon et al., Ann.
Rev. Immunol. 9, 649-678, 1991). Virus-free or “soluble” gpl20 shed from virus or from infected cells in vivo is 10 also an important therapeutic target, since it may otherwise contribute to noninfectious immunopathogenic processes throughout the body, including the central nervous system (Capon et al., 1991, supra; Lipton, Nature 367, 113-114, 1994). Much vaccine research has focused upon gpl20; however, progress has been hampered by hypervariability of the gpl20-neutralizing determinants Sand the consequent extreme strain-dependence of viral sensitivity to gpl20-directed antibodies (Berzofsky, J.
Aca. Immun. Def. Svnd. 4, 451-459, 1991). Relatively little drag discovery and development research has focused specifically upon gpl20. A notable exception is the considerable effort that has been devoted to truncated, recombinant “CD4” proteins (“soluble CD4” or “sCD4”), which bind gpl20 and inhibit HIV infectivity in vitro (Capon et al., 1991, suora; Schooley et al., Ann.
Int. Med. 112, 247-253, 1990; Husson et al., J. Pediatr.
121, 627-633, 1992). However, clinical isolates, in contrast to laboratory strains of HIV, have proven highly resistant to neutralization by sCD4 (Orloff et al., AIDS Res. Hum. Retrovir. 11, 335-342, 1995; Moore et al., J Virol. 66, 235-243, 1992). Initial clinical trials of sCD4 (Schooley et al., 1990, supra; Husson et al., 1992, sura), and of sCD4-coupled immunoglobulins (Langner et al., Arch. Virol. 130, 157-170, 1993), and likewise of sCD4-coupled toxins designed to bind and destroy virusexpressing cells (Davey et al., J. Infect. Dis. 170, 1180-1188, 1994; Ramachandran et al., J. Infect. Dis.
170, 1009-1013, 1994), have been disappointing. Newer gene-therapy approaches to generating sCD4 directly in vivo (Morgan et al., AIDS Res. Hum. Retrovir. 10, 1507- 0 1515, 1994) will likely suffer similar frustrations.
Therefore, new antiviral agents, to be used alone or S”ini- combination with AZT and/or other available antiviral agents, are needed for effective antiviral therapy 0* 0 against AIDS. New agents, which may be used to prevent HIV infection, also are important for prophylaxis. In .both areas of need, the ideal new agents would act as •early as possible in the viral life cycle, be as virusspecific as possible attack a molecular target 00 specific to the virus but not to the infected or infectibie animal host), render the intact virus noninfectious, prevent the death or dysfunction of virusinfected mammalian cells, prevent further production of virus from infected cells, prevent spread of virus infection to uninfected mammalian cells, be highly potent and active against the broadest possible range of strains and isolates of HIV, be resistant to degradation under physiological and rigorous environmental conditions, and be readily and inexpensively produced on a large-scale.
The present invention seeks to provide antiviral proteins and conjugates thereof, which possess at least some of the aforementioned particularly advantageous attributes, as well as compositions comprising same and methods of making and using same. These and other objects of the present invention, as well as additional inventive features, will become apparent from the description provided herein.
BRIEF SUMMARY OF THE INVENTION The present invention provides antiviral agents, in particular antiviral proteins (collectively referred to 10 as cyanovirins) and conjugates thereof. The present invention also provides methods of obtaining a cyanovirin arid a conjugate thereof, nucleic acid molecules encoding cyanovirins and conjugates thereof, host cells containing the aforementioned nucleic acid molecules, a method of using a cyanovirin to target an effector molecule to a virus, and a method of obtaining a substantially pure S”cyanovirin or a conjugate thereof. The cyanovirin, conjugate thereof, and host cells transformed to produce S. a cyanovirin or conjugate thereof can be used in a 00 composition, such as a pharmaceutical composition, which can additionally comprise one or more other antiviral agents. The present invention also provides for the use of cyanovirins, conjugates thereof, host cells transformed to produce a cyanovirin or conjugate thereof, and compositions thereof, alone or in combination with other antiviral agents, in the therapeutic and/or prophylactic treatment of an animal, such as a human, infected or at risk for infection with a virus and in the treatment of inanimate objects, such as medical and laboratory equipment and supplies, as well as suspensions or solutions, such as blood and blood products and -7tissues, to prevent viral infection of an animal, such as a human. The present invention further provides methods of therapeutic or prophylactic treatment of an animal, such as a human, infected or at risk of infection with a virus, comprising the administration or application of one or more cyanovirin(s), conjugate(s), host cell(s) transformed to produce a cyanovirin or conjugate thereof.
According to a first aspect, the present invention provides an isolated and purified antiviral peptide comprising at least nine contiguous amino acids of the amino acid S* sequence of SEQ ID NO:2.
9. According to a second aspect, the present invention provides an isolated and 9 purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate comprising at least nine contiguous amino acids of the amino acid sequence of SEQ ID 9* NO:2, wherein said antiviral protein conjugate or antiviral peptide conjugate is coupled to an effector molecule, a virus, a viral envelope glycoprotein, polyethylene glycol, albumin, dextran, a solid support matrix, or a combination of any of the foregoing.
15 According to a third aspect, the present invention provides an isolated and purified nucleic acid molecule which encodes the peptide, protein conjugate or peptide conjugate of the first or second aspect.
According to a fourth aspect, the present invention provides a pharmaceutical composition comprising an antiviral effective amount of the peptide, protein conjugate or peptide conjugate of the first or second aspect and a pharmaceutically acceptable carrier therefor and, optionally, further comprising an immunostimulant.
According to a fifth aspect, the present invention provides a vector which comprises the nucleic acid molecule of the third aspect.
7a According to a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect.
According to a seventh aspect, the present invention provides an antibody binding the peptide, the protein in the protein conjugate or the peptide in the peptide conjugate of the first or second aspect.
According to an eighth aspect, the present invention provides the use of the peptide, the protein conjugate or the peptide conjugate of the first or second aspect for the manufacture of a medicament for preventing or treating a viral infection of an animal.
According to a ninth aspect, the present invention provides the use of a nucleic acid molecule of the third aspect for the manufacture of a medicament for transforming in vivo host cells to express an antiviral peptide or antiviral peptide conjugate encoded by said nucleic acid molecule in vivo.
According to a tenth aspect, the present invention provides a method of producing 15 a peptide, a protein conjugate or a peptide conjugate, which method comprises 5S expressing a peptide, a protein conjugate or a peptide conjugate in the host cell of the sixth aspect.
According to an eleventh aspect, the present invention provides a method of preventing the spread of viral infection comprising treating an inanimate object, solution, suspension, emulsion or other material with an antiviral effective amount of the peptide, the protein conjugate or the peptide conjugate of the first or second aspect.
According to a twelfth aspect, the present invention provides a method of preventing the spread of viral infection comprising treating ex vivo blood, a blood product, sperm, cells, a tissue or an organ with an antiviral effective amount of the -7bpeptide, the protein conjugate or the peptide conjugate of the first or second aspect.
According to a thirteenth aspect, the present invention provides a method of preventing or treating a viral infection of an animal, which method comprises administering to an animal an antiviral effective amount of the antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate of the first or second aspect.
According to a fourteenth aspect, the present invention provides a method of preventing or treating a viral infection of an animal, which method comprises transforming in vivo host cells with the nucleic acid molecule of the third aspect to express an antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate 10 encoded by said nucleic acid molecule in vivo.
According to a fifteenth aspect, the present invention provides a method of preventing or treating a viral infection of an animal, which method comprises transforming host cells with the nucleic acid molecule of the third aspect and placing said transformed host cells into or onto said animal so as to express an antiviral peptide, 15 antiviral protein conjugate or antiviral peptide conjugate encoded by said nucleic acid molecule.
According to a sixteenth aspect, the present invention provides a method of contacting a virus with an antiviral agent, which method comprises contacting said virus with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.
i~l~lZ~ 7c According to a seventeenth aspect, the present invention provides a method of contacting a virus with an antiviral agent, which method comprises contacting said virus with an antiviral effective amount of the antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate of the first or second aspect.
According to an eighteenth aspect, the present invention provides a method of conjugating a virus or a viral envelope glycoprotein with a cyanovirin, which method comprises contacting said isolated and purified virus or said isolated and purified viral envelope glycoprotein with an isolated and purified antiviral protein or an isolated and purified antiviral peptide, wherein said protein or peptide comprises at least nine o* 10 contiguous amino acids of the amino acid sequence of SEQ ID NO:2, and wherein, upon contacting said isolated and purified virus or said isolated and purified viral envelope glycoprotein with said protein or said peptide, said protein or said peptide binds to said isolated and purified virus or said isolated and purified viral envelope glycoprotein, thereby forming a conjugate.
15 According to a ninteenth aspect, the present invention provides a method of administering an antiviral agent to an animal, which method comprises administering to said animal an antiviral effective amount of the peptide, the protein conjugate or the peptide conjugate of the first or second aspect or a conjugate prepared in accordance with the eighteenth aspect.
According to a twentieth aspect, the present invention provides a method of removing virus from a sample, which method comprises: contacting said sample with a composition comprising an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and 7dpurified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO:2, (ii) said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate is attached to a solid support matrix, and (iii) said at least nine contiguous amino acids bind to said virus, and separating said sample and said composition, whereupon virus is removed from said sample.
According to a twenty-first aspect, the present invention provides a method of 9* removing virus from a sample, which method comprises: 10 contacting said sample with a composition comprising the antiviral peptide, the antiviral protein conjugate or the antiviral peptide conjugate of the first or second aspect, and 9* separating said sample and said composition, whereupon virus is removed from said sample.
15 According to a twenty-second aspect, the present invention provides a method of preventing or treating a viral infection of an animal, which method comprises topically administering to a host an antiviral effective amount of a formulation comprising an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.
1 7e According to a twenty-third aspect, the present invention provides a topical formulation comprising an isolated and purified antiviral agent selected from the group consisting of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said protein, peptide, protein conjugate or peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO: 2.
According to a twenty-fourth aspect, the present invention provides a method of preventing or treating a viral infection of an animal, which method comprises 10 administering to the animal an antiviral effective amount of a virus that has been destroyed or rendered noninfectious by contact with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein !5 conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO: 2.
According to a twenty-fifth aspect, the present invention provides the use of host cells transformed with the nucleic acid molecule of the first or second aspect for the manufacture of a medicament for preventing or treating a viral infection of a mammal wherein said medicament is placed into or onto said animal so as to express an antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate encoded by said nucleic acid molecule.
1 7f- According to a twenty-sixth aspect, the present invention provides the use of a formulation comprising an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the manufacture of a medicament for preventing or treating a viral infection of an animal wherein the medicament is administered topically.
.According to a twenty-seventh aspect, the present invention provides the use of a 10 virus that has been destroyed or rendered non-infectious by contact with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the manufacture of a medicament for preventing or treating a viral infection of an animal.
*o Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a graph of OD (206 nm) versus time (min), which represents an HPLC chromatogram of nonreduced cyanovirin-N.
7g- Figure 1B is a bar-graph of maximum dilution for 50% protection versus HPLC fraction, which illustrates the maximum dilution of each HPLC fraction that provided protection from the cytopathic effects of HIV infection for the nonreduced cyanovirin-N HPLC fractions.
Figure 1C is an SDS-polyacrylamide gel electrophoretogram of nonreduced cyanovirin-N HPLC fractions.
Figure 1D is a graph of OD (206 nm) versus time (min), which represents an HPLC chromatogram of reduced cyanovirin-N.
Figure 1E is a bar graph of maximum dilution for 50% protection versus HPLC t* 10 dilution, which illustrates the maximum dilution of each fraction that provided protection from the cytopathic effects of HIV infection for the reduced cyanovirin-N HPLC fractions.
1~ Figure IF is an SDS-polyacrylamide gel electrophoretogram of reduced cyanovirin-N HPLC fractions.
Figure 2 shows an example of a DNA sequence encoding a synthetic cyanovirin gene.
Figure 3 illustrates a site-directed mutagenesis maneuver used to eliminate codons for a FLAG octapeptide and a Hind III restriction site from the sequence of Figure 2.
10 Figure 4 shows a typical HPLC chromatogram from the 99 o purification of recombinant native cyanovirin-N.
Figure 5A is a graph of k control versus cyanovirin- N concentration which illustrates the antiviral activity of native cyanovirin-N from Nostoc ellipsosporum.
Figure 5B is a graph of control versus cyanovirin- N concentration which illustrates the antiviral activity of recombinant cyanovirin-N.
Figure SC is a graph of control versus cyanovirin- N concentration which illustrates the antiviral activity of recombinant FLAG-cyanovirin-N.
Figure 6A is a graph of control versus cyanovirin- N concentration which depicts the relative numbers of viable CEM-SS cells infected with HIV-1 in a BCECF assay.
Figure 6B is a.graph of control versus cyanovirin- N.concentration which depicts the relative DNA contents of CEM-SS cell cultures infected with HIV-1.
Figure 6C is a graph of control versus cyanovirin- N concentration which depicts the relative numbers of viable CEM-SS cells infected with HIV-1 in an XTT assay.
Figure 6D is a graph of control versus cyanovirin- N concentration which depicts the effect of a range of concentrations of cyanovirin-N upon indices of infectious virus or viral replication.
Figure 7 is a graph of uninfected control versus time-of-addition which shows the results of delayed-addition studies of cyanovirin-N, showing anti- HIV activity in CEM-SS cells infected with HIV-.1 Figure 8A is a graph of OD (450 nm) versus 9 cyanovirin-N concentration (tg/ml), which illustrates cyanovirin/gpl20 interactions defining gpl20 as a S* principal molecular target of 15 cyanovirins.
Figure 8B is a dot-blot of the binding of cyanovirin-N and a gpl20-HRP conjugate, which shows that cyanovirin-N specifically bound a horseradish peroxidase conjugate of gpl20 (gpl20-HRP) in a concentration- S 20 dependent manner.
Figure 9 schematically illustrates a DNA coding sequence comprising a FLAG-cyanovirin-N coding sequence coupled to a Pseudomonas exotoxin coding sequence.
Figure 10 is a graph of OD (450 nM) versus PPE concentration which illustrates selective killing of viral gpl20-expressing (H9/IIIB) cells by a FLAGcyanovirin-N/Pseudomonas exotoxin protein conjugate
(PPE).
Figure 11 is a western-blot from an SDSpolyacrylamide gel elctrophoretogram of lysed COS-7 cells which had been engineered and transformed to express a FLAG-cyanovirin-N; detection was by an anti-FLAG antibody.
Figure 12 is a western-blot from an SDSpolyacrylamide gel electrophoretogram of secreted products, digested with peptide-N4-(N-acetyl-pglucosaminyl) asparagine amidase, from Pichia pastoris engineered and transformed to produce a cyanovirin; detection was by an anti-cyanovirin-N polyclonal antibody.
10 Figure 13 is an SDS-polyacrylamide gel electrophoretogram and a western-blot from a whole-cell lysate from E. coli engineered to produce cyanovirin-N; detection was by an anti-cyanovirin-N polyclonal antibody.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated, at least in part, on the observation that certain extracts from
S
cultured cyanobacteria (blue-green algae) exhibited S 20 antivira activity in an anti-HIV screen. The anti-HIV screen was conceived in 1986 (by M.R. Boyd of the National Institutes of Health) and has been developed and operated at the U.S. National Cancer Institute (NCI) since 1988 (see Boyd, in AIDS, Etiolo~y. Diagnosis, Treatment and Prevention, DeVita et al., eds., Philadelphia: Lippincott, 1988, pp. 305-317).
Cyanobacteria (blue-green algae) were specifically chosen for anti-HIV screening because they had been known to produce a wide variety of structurally unique and biologically active non-nitrogenous and amino acidderived natural products (Faulkner, Nat. Prod. Rec. 11, 355-394, 1994; Glombitza et al., in Alcal and Cvanobacterial Biotechnoloyv, Cresswell, et al.
eds., 1989, pp. 211-218). These photosynthetic procaryotic organisms are significant producers of cyclic and linear peptides (molecular weight generally <3 kDa), which often exhibit hepatotoxic or antimicrobial properties (Okino et al., Tetrahedron Lett. 34, 501-504, 1993; Krishnamurthy PNAS USA 86, 770-774, 1989; Sivonen Chem. Res. Toxicol. 5, 464-469, 1992; 10 Carter J. Orq. 49, 236-241, 1984; Frankmolle Antibiot. 45, 1451-1457, 1992). Sequencing studies of higher molecular weight cyanobacterial proteins have generally focused on those associated with primary metabolic processes ones that 15 can serve as phylogenetic markers (Suter FEBS 217, 279-282, 1987; Rumbeli 221, 1-2, Swanson Biol. 267, 16146- 16154, Michalowski Nucleic Acids 18, 2186, 1990; Sherman in The Cvanobacteria, Fay eds. Elsevier: New York, 1987, pp. 1-33; Rogers, o. The eds., 35-67). In general, antiviral not been sources. The extract leading to the present invention was among many thousands different extracts initially selected randomly and tested blindly anti-HIV screen described above. A number these had determined preliminarily show anti- HIV activity NCI (Patterson J. Phvcol. 29, 125-130, 1993). From this group, an aqueous 1 Ts 12 from Nostoc ellipsosporum, prepared (Patterson, 1993, suDra) showed unusually high potency vitro "therapeutic index" screen, for detailed investigation. specific bioassay-guided strategy used isolate purify a homogenous protein highly active against HIV. In strategy, initial selection fractionation, well decisions concerning overall chemical isolation method be applied, nature individual stips therein, were by interpretation biological testing data. screening assay (see, Boyd, 1988, supra; Weislow Natl. Cancer Inst. 81, 577-586, 1989), guide purification process, measures degree protection human T-lymphoblastoid cells cytopathic effects HIV. Fractions interest are using variety means anda re screen. Active fractions separated further, resulting subfractions likewise screen. This process is repeated times necessary order obtain compound(s), fraction(s) representing pure then subjected analysis structural elucidation. Using ellipsosporum discovered contain protein. It should noted term "protein" herein describe 13 restricted amino acid sequence any particular length includes molecules comprising 100 more acids, less than acids (which sometimes referred "peptides"). The accordingly provides isolated purified specifically known cyanovirin-N. also other cyanovirins. S" "cyanovirin" generically refer *o native ("native cyanovirin") functionally equivalent derivative thereof. In context invention, such thereof (a) contains at least nine (preferably twenty, preferably thirty, most fifty) directly 20 homologous same as) subsequence contiguous contained within cyanovirin (especially cyanovirin-N), antiviral, capable binding virus, primate immunodeficiency HIV-1, HIV-2, SIV, infected host cell expressing one viral antigen(s), envelope glycoprotein, gpl20, respective virus. addition, comprise cvanovirin, particularly cyanovirin-N (see SEQ ID NO:2), 1-20, 1-10, 1, 2, 3, 4, removed both ends, only end, amino-terminal cyanovirin. The inventive comprises substantially cyanovirin, cyanovirins "shbstantially homologous" sufficient homology render characteristic ellipsosporum. There exists about 50% homology, 75% :about 90% homology. Thus, encoded nucleic molecule coding NO:1, NO:3, encoding NO:2, NO:4. The further conjugate, coupled effector molecule(s), toxin immunological reagent. "immunological reagent" antibody, immunoglobulin, recognition element. An elemen: element, peptide, FLAG recombinant cyanovirin-FLAG fusion protein, facilitates, through recognition,>100 >100 >100 >100 >200
N.D.
HIV-2
ROD
MS
CE?-SS
CEM-SS
SIV
Delta,,,, 174xCEM ExamDle 8 This example further illustrates the ConStruCtion of a conjugate DNA coding sequence, and expression thereof, to provide a cyanovirin-toxin protein conjugate that selectively targets and kills HIV-infected cell More snecifically, this example illustrates construction and i ILill–.-;lri-~Li ili~~ iiii.~~~_l-21-~l expression of a conjugate DNA coding sequence for a cyanovirin/Pseudomonas-exotoxin which selectively kills viral gpl20-expressing host cells.
A DNA sequence (SEQ ID NO:3) coding for FLAGcyanovirin-N and a DNA sequence coding for the PE38 fragment of Pseudomonas exotoxin (Kreitman et al., Blood 83, 426-434, 1994) were combined in the pFLAG-1 expression vector. The PE38 coding sequence was excised from a plasmid, adapted, and ligated to the C-terminal 10 position of the FLAG-cyanovirin-N coding sequence using standard recombinant DNA procedures. This construct is illustrated schematically in Figure 9. Transformation of E. coli with this construct, selection of clones, and induction of gene expression with IPTG resulted in production of a conjugate protein with the expected molecular weight and immunoreactivity on western-blot analysis using an anti-FLAG antibody. The chimeric molecule was purified by FLAG-affinity chromatography as in Example 4) and evaluated for toxicity to human lymphoblastoid cells infected with HIV (H9/IIIB cells) as well as their uninfected counterparts (H9 and CEM-SS cells). Cells were plated in 96-well microtitre plates and exposed to various concentrations of the conjugate protein (named PPE). After three days, viability was assessed using the XTT assay (Gulakowski et al., 1991, supra). Figure 10 illustrates the results of this testing. As anticipated, the infected H9/IIIB cells expressing cell-surface gpl20 were dramatically more sensitive to the toxic effects of PPE than were the uninfected H9 or CEM-SS cells. The IC50 values determined from the concentration-effect curves were ^1-I I 0.014 nM for H9/IIIB compared to 0.48 and 0.42 nM for H9 and CEM-SS, respectively.
Example 9 This example illustrates transformation of a mammalian cell to express a cyanovirin therein. A genetic construct suitable for demonstration of expression of a cyanovirin in mammalian cells was prepared by ligating a DNA sequence coding for FLAG- 10 cyanovirin-N into the pFLAG CMV-1 expression vector (IBI- Kodak, Rochester, NY). The FLAG-cyanovirin-N coding sequence (SEQ ID NO:3) was excised from a previously constructed plasmid and ligated to the pFLAG CMV-1 vector using standard recombinant DNA procedures. African green monkey cells (COS-7 cells, obtained from the American Type Culture Collection, Rockville, MD) were transformed by exposure to the construct in DEAE dextran solution.
To assess expression of FLAG-cyanovirin-N, cells were lysed after 72 hours and subjected to PAGE and westernblot analysis. As illustrated in Figure 11, anti-FLAG immunoreactive material was readily detected in transformed COS-7 cells, albeit at an apparent molecular weight substantially greater than native recombinant FLAG-cyanovirin-N produced in E. coli. Diagnostic analyses of digests, performed in the same manner as in Example 10 which follows, indicated that this increased molecular weight was due to post-translational modification (N-linked oligosaccharides) of the FLAGcyanovirin-N.
Example This example illustrates transformation and expression of a cyanovirin in a non-mammalian cell, more specifically a yeast cell.
A genetic construct suitable for demonstration of expression of a cyanovirin in Pichia pastoris was prepared by ligating a DNA sequence coding for cyanovirin-N into the pPIC9 expression vector (Invitrogen Corporation, San Diego, CA). The cyanovirin-N coding 10 sequence (SEQ ID NO:1) was excised from a previously constructed plasmid and ligated to the vector using standard recombinant DNA procedures. Yeast cells were transformed by electroporation and clones were selected for characterization. Several clones were found to express, and to secrete into the culture medium, material reactive with anti-cyanovirin-N polyclonal antibodies (see, Example 11).
Similar to the observations with the mammalian forms described in Example 9, the elevated apparent molecular weight of ‘the yeast-derived product on PAGE and westernblot analysis, suggested that post-translational modification of the cyanovirin-N was occurring in this expression system.
To further define this modification, the secreted products from two clones were digested with peptide-N4- (N-acetyl-P-glucosaminyl) asparagine amidase. This enzyme, obtained from New England Biolabs (Beverly, MA), specifically cleaves oligosaccharide moieties attached to asparagine residues. As illustrated in Figure 12, this treatment reduced the apparent molecular weight of the product to that equivalent to native recombinant 74 cyanovirin-N expressed in E. coli. Inspection of the amino acid sequence of cyanovirin revealed a single recognition motif for N-linked modification (linkage to the asparagine located at position To further establish this as the site of glycosylation, a mutation was introduced at this position to change the asparagine residue to glutamine Expression of this mutant form resulted in production of immunoreactive material with a molecular weight 10 consistent with that of native recombinant FLAGcyanovirin-N.
Example 11 This example further illustrates an antibody specifically binding to a cyanovirin.
Three 2-month old New Zealand White rabbits (1.8-2.2 kg) were subjected to an immunization protocol as follows: A total of 100 4g of cyanovirin-N was dissolved in 100 il of a 1:1 suspension of phosphate-buffered saline (PBS) and Freunds incomplete adjuvant and administered by intramuscular injection at 2 sites on each hind leg; 8-16 months from the initial injection, a final boost of 50 gg of cyanovirin-N per rabbit was dissolved in 1000 1l of a 1:1 suspension of PBS and Freunds incomplete adjuvant and administered by intraperitoneal injection; on days 42, 70, 98 and 122, ml of blood was removed from an ear vein of each rabbit; 14 days after the last intraperitoneal boost, the rabbits were sacrificed and bled out. The IgG fraction of the resultant immune sera from the above rabbits was isolated by protein-A Sepharose affinity chromatography according to the method of Goudswaard et al. (Scand. J. Immunol. 8, 21-28, 1978). The reactivity of this polyclonal antibody preparation for cyanovirin-N was demonstrated by westernblot analysis using a 1:1000 to 1:5000 dilution of the rabbit IgG fractions.
Figure 13 further illustrates that the antibody prepared according to the aforementioned procedure is an antibody specifically binding to a protein of the present 10 invention. SDS-PAGE of a whole-cell lysate, from E. coli strain DH5a engineered to produce cyanovirin-N, was cairied out using 18% polyacrylamide resolving gels and standard discontinuous buffer systems according to Laemmeli (Nature 227, 680-685, 1970). Proteins were visualized by staining with Coomassie brilliant blue (Figure 13A). For western-blot analyses, proteins were electroeluted from the SDS-PAGE gel onto a nitrocellulose .membrane. Non-specific binding sites on the membrane were blocked by washing in a 1% solution of bovine serum albumin (BSA). The membrane was then incubated in a solution of the IgG fraction from the aforementioned rabbit anti-cyanovirin-N immune serum diluted 1:3000 with phosphate buffered saline (PBS). Subsequently, the membrane was incubated in a secondary antibody solution containing goat-antirabbit-peroxidase conjugate (Sigma) diluted 1:10000. The bound secondary antibody complex was visualized by incubating the membrane in a chemiluminescence substrate and then exposing it to x-ray film (Figure 13B).
One skilled in the art additionally will appreciate that, likewise by well-established, routine procedures 76 see Harlow and Lane, 1988, suDra), monoclonal antibodies may be prepared using as the antigen a protein of the present invention, and that such a resulting monoclonal antibody likewise can be shown to be an antibody specifically binding a protein of the present invention.
All of the references cited herein are hereby incorporated in their entireties by reference.
10 While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to ‘those of ordinary skill in the art that variations of the preferred proteins, conjugates, host cells, compositions, methods, and the like can be used and that it is intended that the invention may be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.
1K ‘7 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Boyd, Michael R.
Gustafson, Kirk R.
Shoemaker, Robert H.
McMahon, James B.
(ii) -TITLE OF INVENTION: ANTIVIRAL PROTEINS, DNA CODING SEQUENCES THEREFOR, AND USES TAEREOF (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Leydig, Voit Mayer, Ltd.
0o.. STREET: Two Prudential Plaza, Suite 4900 CITY: Chicago STATE: IL COUNTRY: U.S.A.
ZIP: 60601-6780 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS 5 SOFTWARE: Patent In Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 0. 0(B) FILING DATE: 25-APR-1996 C ‘C CLASSIFICATION: (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/429965 FILING DATE: 27-APR-1995
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: Kilyk, John Jr.
REGISTRATION NUMBER: 30763 REFERENCE/DOCKET NUMBER: 61109 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (312)616-5600 TELEFAX: (312)616-5700 INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 327 base pairs TYPE: nucleic acid 78 STRANDMEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) Cix) FEATURE: CA) NAME/KE-Y: CDS LOCATION: 10.-312 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i: CGATCGAAG CTT GGT AAA TTC TCC CAG ACC TGC TAC AAC TCC GCT ATC Leu Gly Lys Phe Ser Gin Thr Cys Tyr Asn Ser Ala Ile 4 a a.
a a a *4a a a a. a a.
*4 a *4aa 4a a a 4* a 4* a.
CAG GGT Gin Giy is AAC ACC Asn Thr 30 TCC GTT CTG ACC TCC ACC TGC GAA CGT ACC Ser Vai Leu Thr Ser Thr Cys Giu Arg Thr 20 AAC GGT GGT TAC Asn Gly Gly Tyr TCC TCC ATC Ser Ser Ile GAC CTG AAC TCC OTT ATC Asp Leu Asn Ser Val Ile 35 GAA AAC GTT GAC Glu Asn Val Asp 144 192 TCC CTG AAA TG Ser Leu Lys Trp CCG TCC AAC TTC Pro Ser Asn Phe GAA ACC TGC CGT Glu Thr Cys Arg AAC ACC Asn Thr CAG CTG GCT Gin Leu Ala CAG CAG TTC Gin Gin Phe 80 TCC TCC GAA CTG Ser Ser Giu Leu
OCT
Ala 70 GCT GAA TGC AAA Ala Glu Cys Lys ACC CGT OCT Thr Arg Ala ATC GCT AAC Ile Ala Asn 240 GTT TCC ACC AAA ATC AAC CTG GAC GAC Val. Ser Thr Lys Ile Asn Leu Asp Asp 288 ATC GAC Ile Asp GGT ACC CTG Gly Thr Leu AAA TAC GAA TAACTCGAGA TCGTA Lys Tyr Olu 100 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:Z: Leu Oly Lys Phe Ser Gin Thr Cys Tyr Asn Ser Ala Ile Gin Gly Ser 1. 5 10 is Val Leu Thr Ser Thr Cys Giu Arg Thr Asn Gly Gly Tyr Asri Thr Ser Ser Ile Asp Leu Trp Gin Pro Ser Asn Ser Val le Glu Asn Val. Asm e e y Ser Leu Lys Asn ?he Ile Giu Thr Cys Arg 55 Asn Thr Gin Leu Ala Arg Ala Gin Gin Phe Gly Ser Ser Giu Leu Ala Giu Cys Lys Thr Val Ser Thr Lys Ile Asn Leu Asp Asp Ile Ala Asn Ile Asp Gly lo 0 ,00:0 0..
loseS 0000 6:009 Thr Leu Lys Tyr Giu 100 INFORMATION FOR SEQ ID I-O: 3: SEQUENCE CHARACTERISTICS: LENGTH: 327 base pairs TYPE: nucleic acid STRANDEDNESS: doubie TOPOLOGY: iinear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE2: NAME/KEY: CDS LOCATION: 1. .327 (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:3: GAC TAC AAG GAC GAC GAT GAC AAG CTT GGT AAA TTC TCC CAG Asp Tyr Lys -Asp Asp Asp Asp Lys Leu Gly Lys Phe Ser Gin ACC TGC Thr Cys is TAC AAC TCC OCT Tyr Asn Ser Ala ATC CAG GOT TCC Ile Gin Gly Ser CTG ACC TCC ACC Leu. Thr Ser Thr TGC GAA CGT Cys Giu Arg ACC AAC GOT GGT TAC AAC ACC TCC TCC ATC GAC CTG AAC TCC GTT ATC 144 Thr Asn Gly Giy Tyr Asn Thr 5cr Ile Asp Leu Asn 5cr Val Ile GAA AAC Giu Asn so ACC TGC Thr Cys GTT GAC GOT TCC Val Asp Gly Ser
CTG
Leu AAA TOG CAG CCO Lys Trp Gin Pro AAC TTC ATC GAA Asn Phe Ile Glu 192 COT AAC ACC CAG CTG GCT GOT TCC TCC GAA CTG OCT GCT Arg Asn Thr Gin Leu Ala Gly 5cr 5cr Gilu Leu Ala Ala 70 240 TGC AAA ACC CGT OCT CAG CAG TTC GTT TCC ACC AAA ATC AAC CTG GAC 28 Cys Lys Thr Arg Ala Gin Gln Phe Val. Ser Thr Lys le Asn Leu Asp 90 GAC CAC ATC OCT AAC ATC GAC GGT ACC CTG AAA TAC GAA 327 Asp His le Ala Asn Ile Asp Gly Thr Leu Lys Tyr Glu 100 105 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 109 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein see* .00. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: *0*0 Asp Tyr Lys Asp Asp Asp Asp Lys Lea Gly Lys Phe Ser Gin Th~r Cys 1 5 10 .0.0 Tyr Asn Ser Ala Ile Gin Gly Ser Val Leu Thr Ser Thr Cys Gla Arg *20 25 Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile Asp Lea Asn Ser Val Ile “S 35 40 **too:Gia Asn Val Asp Gly Ser Leu Lys Trp Gin Pro Ser Asn Phe le Gia s0 55 Cys Arg Asn Thr Gin Lea Ala Gly Ser Ser Gia Lea Ala Ala Gia St 5G 70 75 s0 Cys Lys Thr A~rrg Ala Gin Gin Phe Val Ser Thr Lys Ile Asn’Leu Asp 90 Asp His Ile Ala Asn Ile Asp Gly Thr Lea Lys Tyr G Iu 100 105

Claims (8)

1. An isolated and purified antiviral peptide comprising at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.

2. An isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate comprising at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, wherein said antiviral protein conjugate or antiviral peptide conjugate is coupled to an effector molecule, a virus, a viral envelope glycoprotein, polyethylene glycol, albumin, dextran, a solid support matrix, or a combination of any of the foregoing.

3. The protein conjugate or peptide conjugate of claim 2, wherein said effector molecule is selected from the group consisting of a toxin and an immunological reagent. 9* 4. The protein conjugate or peptide conjugate of claim 3, wherein said effector molecule is Pseudomonas exotoxin. 15 5. The protein conjugate or peptide conjugate of any of claims 2 to 4, wherein said protein conjugate comprises SEQ ID NO:2.

6. An isolated and purified nucleic acid molecule which encodes the peptide, protein conjugate or peptide conjugate of any of claims 1 to

7. A pharmaceutical composition comprising an antiviral effective amount of the peptide, protein conjugate or peptide conjugate of any of claims 1 to 5 and a pharmaceutically acceptable carrier therefor and, optionally, further comprising an immunostimulant.

8. A vector which comprises the nucleic acid molecule of claim 6.

9. A host cell comprising the vector of claim 8.

82- The host cell of claim 9, wherein said host cell is a mammalian cell. 11. The host cell of claim 9, wherein said host cell is a bacterium. 12. The host cell of claim 11, wherein said bacterium is a lactobacillium. 13. The host cell of claim 9, wherein said host cell is a yeast. 14. An antibody binding the peptide, the protein in the protein conjugate or the peptide in the peptide conjugate of any of claims 1 to The use of the peptide, the protein conjugate or the peptide conjugate of any of claims 1 to 5 for the manufacture of a medicament for preventing or treating a viral infection of an animal. •10 16. The use of a nucleic acid molecule of claim 6 for the manufacture of a medicament for transforming in vivo host cells to express an antiviral peptide or antiviral peptide oconjugate encoded by said nucleic acid molecule in vivo. 17. A method of producing a peptide, a protein conjugate or a peptide conjugate, which method comprises expressing a peptide, a protein conjugate or a peptide conjugate in the host cell of any of claims 9 to 13. •18. A method of preventing the spread of viral infection comprising treating an inanimate object, solution, suspension, emulsion or other material with an antiviral effective amount of the peptide, the protein conjugate or the peptide conjugate of any of claims 1 to 19. A method of preventing the spread of viral infection comprising treating ex vivo blood, a blood product, sperm, cells, a tissue or an organ with an antiviral effective amount of the peptide, the protein conjugate or the peptide conjugate of any of claims 1 to -83- A method of preventing or treating a viral infection of an animal, which method comprises administering to an animal an antiviral effective amount of the antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate of any of claims 1 to 21. A method of preventing or treating a viral infection of an animal, which method comprises transforming in vivo host cells with the nucleic acid molecule of claim 6 to express an antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate encoded by said nucleic acid molecule in vivo. •22. A method of preventing or treating a viral infection of an animal, which method comprises transforming host cells with the nucleic acid molecule of claim 6 and placing l0 said transformed host cells into or onto said animal so as to express an antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate encoded by said nucleic acid molecule. 23. A method of contacting a virus with an antiviral agent, which method comprises contacting said virus with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2. 24. A method of contacting a virus with an antiviral agent, which method comprises contacting said virus with an antiviral effective amount of the antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate of any of claims 1 to A method of conjugating a virus or a viral envelope glycoprotein with a cyanovirin, which method comprises contacting said isolated and purified virus or said _I~ -84- isolated and purified viral envelope glycoprotein with an isolated and purified antiviral protein or an isolated and purified antiviral peptide, wherein said protein or peptide comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, and wherein, upon contacting said isolated and purified virus or said isolated and purified viral envelope glycoprotein with said protein or said peptide, said protein or said peptide binds to said isolated and purified virus or said isolated and purified viral envelope glycoprotein, thereby forming a conjugate. S•26. A method of administering an antiviral agent to an animal, which method comprises administering to said animal an antiviral effective amount of the peptide, the 10 protein conjugate or the peptide conjugate of any of claims I to 5 or a conjugate prepared in accordance with claim 27. A method of removing virus from a sample, which method comprises: contacting said sample with a composition comprising an isolated and ••purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide S conjugate, wherein said antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO:2, (ii) said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate is attached to a solid support matrix, and (iii) said at least nine contiguous amino acids bind to said virus, and separating said sample and said composition, whereupon virus is removed from said sample. 28. A method of removing virus from a sample, which method comprises: contacting said sample with a composition comprising the antiviral peptide, the antiviral protein conjugate or the antiviral peptide conjugate of any of claims 1 to 5, and separating said sample and said composition, whereupon virus is removed from said sample. 29. The method of claim 27 or claim 28, wherein said sample is blood, a component of blood, sperm, cells, a tissue or an organ. 30. The method of any of claims 27 to 29, wherein said sample is a vaccine formulation and the virus that is removed is infectious. o 10 31. A method of preventing or treating a viral infection of an animal, which method comprises topically administering to a host an antiviral effective amount of a formulation comprising an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino S” acids of the amino acid sequence of SEQ ID NO:2. 32. The method of claim 31, wherein said formulation is topically administered to the vagina, the penis, the rectum or the mouth of the animal. 33. The method of any of claims 23, 25, 27 or 31, wherein said antiviral protein or antiviral protein conjugate comprises SEQ ID NO:2. 34. A topical formulation comprising an isolated and purified antiviral agent selected from the group consisting of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said protein, peptide, protein -86- conjugate or peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO: 2. The topical formulation of claim 34, wherein said antiviral protein or antiviral protein conjugate comprises the amino acid sequence of SEQ ID NO:2. 36. The topical formulation of claim 34 or claim 35, wherein said formulation is an emulsion, a suspension, a solution, a gel, a cream, a paste, a foam, a lubricant, a spray, a suppository, a pessary, or a tampon. 37. The topical formulation of any of claims 34 to 36, wherein said formulation is in or on a contraceptive device. 10 38. The topical formulation of claim 37, wherein said contraceptive device is a condom. 39. The topical formulation of any of claims 34 to 38, wherein said formulation further comprises an agent that inhibits conception. The topical formulation of claim 39, wherein said agent that inhibits conception is nonoxynol-9. 41. The topical formulation of any of claims 34 to 40, wherein said formulation further 9* comprises another antiviral agent. 42. The topical formulation of any of claims 34 to 41, wherein said formulation further comprises an antibiotic agent. 43. The topical formulation of claim 42, wherein said antibiotic agent is an antifungal agent or an antibacterial agent. 44. A method of preventing or treating a viral infection of an animal, which method comprises administering to the animal an antiviral effective amount of a virus that has been destroyed or rendered noninfectious by contact with an antiviral effective amount -87- of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO: 2. The method of claim 44, wherein said antiviral protein or antiviral protein conjugate comprises the amino acid sequence of SEQ ID NO:2. o 46. The use of host cells transformed with the nucleic acid molecule of claim 6 for the manufacture of a medicament for preventing or treating a viral infection of a mammal :0 wherein said medicament is placed into or onto said animal so as to express an antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate encoded by said nucleic acid molecule. a. 47. The use of a formulation comprising an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the manufacture of a medicament for preventing or treating a viral infection of an animal wherein the medicament is administered topically. 48. The use of a virus that has been destroyed or rendered non-infectious by contact with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises -88- at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the manufacture of a medicament for preventing or treating a viral infection of an animal. 49. An isolated and purified antiviral peptide, substantially as herein described with reference to any one of the examples but excluding comparative examples. An isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. 51. An isolated and purified nucleic acid molecule which encodes a peptide, protein 10 conjugate or peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. 52. A pharmaceutical composition comprising an antiviral effective amount of a peptide, protein conjugate or peptide conjugate, substantially as herein described with °°oo reference to any one of the examples but excluding comparative examples. 53. A vector which comprises an isolated and purified nucleic acid molecule which encodes a peptide, protein conjugate or peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. 54. A host cell comprising a vector which comprises an isolated and purified nucleic acid molecule which encodes a peptide, protein conjugate or peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. -89- An antibody binding a peptide, a protein or a protein conjugate or a peptide in a peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. 56. The use of a peptide, a protein conjugate or a peptide conjugate, for the manufacture of a medicament for preventing or treating a viral infection of an animal, substantially as herein described with reference to any one of the examples but excluding comparative examples. 57. The use of an isolated and purified nucleic acid molecule which encodes a peptide, protein conjugate, or peptide conjugate for the manufacture of a medicament for transforming in vivo host cells to express an antiviral peptide or antiviral peptide conjugate encoded by said nucleic acid molecule in vivo, substantially as herein described with reference to any one of the examples but excluding comparative examples. 58. A method of producing a peptide, a protein conjugate or peptide conjugate, substantially as herein described with reference to any one of the examples but excluding comparative examples. S. ;59. A method of preventing the spread of viral infection, substantially as herein described with reference to any one of the examples but excluding comparative examples. A method of preventing or treating a viral infection of an animal, substantially as herein described with reference to any one of the examples but excluding comparative examples. 61. A method of contacting a virus with an antiviral agent, substantially as herein described with reference to any one of the examples but excluding comparative examples. 62. A method of conjugating a virus or viral envelope glycoprotein with a cyanovirin, substantially as herein described with reference to any one of the examples but excluding comparative examples. 63. A method of administering an antiviral agent to an animal, substantially as herein described with reference to any one of the examples but excluding comparative examples. 64. A method of removing virus from a sample, substantially as herein described with reference to any one of the examples but excluding comparative examples. A topical formulation, substantially as herein described with reference to any one 10 of the examples but excluding comparative examples. 66. Use of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the manufacture of a medicament, i substantially as herein described with reference to any one of the examples but excluding comparative examples. 67. Use of a virus that has been destroyed or rendered non-infectious by contact with an antiviral effective amount of an isolated and purified antiviral protein, an isolated and purified antiviral peptide, an isolated and purified antiviral protein conjugate or an isolated and purified antiviral peptide conjugate, wherein said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprises at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, for the -91 manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples. DATED this 22nd day of October 1999 THE UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Attorney: IVAN A. RAJKOVIC Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS go o* Gs** 0* e go

AU56039/99A
1995-04-27
1999-10-22
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Ceased

AU746809B2
(en)

Priority Applications (1)

Application Number
Priority Date
Filing Date
Title

AU56039/99A

AU746809B2
(en)

1995-04-27
1999-10-22
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Applications Claiming Priority (2)

Application Number
Priority Date
Filing Date
Title

US08/429965

1995-04-27

AU56039/99A

AU746809B2
(en)

1995-04-27
1999-10-22
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Related Parent Applications (1)

Application Number
Title
Priority Date
Filing Date

AU56691/96A
Division

AU707781B2
(en)

1995-04-27
1996-04-26
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Publications (2)

Publication Number
Publication Date

AU5603999A
true

AU5603999A
(en)

2000-01-06

AU746809B2

AU746809B2
(en)

2002-05-02

Family
ID=3741661
Family Applications (1)

Application Number
Title
Priority Date
Filing Date

AU56039/99A
Ceased

AU746809B2
(en)

1995-04-27
1999-10-22
Antiviral proteins, DNA coding sequences therefor, and uses thereof

Country Status (1)

Country
Link

AU
(1)

AU746809B2
(en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

US5843882A
(en)

1995-04-27
1998-12-01
The United States Of America As Represented By The Department Of Health And Human Services
Antiviral proteins and peptides

1999

1999-10-22
AU
AU56039/99A
patent/AU746809B2/en
not_active
Ceased

Also Published As

Publication number
Publication date

AU746809B2
(en)

2002-05-02

Contact information