AU2003252207B2 - Cyanovirin conjugates, matrix -anchored cyanovirin and anti-cyanovirin antibody, and related compositions and methods of use - Google Patents

Description

CYANOVIIUN CONJUGATES, MATRIX-ANCHORED CYANOVIRIN AND ANTI-CYANOVIRIN ANTIBODY, AND RELATED COMPOSITIONS AND METHODS OF USE TECHNICAL FIELD OF THE INVENTION The present invention relates to cyanovirins and conjugates thereof, matrixanchored cyanovirins and conjugates thereof, matrix-anchored anti-cyanovirin antibodies, compositions comprising same, methods of using same to remove virus from a sample and to inhibit prophylactically or therapeutically a viral infection of a host, as well as methods of inducing an immune response to a virus in an animal and compositions for use in such methods.

BACKGROUND OF THE INVENTION Acquired immune deficiency syndrome (AIDS) is a fatal disease, reported cases of which have increased dramatically within the past several years. The AIDS virus 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 is also known as lymphadenopathyassociated virus (LAV), AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct families of HIV have been described to date, namely HIV-I and HIV-2. The acronym HIV is used herein to refer to human immunodeficiency viruses generically.

HIV exerts profound cytopathic effects on the CD4+ helper/inducer T-cell s, 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 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, 2 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; and 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. Acquir. Immun. Def. Synd. 4,207-218, 1991; and Mitsuya et al., Science 249, 1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit the viral reverse transcriptase, are the only clinically active agents that are currently available commercially for anti-HIV therapy. Although very useful in some patients, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy. Also, given the recent revelations about the true dynamics of HV infection (Coffin, Science 267, 483-489, 1995; and Cohen, Science 267, 179, 1995), it is now increasingly apparent that agents acting as early as possible in the viral replicative cycle are needed to inhibit infection of newly produced, uninfected immune cells generated in the body in response to the virus-induced 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 "gpl 120") with the cell-surface receptor CD4, followed by fusion and entry (Sattentau, AIDS 2, 101-105, 1988; and Koenig et al., EmPNAS USA 86, 2443-2447, 1989). Productively infected, virus-producing cells express gp 120 at the cell surface; interaction of gp 120 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 gpl 20/CD4 interaction is a particularly attractive target for interruption of HIV infection and cytopathogenesis, 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 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; and Lipton, Nature 367, 113-114, 1994). Much vaccine research has focused upon however, progress has been hampered by hypervariability of the neutralizing determinants, Ad consequent extreme strain-dependence of viral sensitivity to gpl20-directed antibodies (Berzofsky, J. Acq. Immun. Def. Synd. 4, 451-459, 1991). Relatively little drug 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, supra; Schooley et al., Ann. Int. Med. 112, 247-253, 1990; and 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 (Orloffet al., AIDS Res. Hum. Retrovir. 11, 335-342, 1995; and Moore et al., J.

Virol. 66, 235-243, 1992). Initial clinical trials of sCD4 (Schooley et al., 1990, supra; and Husson et al., 1992, supra), and ofsCD4-coupled immnunoglobulins (Langner et al., Arch. Virol. 130, 157-170, 1993), and likewise of sCD4-coupled toxins designed to bind and destroy virus-expressing cells (Davey et al., J. Infect.

Dis. 170, 1180-1188, 1994; and Ramachandran et al., J. Infect. Dis. 170, 1009- 1113, 1994), have been disappointing. Newer gene-therapy approaches to generating sCD4 directly in vivo (Morgan et al., AIDS Res. Hum. Retrovir. 1507-15 15, 1994) will likely suffer similar frustrations.

In this regard, it would be advantageous to develop methods and compositions to protect cells against initial infection of a virus. To date, no practical protocols have been developed to produce immunological protection against lIHV. The maj or hurdles associated with the induction of protective immunity against the virus include the inability to separate iunfectious and noninfectious viral particles for use in a composition that can induce an immune response, such as a vaccine composition. The presence of such non-infectious viral particles in serum samples has been observed and are presumably the result of error-prone replication processes for many viruses. The use of geneticallyengineered replication- incompetent virus as a vaccine composition has been proposed to obviate the need to separate infectious and non-infectious viral particles. However, it is unclear how manipulation of the viral genome affects the presentation of antigens on the viral surface. It is entirely possible that the repertoire of viral antigens on a genetically modified replication-incompetent virus would not be characteristic of a naturally occurring virus. It also has been proposed to use incomplete particles to induce an immnune response against intact virus. Yet, an immune response to mere pieces of virus cannot be as comprehensive or complete as an immune response to an intact, naturally occurring virus.

Therefore, new classes of antiviral agents, to be used alone or in combination with AZT and/or other available antiviral agents, are needed for effective antiviral therapy against AIDS. New agents, which may be used to prevent HIV infection, are also important for prophylaxis. In both areas of need, the ideal new agent(s) would act as early as possible in the viral life cycle; be as virus-specific as possible attack a molecular target specific to the virus but not the host); render the intact virus noninfectious; prevent the death or dysfunction of virus-infected cells; prevent further production of virus from infected cells; prevent spread of virus infection to uninfected 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 basis.

According to a first broad form of the invention there is provided a method of inhibiting prophylactically or therapeutically a viral infection of a host, which method comprises administering to said host an antiviral effective amount of a conjugate comprising an isolated and purified antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2, wherein said at least nine contiguous amino acids bind to a virus, chemically coupled or fused to an anti-cyanovirin antibody, whereupon administration of said antiviral effective amount of said conjugate, said viral inhibition is inhibited.

According to a further broad form of the invention there is provided a method of inhibiting prophylactically or therapeutically a viral infection of a host, which method comprises topically administering to a host an antiviral effective amount of a composition comprising an isolated and purified antiviral agent selected from the group consisting of an antiviral protein, an antiviral peptide, an antiviral protein conjugate, and an antiviral peptide conjugate, wherein said antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2, (ii) said at least nine contiguous amino acids of SEQ ID NO: 2 have antiviral activity, and (iii) said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate is attached to a solid support matrix, whereupon administration of said antiviral effective amount of said composition, said viral infection is inhibited.

According to yet a further broad form of the invention there is provided a matrixanchored anti-cyanoviran antibody, wherein said antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 and wherein said antibody is attached to a magnetic bead or a flow-through matrix.

According to another broad form of the invention there is provided a matrixanchored anti-cyanoviran antibody, wherein said antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 and wherein said antibody is attached to a solid support matrix via streptavidin/biotin coupling or coupling through polyethylene glycol, albumin or dextran.

According to a further broad form of the invention there is provided a composition comprising the matrix-anchored anti-cyanovirin antibody described above.

It will be appreciated that minor modifications may be made to the invention as described and claimed herein without departing from the spirit and scope thereof.

6 BRIEF SUMMARY OF THE INVENTION The present invention provides, among other things, a method of removing virus from a sample. The method comprises contacting a sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, any one of which i s attached to a solid support matrix. The antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2 tharbind to the virus. The method further comprises separating the sample and the composition, whereupon the virus is removed from the sample.

The present invention further provides a composition comprising a solid support matrix to which is attached an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, in which the antiviral protein or antiviral peptide conriprises at least nine contiguous amino acids of SEQ ID NO: 2 that bind to a virus. In this regard, the present invention also provides a method of administering such a composition to a host to inhibit prophylactically or therapeutically a viral infection.

A conjugate comprising an isolated and purified antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 that bind to a virus, coupled to an anti-cyanovirin antibody or at least one effector component, which can be the same or different, selected from the group consisting of polyethylene glycol, dextran and albumin, and a composition comprising such a conjugate are also provided. Also provided is a method of administering such a conjugate to a host to inhibit prophylactically or therapeutically a viral infection.

A matrix-anchored anti-cyanovirin antibody is also provided. The antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2. The antibody is attached to a solid support matrix. In this regard, the present invention also provides a method of removing virus from a sample. -The method comprises contacting the sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate in which the antiviral peptide or antiviral protein comprises at least nine contiguous amino acids of SEQ ID NO; 2 that bind to a virus. The method further comprises contacting the sample with the matrix-anchored anti-cyanovirin antibody and then separating the matrix-anchored anti-cyanovirin antibody and sample, whereupon the virus is removed from the sample.

Still further provided by the present invention is a composition comprising naturally occuring, non-infectious virus. The composition desirably is obtained in accordance with the methods provided. The composition can comprise naturally -occurring, non-infectious HIV comprising gpl20. In this regard, the present invention further provides a method of inducing an immune response to a virus in an animal by administering an immune-response inducing amount of such a composition.

BRIEF DESCRIPTION OF THE DRAWINGS Figure IA is a graph of OD (206 nm) versus time (min), which represents an HPLC chromatogram of nonreduced cyanovirin.

Figure IB is a bar graph of maximum dilution for 50% protection versus HPLC fraction, which illustrates the maximum dilution of each HPLC fraction that provided 50% protection from the cytopathic effects of HIV infection for the nonreduced cyanovirin HPLC fractions.

Figure 1C is a graph ofOD (206) nm versus time (min), which represents an HPLC chromatogram of reduced cyanovirin.

Figure ID is a bar graph of maximum dilution for 50% protection versus HPLC dilution, which illustrates the maximum dilution of each fraction that provided 50% protection from the eytopathic effects of HIV infection for the reduced cyanovirin HPLC fractions.

Figure 2 shows an example of a DNA sequence encoding a synthetic cyanovirin gene (SEQ ID NOS: 1-4).

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.

Figure 4 shows a typical HPLC chromatogram during the purification of a recombinant native cyanovirin.

Figure 5A is a graph of control versus concentration which illustrates the antiviral activity of native cyanovirin from Nostoc ellipsosporum.

Figure 5B is a graph of% control versus concentration which illustrates the antiviral activity of recombinant cyanovirin.

Figure 5C is a graph of control versus concentration which illustrates the antiviral activity of recombinant FLAG-fusion cyanovirin.

Figure 6A is a graph of% control versus concentration which depicts the relative numbers of viable CEM-SS cells infected with HIV-i in a BCECF assay.

Figure 6B is a graph of control versus concentration which depicts the relative DNA contents of CEM-SS cell cultures infected with

HIV-.

Figure 6C is a graph of control versus 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 concentration which depicts the effect of a range of concentration of cyanovirin upon indices of infectious virus or viral replication.

Figure 7 is a graph of% uninfected control versus time of addition (hrs), which shows results of time-of-addition studies of a cyanovirin, showing anti-HIV activity in CEM-SS cells infected with HIV-1RF.

Figure 8 is a graph of OD (450 nm) versus cyanovirin concentration which illustrates cyanovirin/gpl20 interactions defining gpl20 as a principal molecular target of cyanovirin.

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 (nM), which illustrates selective killing of viral gpl20-expressing (H9/IIIB) cells by a FLAG-cyanovirin-N/Pseudomonas exotoxin protein conjugate (PPE).

Figure 11 is a flowchart of the synthesis of a cyanovirin gene.

Figure 12 is a flowchart of the expression of synthetic cyanovirin.

DETAILED DESCRIPTION OF THE INVENTION The principal overall objective of the present invention is to provide antiviral proteins, peptides and derivatives thereof, and broad medical uses thereof, including prophylactic and/or therapeutic applications against viruses, such as retroviruses, in particular a human immunodeficiency virus, specifically HIV-1 or HIV-2.

An initial observation, which led to the present invention, was antiviral activity in certain extracts from cultured cyanobacteria (blue-green algae) tested in an anti-MV screen. The screen is one that 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, Etiology.

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 acid-derived natural products (Faulkner, Nat. Prod. Rep. 11, 355-394, 1994; arid Glombitza et al., in Algal and Cyanobacterial Biotechnology, 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 et al., PNAS USA 86, 770-774, 1989; Sivonen et al., Chem. Res. Toxicol. 5, 464-469, 1992; Carter et al., J. Org. Chem. 49, 236- 241, 1984; and Frankmolle ct al., J. Antibiot, 45, 1451-1457, 1992). Sequencing studies of higher molecular weight cyanobacterial peptides and proteins have generally focused on those associated with primary metabolic processes or ones that can serve as phylogenetic markers (Suter et al., FEBS Lett. 217, 279-282, 1987; Rumbeli et al., FEBS Lett. 221, 1-2, 1987; Swanson et al., J. Biol. Chem.

267, 16146-16154, 1992; Michalowski et al., Nucleic Acids Res. 18, 2186, 1990; Sherman et al., in The Canobactria, Fay et al-, eds., Elsevier: New York, 1987, pp. 1-33; and Rogers, in The Cvanobacteria, Fay et al., eds., Elsevier: New York, 1987, pp. 35-67). In general, proteins with antiviral properties have not been associated with cyanobacterial sources.

The cyanobacterial extract leading to the present invention was among many thousands of different extracts initially selected randomly and tested blindly in the anti-HIV screen described above. A number of these extracts had been determined preliminarily to show anti-HIV activity in the NCI screen (Patterson et al., 1. Phycol. 29, 125-130, 1993). From this group, an aqueous extract from Nostoc ellipsosporum, which had been prepared as described (Patterson, 1993, supra) and which showed an unusually high anti-HIV potency and in vitro "therapeutic index" in the NCI primary screen, was selected for detailed investigation. A specific bioassay-guided strategy was used to isolate and purify a homogenous protein highly active against HIV.

In the bioassay-guided strategy, initial selection of the extract for fractionation, as well as the decisions concerning the overall chemical isolation method to be applied, and the nature of the individual steps therein, were determined by interpretation of biological testing data. The anti-HIV screening assay see Boyd, 1988, supr; Weislow et al., J. Natl. Cancer Inst. 81, 577- 586, 1989), which was used to guide the isolation and purification process, measures the degree of protection of human T-lymphoblastoid cells from the cytopathic effects of HIV. Fractions of the extract of interest are prepared using a variety of chemical means and are tested blindly in the primary screen. Active fractions are separated further, and the resulting subfractions are likewise tested blindly in the screen. This process is repeated as many times as necessary in order to obtain the active compound(s), antiviral fraction(s) representing pure compound(s), which then can be subjected to detailed chemical analysis and structural elucidation.

Using this strategy, aqueous extracts of Nostoc ellipsosporum were shown to contain an antiviral protein. Accordingly, the present invention provides an isolated and purified antiViral protein, named cyanovirin-N, from Nostoc ellipsosporum. Herein the term "cyanovirin" is used generically to refer to a native cyanovirin or any related, functionally equivalent protein, peptide or derivative thereof. By definition, in this context, a related, functionally equivalent protein, peptide or derivative thereof a) contains a sequence of at least nine amino acids directly homologous with any sub-sequence of nine contiguous amino acids contained within a native cyanovirin, and, b) is capable of specifically binding to virus, in particular a retrovirus, more specifically a primate immunodeficiency virus, more specifically HIV-1, HIV-2 or SIV, or to an infected host cell expressing one or more viral antigen(s), more specifically an envelope glycoprotein, such as gpl20, of the respective virus. Herein, the term "protein" refers to a sequence comprising 100 or more amino acids, whereas "peptide" refers to a sequence comprising less than 100 amino acids. In addition, such a functionally equivalent protein or derivative thereof can comprise the amino acid sequence of a native cyanovirin, particularly cyanovirin-N (see SEQ ID NO:2), in which 1-20, preferably 1-10, more preferably 1, 2, 3, 4, or 5, and most preferably 1 or 2, amino acids have been removed from one or both ends, preferably from only one end, and most preferably from the amino-terminal end, of the native cyanovirin.

Preferably, the protein, peptide or derivative thereof comprises an amino acid sequence that is substantially homologous to that of an antiviral protein from Nostoc ellipsosporum. By "substantially homologous" is meant sufficient homology to render the protein, peptide or derivative thereof antiviral, with antiviral activity characteristic of an antiviral protein isolated from Nostoc ellipsosporum. At least about 50% homology, preferably at least about homology, and most preferably at least about 90% homology should exist. A cyanovirin conjugate comprises a cyanovirin coupled to one or more selected effector molecule(s), such as a toxin or immunological reagent. "Immunological reagent" will be used to refer to an antibody, an immunoglobulin, and an immunological recognition element. An immunological recognition element is an element, such as a peptide, the FLAG sequence of the recombinant cyanovirin-FLAG fusion protein, which facilitates, through immunological recognition, isolation and/or purification and/or analysis of the protein or peptide to which it is attached. A cyanovirin fusion protein is a type of cyanovirin conjugate, wherein a cyanovirin is coupled to one or more other protein(s) having any desired properties or effector functions, such as cytotoxic or immunological properties, or other desired properties, such as to facilitate isolation, purification or analysis of the fusion protein.

Accordingly, the present invention provides an isolated and purified protein encoded by a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, a nucleic acid molecule comprising a sequence of SEQ ID NO:3, a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, or a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4. Preferably, the aforementioned nucleic acid molecules encode at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.

The present invention also provides a method of obtaining a cyanovirin from Nosioc ellipsosporum. Such a method comprises identifying an extract of Nostoc ellipsosporum containing antiviral activity, optionally removing high molecular weight biopolymers from the extract, antiviral bioassay-guided fractionating the extract to obtain a crude extract of cyanovirin, and purifying the crude extract by reverse-phase HPLC to obtain cyanovirin (see, also, Example More specifically, the method involves the use of ethanol to remove high 13 molecular weight biopolymers from the extract and the use of an anti-HIV bioassay to guide fractionation of the extract.

Cyanovirin-N, which was isolated and purified using the aforementioned method, was subjected to conventional procedures typically used to determine the amino acid sequence of a given pure protein. Thus, the cyanovirin was initially sequenced by N-terminal Edman degradation of intact protein and numerous overlapping peptide fragments generated by endoproteinase digestion. Amino acid analysis was in agreement with the deduced sequence. ESI mass spectrometry of reduced, HPLC-purified cyanovirin-N showed a molecular ion consistent with the calculated value. These studies indicated that cyanovirin-N from Nostoc ellipsosporum was comprised of a unique sequence of 101 amino acids having little or no significant homolbgy to previously described proteins or transcription products of known nucleotide sequences. No more than eight contiguous amino acids from cyanovirin were found in any amino acid sequences from known proteins, nor were there any known proteins from any source containing greater than 13% sequence homology with cyanovirin-N. Given the chemically deduced amino acid sequence of cyanovirin-N, a corresponding recombinant cyanovirin-N (r-cyanovirin-N) was created and used to definitively establish that the deduced amino acid sequence was, indeed, active against virus, such as HIV (Boyd et al., 1995, supra; also, see Examples Accordingly, the present invention provides isolated and purified nucleic acid molecules and synthetic nucleic acid molecules, which comprise a coding sequence for a cyanovirin, such as an isolated and purified nucleic acid molecule comprising a sequence of SEQ ID NO:1, an isolated and purified nucleic acid molecule comprising a sequence of SEQ ID NO:3, an isolated and purified nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, an isolated and purified nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4, and a nucleic acid molecule that is substantially homologous to any one or more of the aforementioned nucleic acid molecules. By "substantially homologous" is meant sufficient homology to render the protein, peptide or derivative thereof antiviral, with antiviral activity characteristic of an antiviral protein isolated from Nostoc ellipsosporum. At least about 50% homology, preferably at least about homology, and most preferably at least about 90% homology should exist.

More specifically, the present invention provides one of the aforementioned nucleic acid molecules, which comprises a nucleic acid sequence encoding at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2.

The present inventive nucleic acid molecule desirably comprises a nucleic acid sequence encoding at least nine (preferably at least twenty, more preferably at least thirty, and most preferably at least fifty) contiguous amino acids of the amino acid sequence of SEQ ID NO:2. The present inventive nucleic acid molecule also desirably comprises a nucleic acid sequence encoding a protein comprising the amino acid sequence of a native cyanovirin, particularly cyanovirin-N, in which 1preferably 1-10, more preferably 1, 2, 3, 4, or 5, and most preferably 1 or 2, amino acids have been removed from one or both ends, preferably from only one end, and most preferably from the amino-terminal end, of the native cyanovirin.

Given the present disclosure, it will be apparent to one skilled in the art that a partial cyanovirin-N gene codon sequence will likely suffice to code for a fully functional, antiviral, such as anti-HIV, cyanovirin. A minimum essential DNA coding sequence(s) for a functional cyanovirin can readily be determined by one skilled in the art, for example, by synthesis and evaluation of sub-sequences comprising the native cyanovirin, and by site-directed mutagenesis studies of the cyanovirin-N DNA coding sequence.

Using an appropriate DNA coding sequence, a recombinant cyanovirin can be made by genetic engineering techniques (for general background see, e.g., Nicholl, in An Introduction to Genetic Engineering, Cambridge University Press: Cambridge; 1994, pp. 1-5 127-130; Steinberg et al., in Recombinant DNA Technology Concepts and Biomedical Applications, Prentice Hall: Englewood Cliffs, NJ, 1993, pp. 81-124 150-162; Sofer in Introduction to Genetic Engineering, Butterworth-Heinemann, Stoneham, MA, 1991, pp. 1-21 103-126; Old et al., in Principles of Gene Manipulation, Blackwell Scientific Publishers:

I

London, 1992, pp. 1-13 108-221; and Emtage, in Delivery Systems for Peptide Drugs, Davis et al., eds., Plenum Press: New York, 1986, pp. 23-33). For example, a Nostoc ellipsosporum gene or cDNA encoding a cyanovirin can be identified and subcloned. The gene or cDNA can then be incorporated into an appropriate expression vector and delivered into an appropriate proteinsynthesizing organism E. coli, S, cerevisiae, P. pastoris, or other bacterial, yeast, insect or mammalian cells, where the gene, under the control of an endogenous or exogenous promoter, can be appropriately transcribed and translated. Such expression vectors (including, but not limited to, phage, cosmid, viral, and plasmid vectors) are known to those skilled in the art, as are reagents and techniques appropriate for gene transfer transfection, electroporation, transduction, micro-injection, transformation, etc.). Subsequently, the recombinantly produced protein can be isolated and purified using standard techniques known in the art chromatography, centrifugation, differential solubility, isoelectric focusing, etc.), and assayed for antiviral activity.

Alternatively, a native cyanovirin can be obtained from Nostoc ellipsosporum by non-recombinant methods see Example 1 and above), and sequenced by conventional techniques. The sequence can then be used to synthesize the corresponding DNA, which can be subcloned into an appropriate expression vector and delivered into a protein-producing cell for en mass recombinant production of the desired protein.

In this regard, the present invention also provides a vector comprising a DNA sequence, a Nostoc ellipsosporum gene sequence for cyanovirin, a cDNA encoding a cyanovirin, or a synthetic DNA sequence encoding cyanovirin, a host cell comprising the vector, and a method of using such a host cell to produce a cyanovirin.

The DNA, whether isolated and purified or synthetic, or cDNA encoding a cyanovirin can encode for either the entire cyanovirin or a portion thereof. Where the DNA or cDNA does not comprise the entire coding sequence of the native cyanovirin, the DNA or cDNA can be subcloned as part of a gene fusion. In a 16 transcriptional gene fusion, the DNA or cDNA will contain its own control sequence directing appropriate production of protein ribosome binding site, translation initiation codon, etc.), and the transcriptional control sequences promoter elements and/or enhancers) will be provided by the vector. In a translational gene fusion, transcriptional control sequences as well as at least some of the translational control sequences the translational initiation codon) will be provided by the vector. In the case of a translational gene fusion, a chimeric protein will be produced.

Genes also can be constructed for specific fusion proteins containing a functional cyanovirin component plus a fusion component conferring additional desired attribute(s) to the composite protein. For example, a fusion sequence for a toxin or immunological reagent, as defined above, can be added to facilitate purification and analysis of the functional protein such as the FLAGcyanovirin-N fusion protein detailed within Examples Genes can be specifically constructed to code for fusion proteins, which contain a cyanovirin coupled to an effector protein, such as a toxin or immunological reagent, for specific targeting to viral-infected, HIV and/or HIV-infected, cells. In these instances, the cyanovirin moiety serves not only as a neutralizing agent but also as a targeting agent to direct the effector activities of these molecules selectively against a given virus, such as HIV. Thus, for example, a therapeutic agent can be obtained by combining the HIV-targeting function of a functional cyanovirin with a toxin aimed at neutralizing infectious virus and/or by destroying cells producing infectious virus, such as HIV. Similarly, a therapeutic agent can be obtained, which combines the viral-targeting function of a cyanovirin with the multivalency and effector functions of various immunoglobulin subclasses. Example 6 further illustrates the viral-targeting, specifically targeting, properties of a cyanovirin.

Similar rationales underlie extensive developmental therapeutic efforts exploiting the HIV gpl20-targeting properties of sCD4. For example, sCD4-toxin conjugates have been prepared in which sCD4 is coupled to a Pseudomonas exotoxin component (Chaudhary et al., in The Human Retrovirus, Gallo et al., eds., Academic Press: San Diego, 1991, pp. 379-387; and Chaudhary et al., Nature 335, 369-372,1988), or to a diphtheria toxin component (Aullo et al., EMBO J.

11, 575-583, 1992) or to aricin A-chain component (Till et al., Science 242, 1166- 1167, 1988). Likewise, sCD4-immunoglobulin conjugates have been prepared in attempts to decrease the rate of in vivo clearance of functional sCD4 activity, to enhance placental transfer, and to effect a targeted recruitment of immunological mechanisms of pathogen elimination, such as phagocytic engulfment and killing by antibody-dependent cell-mediated cytotoxicity, to kill and/or remove IVinfected cells and virus (Capon et al., Nature 337, 525-531, 1989; Traunecker et al., Nature 339, 68-70, 1989; and Langner et al., 1993, supra). While such CD4immunoglobulin conjugates (sometimes called "immunoadhesins") have, indeed, shown advantageous pharmacokinetic and distributional attributes in vivo, and anti-HIV effects in vitro, clinical results have been discouraging (Schooley et al., 1990, supra; Husson et al., 1992, supra; and Langner et al., 1993, sura). This is not surprising since clinical isolates of HIV, as opposed to laboratory strains, are highly resistant to binding and neutralization by sCD4 (Orloff et al., 1995, suPra; and Moore et al., 1992, supra). Therefore, the extraordinarily broad targeting properties of a functional cyanovirin to viruses, primate retroviruses, in general, and clinical and laboratory strains, in particular (Boyd et al., 1995, supra; and Gustafson et al., 1995, supra), can be especially advantageous for combining with toxins, immunoglobulins and other selected effector proteins.

Viral-targeted conjugates can be prepared either by genetic engineering techniques (see, for example, Chaudhary et al., 1988, supra) or by chemical coupling of the targeting component with an effector component. The most feasible or appropriate technique to be used to construct a given cyanovirin conjugate or fusion protein will be selected based upon consideration of the characteristics of the particular effector molecule selected for coupling to a cyanovirin. For example, with a selected non-proteinaceous effector molecule, chemical coupling, rather than genetic engineering techniques, may be the only feasible option for creating the desired cyanovirin conjugate.

Accordingly, the present invention also provides nucleic acid molecules encoding cyanovirin fusion proteins. In particular, the present invention provides a nucleic acid molecule comprising SEQ ID NO:3 and substantially homologous sequences thereof. Also provided is a vector comprising a nucleic acid sequence encoding a cyanovirin fusion protein and a method of obtaining a cyanovirin fusion protein by expression of the vector encoding a cyanovirin fusion protein in a protein-synthesizing organism as described above. Accordingly, cyanovirin fusion proteins are also provided.

In view of the above, the present invention further provides an isolated and purified nucleic acid molecule, which comprises a cyanovirin coding sequence, such as one of the aforementioned nucleic acids, namely a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4, a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, or a nucleic acid molecule comprising a sequence of SEQ ID NO:3, coupled to a second nucleic acid encoding an effector protein. The first nucleic acid preferably comprises a nucleic acid sequence encoding at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2, which encodes a functional cyanovirin, and the second nucleic acid preferably encodes an effector protein, such as a toxin or immunological reagent as described above.

Accordingly, the present invention also further provides an isolated and purified protein encoded by a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, a nucleic acid molecule comprising a sequence of SEQ ID NO:3, a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2, or a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:4.

Preferably, the aforementioned nucleic acid molecules encode at least nine contiguous amino acids of the amino acid sequence of SEQ ID NO:2 coupled to an effector molecule, such as a toxin or immunological reagent as described above.

Preferably, the effector molecule targets a virus, more preferably HIV, and, most preferably glycoprotein gpl20. The coupling can be effected at the DNA level or by chemical coupling as described above. For example, a cyanovirin-effector protein conjugate of the present invention can be obtained by selecting a desired effector protein or peptide; synthesizing a composite DNA coding sequence comprising a first DNA coding sequence comprising one of the aforementioned nucleic acid sequences, which codes for a functional cyanovirin, coupled to a second DNA coding sequence for an effector protein or peptide, e.g., a toxin or immunological reagent; expressing said composite DNA coding sequence in an appropriate protein-synthesizing organism; and purifying the desired fusion protein or peptide to substantially pure form. Alternatively, a cyanovirin-effector molecule conjugate of the present invention can be obtained by selecting a desired effector molecule and a cyanovirin or cyanovirin fusion protein; chemically coupling the cyanovirin or cyanovirin fusion protein to the effector molecule; and purifying the desired cyanovirin-effector molecule conjugate to substantially pure form.

Conjugates comprising a functional cyanovirin an antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO:2, such as SEQ ID NO: 2, wherein the at least nine contiguous amino acids bind to a virus, in particular an infectious virus, such as HIV, in which case the cyanovirin binds to gpl20) coupled to an anti-cyanovirin antibody or at least one effector component, which can be the same or different, such as a toxin, an immunological reagent, or other functional reagent, can be designed even more specifically to exploit the unique viral targeting, gp 120-targeting properties, of cyanovirins.

Other functional reagents that can be used as effector components in the present inventive conjugates can include, for example, polyethylene glycol, dextran, albumin and the like, whose intended effector functions may include one or more of the following: to improve stability of the conjugate; to increase the half-life of the conjugate; to increase resistance of the conjugate to proteolysis; to decrease the immunogenicity of the conjugate; to provide a means to attach or immobilize a functional cyanovirin onto a solid support matrix see, for example, Harris, in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, Harris, ed., Plenum Press: New York (1992), pp. 1-14).

Conjugates furthermore can comprise a functional cyanovirin coupled to more than one effector molecule, each of which, optionally, can have different effector functions such as a toxin molecule (or an immunological reagent) and a polyethylene glycol (or dextran or albumin) molecule). Diverse applications and uses of functional proteins and peptides, such as in the present instance a functional cyanovirin, attached to or immobilized on a solid support matrix, are exemplified more specifically for poly(ethylene glycol) conjugated proteins or peptides in a review by Holmberg et al. (In Polv(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications. Harris, ed., Plenum Press: New York, 1992, pp. 303-324). Preferred examples of solid support matrices include magnetic beads, a flow-through matrix, and a matrix comprising a contraceptive device, such as a condom, a diaphragm, a cervical cap, a vaginal ring or a sponge: Example 6 reveals novel gp 120-directed effects of cyanovirins. Additional insights have been reported by Esser et al. Virol. 73: 4360-4371, 1999). They described a series of studies that further confirmed that cyanovirins block mediated binding and fusion of intact HIV-1 virions to host cells. They also provided additional confirmation that cyanovirins inhibit envelope glycoproteinmediated infectivity of other viruses, including feline immunodeficiency virus

(FIV).

The range of antiviral activity (Boyd et al., 1995, supra) of cyanovirin against diverse CD4+-tropic immunodeficiency virus strains in various target cells is remarkable; all tested strains of HIV-1, HIV-2 and SIV were similarly sensitive to cyanovirin; clinical isolates and laboratory strains showed essentially equivalent sensitivity. Cocultivation of chronically infected and uninfected CEM-SS cells with cyanovirin did not inhibit viral replication, but did cause a concentrationdependent inhibition of cell-to-cell fusion and virus transmission; similar results from binding and fusion inhibition assays employing HeLa-CD4-LTRgalactosidase cells were consistent with cyanovirin inhibition of virus-cell and/or cell-cell binding.

The anti-viral, anti-HIV, activity of the cyanovirins and conjugates thereof of the present invention can be further demonstrated in a series of interrelated in vitro antiviral assays (Gulakowski et al., J. Virol. Methods 33, 87- 100, 1991), which accurately predict for antiviral activity in humans. These assays measure the ability of compounds to prevent the replication of HIV and/or the cytopathic effects of HIV on human target cells. These measurements directly correlate with the pathogenesis of HIV-induced disease in vivo. The results of the analysis of the antiviral activity of cyanovirins or conjugates, as set forth in Example 5 and as illustrated in Figures 8, 9 and 10, are believed to predict accurately the antiviral activity of these products in vivo in humans and, therefore, establish the utility of the present invention. Furthermore, since the present invention also provides methods of ex vivo use of cyanovirins and conjugates see results set forth in Example 5, and in Figures 6 and the utility of cyanovirins and conjugates thereof is even more certain.

The cyanovirins and conjugates thereof of the present invention can be shown to inhibit a virus, specifically a retrovirus, more specifically an immunodeficiency virus, such as the human immunodeficiency virus, HIV-1 or HIV-2. The cyanovirins and conjugates of the present invention can be used to inhibit other retroviruses as well as other viruses. Examples of viruses that may be treated in accordance with the present invention include, but are not limited to, Type C and Type D retroviruses, HTLV-1, HTLV-2, HIV, FIV, FLV, SIV, MLV, BLV, BIV, equine infectious virus, anemia virus, avian sarcoma viruses, such as Rous sarcoma virus (RSV), hepatitis type A, B, non-A and non-B viruses, arboviruses, varicella viruses, human herpes virus HHV-6), measles, mumps and rubella viruses.

Cyanovirins and conjugates thereof collectively comprise proteins and peptides, and, as such, are particularly susceptible to hydrolysis of amide bonds catalyzed by peptidases) and disruption of essential disulfide bonds or 22 formation of inactivating or unwanted disulfide linkages (Carone et al., J. Lab.

Clin. Med. 100, 1-14, 1982). There are various ways to alter molecular structure, if necessary, to provide enhanced stability to the cyanovirin or conjugate thereof (Wunsch, Biopolvmers 22, 493-505, 1983; and Samanen, in Polymeric Materials in Medication, Gebelein et al., eds., Plenum Press: New York, 1985, pp. 227-242), which may be essential for preparation and use of pharmaceutical compositions containing cyanovirins or conjugates thereof for therapeutic or prophylactic applications against viruses, HIV. Possible options for useful chemical modifications of a cyariovirin or conjugate include, but are not limited to, the following (adapted from Samanen, 1985, supra): olefin substitution, (b) carbonyl reduction, D-amino acid substitution, N-methyl substitution, (e) C-methyl substitution, C-C'-methylene insertion, dehydro amino acid insertion, retro-inverso modification, N-terminal to C-terminal cyclization, and thiomethylene modification. Cyanovirins and conjugates thereof also'can be modified by covalent attachment of carbohydrate and polyoxyethylene derivatives, which are expected to enhance stability and resistance to proteolysis (Abuchowski et al., in Enzymes as Drugs, Holcenberg et al., eds., John Wiley: New York, 1981, pp. 367-378).

Other important general considerations for design of delivery strategy systems and compositions, and for routes of administration, for protein and peptide drugs, such as cyanovirins and conjugates thereof (Eppstein, CRC Crit. Rev.

Therapeutic Drug Carrier Systems 5, 99-139, 1988; Siddiqui et al., CRC Crit. Rev.

Therapeutic Drug Carrier Systems 3, 195-208, 1987); Banga et al., Int. J.

Pharmaceutics 48, 15-50, 1988; Sanders, Eur. J. Drug Metab. Pharmacokinetics 15, 95-102, 1990; and Verhoef, Eur. J. Drug Metab. Pharmacokinetics 15, 83-93, 1990), also apply. The appropriate delivery system for a given cyanovirin or conjugate thereof will depend upon its particular nature, the particular clinical application, and the site of drug action. As with any protein or peptide drug, oral delivery of a cyanovirin or a conjugate thereof will likely present special problems, due primarily to instability in the gastrointestinal tract and poor 23 absorption and bioavailability of intact, bioactive drug therefrom. Therefore, especially in the case of oral delivery, but also possibly in conjunction with other routes of delivery, it will be necessary to use an absorption-enhancing agent in combination with a given cyanovirin or conjugate thereof. A wide variety of absorption-enhancing agents have been investigated and/or applied in combination with protein and peptide drugs for oral delivery and for delivery by other routes (Verhoef, 1990, supra; van Hoogdalem, Pharmac. Ther. 44, 407-443, 1989; Davis, J. Pharm. Pharmacol. 44(Suppl. 186-190, 1992). Most commonly, typical enhancers fall into the general categories of chelators, such as EDTA, salicylates, and N-acyl derivatives of collagen, surfactants, such as lauryl sulfate and polyoxyethylene-9-lauryl ether, bile salts, such as glycholate and taurocholate, and derivatives, such as taurodihydrofusidate, fatty acids, such as oleic acid and capric acid, and their derivatives, such as acylcamitines, monoglycerides and diglycerides, non-surfactants, such as unsaturated cyclic ureas, saponins, cyclodextrins, and phospholipids.

Other approaches to enhancing oral delivery of protein and peptide drugs, such as the cyanovirins and conjugates thereof, can include aforementioned chemical modifications to enhance stability to gastrointestinal enzymes and/or increased lipophilicity. Alternatively, or in addition, the protein or peptide drug can be administered in combination with other drugs or substances, which directly inhibit proteases and/or other potential sources of enzymatic degradation of proteins and peptides. Yet another alternative approach to prevent or delay gastrointestinal absorption of protein or peptide drugs, such as cyanovirins or conjugates, is to incorporate them into a delivery system that is designed to protect the protein or peptide from contact with the proteolytic enzymes in the intestinal lumen and to release the intact protein or peptide only upon reaching an area favorable for its absorption. A more specific example of this strategy is the use of biodegradable microcapsules or microspheres, both to protect vulnerable drugs from degradation, as well as to effect a prolonged release of active drug (Deasy, in Microencapsulation and Related Processes, Swarbrick, ed., Marcell Dekker, Inc.: 24 New York, 1984, pp. 1-60, 88-89, 208-211). Microcapsules also can provide a useful way to effect a prolonged delivery of a protein and peptide drug, such as a cyanovirin or conjugate thereof, after injection (Maulding, J. Controlled Release 6, 167-176, 1987).

Given the aforementioned potential complexities of successful oral delivery of a protein or peptide drug, it is fortunate that there are numerous other potential routes of delivery of a protein or peptide drug, such as a cyanovirin or conjugate thereof. These routes include intravenous, intraarterial, intrathecal, intracisternal, buccal, rectal, nasal, pulmonary, transdermal, vaginal, ocular, and the like (Eppstein, 1988, supra; Siddiqui et al., 1987, supra; Banga et al., 1988, sura; Sanders, 1990, supra; Verhoef, 1990, supra; Barry, in Delivery Systems for Peptide Drugs, Davis et al., eds., Plenum Press: New York, 1986, pp. 265-275; and Patton et al., Adv. Drug Delivery Rev. 8, 179-196, 1992). With any of these routes, or, indeed, with any other route of administration or application, a protein or peptide drug, such as a cyanovirin or conjugate thereof, may initiate an immunogenic reaction. In such situations it may be necessary to modify the molecule in order to mask immunogenic groups. It also can be possible to protect against undesired immune responses by judicious choice of method of formulation and/or administration. For example, site-specific delivery can be employed, as well as masking of recognition sites from the immune system by use or attachment of a so-called tolerogen, such as polyethylene glycol, dextran, albumin, and the like (Abuchowski et al., 1981, supra; Abuchowski et al., J. Biol. Chem. 252, 3578- 3581, 1977; Lisi et al., J. Appl. Biochem. 4, 19-33, 1982; and Wileman et al., L Pharm. Pharmacol. 38, 264-271, 1986). Such modifications also can have advantageous effects on stability and half-life both in vivo and ex vivo.

Procedures for covalent attachment of molecules, such as polyethylene glycol, dextran, albumin and the like, to proteins, such as cyanovirins or conjugates thereof, are well-known to those skilled in the art, and are extensively documented in the literature see Davis et al., In Peptide and Protein Drug Delivery, Lee, ed., Marcel Dekker: New York, 1991, pp. 831-864).

Other strategies to avoid untoward immune reactions can also include the induction of tolerance by administration initially of only low doses. In any event, it will be apparent from the present disclosure to one skilled in the art that for any particular desired medical application or use of a cyanovirin or conjugate thereof, the skilled artisan can select from any of a wide variety of possible compositions, routes of administration, or sites of application, what is advantageous.

Accordingly, the antiviral cyanovirins and conjugates thereof of the present invention can be formulated into various compositions for use, for example, either in therapeutic treatment methods for infected individuals, or in prophylactic methods against viral, HIV, infection of uninfected individuals.

The present invention also provides a composition, such as a pharmaceutical composition, which comprises an isolated and purified cyanovirin, a cyanovirin conjugate, a matrix-anchored cyanovirin or a matrix-anchored cyanovirin conjugate, such as an antiviral effective amount thereof. The composition can further comprise a carrier, such as a pharmaceutically acceptable carrier. The composition can further comprise at least one additional antiviral compound other than a cyanovirin or conjugate thereof, such as in an antiviral effective amount. Suitable antiviral compounds include AZT, ddl, ddC, gancyclovir, fluorinated dideoxynucleosides, nevirapine, R82913, Ro 31-8959, BI-RJ-70, acyclovir, a-interferon, recombinant sCD4, michellamines, calanolides, nonoxynol-9, gossypol and derivatives thereof, and gramicidin. If the composition is to be used to induce an inmmune response, it comprises an immune response-inducing amount of a cyanovirin or conjugate thereof and can further comprise an immunoadjuvant, such as polyphosphazene polyelectrolyte. The cyanovirin used in the composition, pharmaceutical composition,'can be isolated and purified from nature or genetically engineered.

Similarly, the cyanovirin conjugate can be genetically engineered or chemically coupled.

The present inventive compositions can be administered to a host, such as a human, so as to inhibit viral infection in a prophylactic or therapeutic method. The compositions of the present invention are particularly useful in inhibiting the growth or replication of a virus, such as a retrovirus, in particular an immunodeficiency virus, such as HIV, specifically HIV-1 and HIV-2. The compositions are useful in the therapeutic or prophylactic treatment of animals, such as humans, who are infected with a virus or who are at risk for viral infection, respectively. The compositions also can be used to treat objects or materials, such as medical equipment, supplies, or fluids, including biological fluids, such as blood, blood products and vaccine formulations, cells, tissues and organs, to remove or inactivate virus in an effort to prevent or treat viral infection of an animal, such as a human: Such compositions also are useful to prevent sexual transmission of viral infections, HIV, which is the primary way in which the world's AIDS cases are contracted (Merson, 1993, supa).

Potential virucides used or being considered for use against sexual transmission of HIV are very limited; present agents in this category include nonoxynol-9 (Bird, AIDS 5, 791-796, 1991), gossypol and derivatives (Polsky et al., Contraception 39, 579-587, 1989; Lin, Antimicrob. Agents Chemother. 33, 2149-2151, 1989; and Royer, Pharmacol. Res. 24, 407-412, 1991), and gramicidin (Bourinbair, Life Sci./Pharmacol. Lett. 54, PL5-9, 1994; and Bourinbair et al., Contraception 49, 131-137, 1994). The method of prevention of sexual transmission of viral infection, HIV infection, in accordance with the present invention comprises vaginal, rectal, oral, penile or other topical treatment with an antiviral effective amount of a cyanovirin and/or cyanovirin conjugate, alone or in combination with another antiviral compound as described above.

In a novel approach to anti-HIV prophylaxis currently being initiated under the auspices of the U.S. National Institute of Allergy and Infectious Diseases (NIAID) as conveyed by Painter, USA Today, February 13, 1996), the vaginal suppository instillation of live cultures oflactobacilli is being evaluated in a 9 0 0 -woman study. This study is based especially upon observations of anti-HIV effects of certain H202-producing lactobacilli in vitro see published abstract by Hilier, from NIAID-sponsored Conference on "Advances in AIDS Vaccine Development", Bethesda, MD, February 11-15, 1996). Lactobacilli readily populate the vagina, and indeed are a predominant bacterial population in most healthy women (Redondo-Lopez et al., Rev. Infect. Dis. 12, 856-872, 1990; Reid et al., Clin. Microbiol. Rev. 3, 335-344, 1990; Bruce and Reid, Can. J. Microbiol.

34, 339-343, 1988;rcu et al., J. Infect. Dis. 171, 1237-1243, 1995; Hilier et al., Clin. Infect. Dis. 16(Suppl S273-S281; Agnew et al., Sex. Transm. Dis. 22, 269-273, 1995). Lactobacilli are also prominent, nonpathogenic inhabitants of other body cavities such as the mouth, nasopharynx, upper and lower gastrointestinal tracts, and rectum.

It is well-established that lactobacilli can be readily transformed using available genetic engineering techniques to incorporate a desired foreign DNA coding sequence, and that such lactobacilli can be made to express a corresponding desired foreign protein (see, Hols et al., Appl. and Environ. Microbiol. 1401-1413, 1994). Therefore, within the context of the present disclosure, it will be appreciated by one skilled in the art that viable host cells containing a DNA sequence or vector of the present invention, and expressing a protein of the present invention, can be used directly as the delivery vehicle for a cyanovirin or conjugate thereof to the desired site(s) in vivo. Preferred host cells for such delivery of cyanovirins or conjugates thereof directly to desired site(s), such as, for example, to a selected body cavity, can comprise bacteria. More specifically, such host cells can comprise suitably engineered strain(s) of lactobacilli, enterococci, or other common bacteria, such as E. coli, normal strains of which are known to commonly populate body cavities. More specifically yet, such host cells can comprise one or more selected nonpathogenic strains of lactobacilli, such as those described by Andreu et al. (1995, sunra), especially those having high adherence properties to epithelial cells, such as, for example, adherence to vaginal epithelial cells, and suitably transformed using the DNA sequences of the present invention.

As reviewed by McGroarty (FEMS Immunol. Med. Microbiol. 6, 251-264, 1993) the "probiotic" or direct therapeutic application of live bacteria, particularly bacteria that occur normally in nature, more particularly lactobacilli, for treatment or prophylaxis against pathogenic bacterial or yeast infections of the urogenital 28 tract, in particular the female urogenital tract, is a well-established concept.

Recently, the use of a conventional probiotic strategy, in particular the use of live lactobacilli, to inhibit sexual transmission of HIV has been suggested, based specifically upon the normal, endogenous production of virucidal levels of H202 and/or lactic acid and/or other potentially virucidal substances by certain normal strains of lactobacilli Hilier, 1996, supra). However, the present inventive use of non-mammalian cells, particularly bacteria, more particularly lactobacilli, specifically engineered with a foreign gene, more specifically a cyanovirin gene, to express an antiviral substance, more specifically a protein, and even more specifically a cyanovirin, is heretofore unprecedented as a method of treatment of an animal, specifically a human, to prevent infection by a virus, specifically a retrovirus, more specifically HIV-I or HIV-2.

Elmer et al. (JAMA 275, 870-876, 1996) have recently speculated that "genetic engineering offers the possibility of using microbes to deliver specific actions or products to the colon or other mucosal surfaces other fertile areas for future study include defining the mechanisms of action of various biotherapeutic agents with the possibility of applying genetic engineering to enhance activities." Elmer et al. (1996, supra) further point out that the terms "probiotic" and "biotherapeutic agent" have been used in the literature to describe microorganisms that have antagonistic activity toward pathogens in vivo; those authors more specifically prefer the term "biotherapeutic agent" to denote "microorganisms having specific therapeutic properties.

In view of the present disclosure, one skilled in the art will appreciate that the present invention teaches an entirely novel type of "probiotic" or "biotherapeutic" treatment using specifically engineered strains of microorganisms provided herein which do not occur in nature. Nonetheless, available teachings concerning selection of optimal microbial strains, in particular bacterial strains, for conventional probiotic or biotherapeutic applications can be employed in the context of the present invention. For example, selection of optimal lactobacillus strains for genetic engineering, transformation, direct expression of cyanovirins or 29 conjugates thereof, and direct probiotic or biotherapeutic applications, to treat or prevent HIV infection, can be based upon the same or similar criteria, such as those described by Elmer et al. (1996, supra), typically used to select normal, endogenous or "nonengineered" bacterial strains for conventional probiotic or biotherapeutic therapy. Furthermore, the recommendations and characteristics taught by McGroarty, particularly for selection of optimal lactobacillus strains for conventional probiotic use against female urogenital infections, are pertinent to the present invention: lactobacilli chosen for incorporation into probiotic preparations should be easy and, if possible, inexpensive to cultivate strains should be stable, retain viability following freeze-drying and, of course, be nonpathogenic to the host it is essential that lactobacilli chosen for use in probiotic preparations should adhere well to the vaginal epithelium ideally, artificially implanted lactobacilli should adhere to the vaginal epithelium, integrate with the indigenous microorganisms present, and proliferate" (McGroarty, 1993 supra).

While McGroarty's teachings specifically address selections of"normal" lactobacillus strains for probiotic uses against pathogenic bacterial or yeast infections of the female urogenital tract, similar considerations will apply to the selection of optimal bacterial strains for genetic engineering and "probiotic" or "biotherapeutic" application against viral infections as particularly encompassed by the present invention.

Accordingly, the method of the present invention for the prevention of sexual transmission of viral infection, HIV infection, comprises vaginal, rectal, oral, penile, or other topical, insertional, or instillational treatment with an antiviral effective amount of a cyanovirin, a cyanovirin conjugate, a matrixanchored cyanovirin or conjugate thereof, and/or viable host cells transformed to express a cyanovirin or conjugate thereof, alone or in combination with one or more other antiviral compound as described above).

Compositions for use in the prophylactic or therapeutic treatment methods of the present invention comprise one or more cyanovirin(s) or conjugate(s) thereof, either one of which can be matrix-anchored, and desirably a carrier therefor, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well-known to those who are skilled in the art, as are suitable methods of administration. The choice of carrier will be determined in part by the particular cyanovirin or conjugate thereof, as well as by the particular method used to administer the composition.

One skilled in the art will appreciate that various routes of administering a drug are available, and, although more than one route can be used to administer a particular drug, a particular route can provide a more immediate and more effective reaction than aiother route. Furthermore, one skilled in the art will appreciate that the particular pharmaceutical carrier employed will depend, in part, upon the particular cyanovirin or conjugate thereof employed, and the chosen route of administration. Accordingly, there is a wide variety of suitable formulations of the composition of the present invention.

Formulations suitable for oral administration can consist of liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solid, granules or freeze-dried cells; solutions or suspensions in an aqueous liquid; and oil-in-water emulsions or water-in-oil emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Suitable formulations for oral delivery can also be incorporated into synthetic and natural polymeric microspheres, or other means to protect the agents of the present invention from degradation within the gastrointestinal tract (see, for example, Wallace et al., Science 260, 912-915, 1993).

The cyanovirins or conjugates thereof, alone or in combination with other antiviral compounds, can be made into aerosol formulations to be administered via

II~

31 inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethanc, propane, nitrogen and the like.

The cyanovirins or conjugates thereof, alone or in combinations with other antiviral compounds or absorption modulators, can be made into suitable formulations for transderznal application and absorption (Wallace et al., 1993, suna) Transdermal electroporation or iontophoresis also can be used to promote and/or control the systemic delivery of the compounds and/or compositions of the present invention through the skin see Theiss et al., Meth. Find. Exp. Clin.

Pharmacol. 13, 353-359,-1991).

Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient mna suitable liquid carrier; as well as creams, emulsions, gels and the like containing, in addition to the active ingredient, such as, for example, freeze-dried lactobacilli genetically engineered to directly produce a cyanovirin or conjugate thereof of the present invention, such carriers as are known in the art.

Formulations for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa hutter or a salicylate.

Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such as, for example, freeze-dried lactobacilli genetically engineered to directly produce a cyanovirin or conjugate thereof of the present invention) such carriers as are known in the art to be appropriate. Similarly, the active ingredient can be combined with a lubricant as a coating on a condom.

Indeed, preferably, the active ingredient is applied to any contraceptive device, including, but not limited to, a condom, a diaphragm, a cervical cap, a vaginal ring and a sponge.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, 32 buffers, bacteriostats, and salutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending ageints, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid cardier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and. tablets of the kind previously described.

Formulations comprising a cyanovirin or cyanovirin conjugate suitable for virucidal HIV) sterilization of inanimate objects, such as medical supplies or equipment, laboratory equipment and supplies, instruments, devices, and the like, can, for example, be selected or adapted as appropriate, by one skilled in the art, from any of the aforementioned compositions or formulations. Preferably, the cyanovirin is produced by recombinant DNA technology. The cyanovirin conjugate can be produced by recombinant DNA technology or by chemical coupling of a cyanovirin with an effector molecule as described above. Similarly, formnulations. suitable for ex vivo sterilization or removal of virus, such as 'infectious virus, from. a sample,. such as blood, blood products, sperm, or other bodily products, such as a fluid, cells, a tissue or an organ, or any other solution, suspension, emulsion, vaccine formulation (such as in the removal of infectious virus), or any other material which can be administered to a patient in a medical procedure, can be selected or adapted as appropriate by one skilled in the art, fromany of the aforementioned compositions or formulations. However, suitable formulations for ex vivo sterilization or removal of virus from a sample or on an inanimate object are by no means limited to any of the aforementioned formulations or compositions. For example, such formulations or compositions can comprise a functional cyanovirin, such as that which is encoded by SEQ ID NO: 2, or antiviral fragment thereat, such as a fragment comprising at least nine contiguous amino acids of SEQ ID NO:2, wherein the at least nine contiguous 33 amino acids bind- to a virus, or a conjugate of either of the foregoing, attached to a solid support matrix, to facilitate contacting or binding infectious virus in a sample or removing ilifectious virus from a sample as described above, e.g a bodily product such as a fluid, cells, a tissue or an organ from an organism, in particular a mammal, such as a human, including, for example, blood, a component of blood, or sp erm. Preferably, the antiviral protein comprises SEQ ID NO: 2. Also preferably, the at least nine contiguous amino acids bind gpl2O of MVy, in particular infectious HIV. As a more specific example, such a formulation or composition can comprise a functional cyanovirin, or conjugate thereof, attached to (eg, coupled to or immobilized on) a solid support matrix comprising magnetic beads, to facilitate contacting, binding and removal of infectious virus, and to enable magnet-assisted removal of the virus from a samrple as described above, a bodily product such as a fluid, cells, a tissue or an organ, blood, a component of blood, or sperm. Alternatively, and also preferably, the solid support matrix comprises a contraceptive device, such as a condom, a diaphragm, a cervical cap, a vaginal ring or a sponge, As an even more specific illustration, such a composition for ex vivo) can comprise a functional gp 120-binding, HI V- inactivating) cyanovirin, or conjugate thereof, attached to a solid support matrix, such as magnetic beads or a flow-through matrix, by means of an anti-cyanovirin antibody or at least one effector component, which can be the same or different, such as polyethylene glycol, albumin or dextran. The conjugate can further comprise at least one effector component, which can be the same or different, selected from the group consisting of an immunological reagent and a toxin. A flow-through matrix would comprise, for instance, a configuration similar to an affinity column. The cyanovirin can be covalently coupled to a solid support matrix via an anticyanovirin antibody, described below. Methods of attaching an antibody to a solid support matrix are well-known in the art (see, for example, Harlow and Lane.

Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory, Cold Spring Harbor, NY, 1988). Alternatively, the solid support matrix, such as magnetic beads, can be coated with streptavidin, in which case the cyanovirin or fragment thereof (which comprises at least nine contiguous amino acids of SEQ ID NO: 2), or a conjugate of either one, is biotinylated. Preferably, the at least nine contiguous amino acids of SEQ ID NO: 2 bind gp120 of HIV, which preferably is infectious. Preferably, the antiviral protein comprises SEQ ID NO: 2. Such a composition can be prepared, for example, by biotinylating the cyanovirin, or conjugate thereof, and then contacting the biotinylated protein or peptide with a (commercially available) solid support matrix, such as magnetic beads, coated with streptavidin. The use of biotinylation as a means to attach a desired biologically active protein or peptide to a streptavidin-coated support matrix, such as magnetic beads, is well-known in the art. Example 7 specifically describes a procedure applicable to biotinylation of cyanovirin-N and attachment of the biotinylated protein to streptavidin-coated magnetic beads. Example 7 also illustrates another important principle that is critical to the present invention: for any given formulation or composition comprising a functional cyanovirin, or conjugate thereof, attached to or immobilized on a solid support matrix, it is 'essential that the formulation or composition retain the desired in this instance, the virus binding gpl20-binding) and virus-inactivating HIV)) properties of the functional cyanovirin, or conjugate thereof, itself.

One skilled in the art will appreciate that a suitable or appropriate formulation can be selected, adapted or developed based upon the particular application at hand.

For ex vivo uses, such as virucidal treatments of inanimate objects or materials, blood or blood products, or tissues, the amount of cyanovirin, or conjugate or composition thereof, to be employed should be sufficient that any virus or virus-producing cells present will be rendered noninfectious or will be destroyed. For example, for HIV, this would require that the virus and/or the virus-producing cells be exposed to concentrations of cyanovirin-N in the range of 0.1-1000 nM. Similar considerations apply to in vivo applications. Therefore, the designation of "antiviral effective amount" is used generally to describe the amount of a particular cyanovirin, conjugate or composition thereof required for antiviral efficacy in any given application.

In view of the above, the present invention also provides a method of inhibiting prophylactically or therapeutically a viral infection of a host. The method comprises administering to the host an antiviral effective amount of an above-described conjugate. Upon administration of the antiviral effective amount of the conjugate, the viral infection is inhibited. Alternatively, a composition comprising a solid support matrix to which is attached an isolated and purified antiviral protein, antivirid peptide, antiviral protein conjugate or antiviral peptide conjugate as described above can be administered.

The present invention additionally provides a method of prophylactically or therapeutically inhibiting a viral infection of a host. The method comprises topically administering to a host an antiviral effective amount of a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprising at least nine contiguous amino acids of SEQ ID NO: 2 having antiviral activity and attached to a solid support matrix. Upon administration of the antiviral effective amount of the composition, the viral infection is inhibited. Preferably, the solid support matrix is a contraceptive device, such as a condom, diaphragm, cervical cap, vaginal ring or sponge. In an alternative embodiment, a solid support matrix can be surgically implanted and later removed.

For in vivo uses, the dose of a cyanovirin, or conjugate or composition thereof, administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a prophylactic or therapeutic response in the individual over a reasonable time frame. The dose used to achieve a desired antiviral concentration in vivo 0. 1- 1000 JIM) will be determined by the potency of the particular cyanovi rin or conjugate employed, the severity of the disease state of infected individuals, as well as, in die case of systemic administration;- the body weight and age of the infected individual. The size of the dose also will be deternined by the existence of any adverse side effects that may accompany the particular cyanovirin, or conjugate or composition thereof, employed. It is always desirable, whenever possible, to keep adverse side effects to a midnimum.

The present invention also provides a method of removing virus, such as infectious virus, from a sample. The method comprises contacting the sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate, or antiviral peptide conjugate, comprising at least nine contiguous amino acids of SEQ ID NO:2, wherein the at least nine contiguous amino acids bind to the virus and the antiviral protein or antiviral peptide (or conjugate of either of the foregoing) is attached to a solid support matrix, such as a magnetic bead. "Attached" is used herein to refer to attachment to (or coupling to) and immobilization in or on a solid support matrix. While any means of attachment can be used, preferably, attachment is by covalent bonds.

The method further comprises separating the sample and the composition by any suitable means, whereupon the virus, such as infectious virus, is removed from the sample. Preferably, the antiviral protein comprises SEQ JD NO:2. In one embodiment, the antiviral protein or antiviral peptide is conjugated with an anticyanovirin antibody or at least one effector component, which can be the same or different, selected from polyethylene glycol, dextran and albumin, in which case the antiviral protein or antiviral peptide is desirably attached to the solid support matrix through at~least one effector component The antiviral protein or antiviral peptide can be further conjugated with at least one eff'ector component, which can be the same or different, selected from the group consisting of an immunological reagent and a toxin. In another embodiment, the solid support matrix is coated with streptavidin and the antiviral protein or antiviral'peptide is biotinylated.

Through biotin, the biotinylated antiviral proteinlpeptide is attached to the streptavidin-coated solid support matrix. Other types of nicani, as are known in the art, can be used to attach a tbnctional cyanovirin an antiviral protein or peptide or conjugate of either of the foregoing as described above) to a solid support matrix, such as a magnetic bead, in which case contact with a magnet is used to separate the sample and the composition. Similarly, other types of solid support matrices can be used, such as a matrix comprising a porous surface or membrane, over or through which a sample is flowed or percolated, thereby selectively entrapping or removing infectious virus firom the sample. The choice of solid support matrix, means of attachment of the functional cyanovirin to the solid support matrix, and means of separating the sample and the matrix-anchored cyanovirin will depend, in part on the sample fluid vs. tissue) and the virus to be removed. It is expected that the use of a selected coupling molecule can confer certain desired properties to a matrix, comprising a functional cyanovirin coupled therewith, that may have particularly advantageous properties in a given situation. Preferably, the sample is blood, a component of blood, sperm, cells, tissue or an organ. Also, preferably the sample is a vaccine formulation, in which case the virus that is removed is infectious, such as IMV, although i-nv, in particular infectious HIV, can be removed from other samples in accordance with this method. Such methods also have utility in real time ax vivo removal of virus or virus infected cells from a bodily fluid, such as blood, in the treatment of viral infection, or in the removal of virus from blood or a component of blood, for transfusion, in the inhibition or prevention of viral infection. Such methods also have potential utility in dialysis, such as kidney dialysis, and in removing virus from sperm obtained from a donor for in vitro and in vivo fertilization. The methods also have applicability in the context of tissue and organ transplantations.

For instance, the skilled practitioner might select a poly(ethylene glycol) molecule for attaching a functional cyanovirin to a solid support matrix, thereby to provide a matrix-anchored cyanovirin, wherein the cyanovirin is attached to the matrix by a longer "tether" than would be feasible or possible for other attachment methods, such as biotinylation/streptavidin coupling. A cyanovirin coupled by a poly(ethylene glycol) "tether" to a solid support matrix (such as magnetic beads, porous surface or membrane, and the like) can permit optimal exposure of a binding surface, epitope, hydrophobic or hydrophobic focus, and/or the like, on a functional cyanovirin in a manner that, in a given situation and/or for a particular virus, facilitates the binding and/or inactivation of the virus. A preferred solid support matrix is a magnetic bead such that separation of the sample and the composition is effected by a magnet. In a preferred embodiment of the method, the at least nine contiguous amino acids bind gpl20 of HIV and IIV is removed from the-sample.

In summary, a cyanovirin attached to a solid support matrix, such as a magnetic bead, can be used to remove virus, in particular infectious virus, including immunodeficieiicy virus, such as HIV, [HIV- I or HIV-2, from a sample, such as a sample comprising both infectious and noninfectibus virus. In contrast to previously disclosed methods of inactivating or destroying virus in a sample, the present inventive method utilizes cyanovirins, which bind irreversibly to virus, such as infectious virus, in particular infectious immunodeficiency virus, HIV, specifically HTV-I or HIV-2. The infectious viral particle is permanently fixed to the antiviral agent and unable to infect host cells. The present inventive method enables separation of infectious from non-infectious virus and, therefore, is advantageous over other methods currently available in the art. In this regard, it has been unexpectedly observed that a cyanovirin recognizes a conformation or a portion of gp120 characteristic of infectious HIV. Therefore, only infectious virus is bound by the cyanovirin, while non-infectious virus remains in the sample. One of ordinary skill in the art will appreciate the advantages of using the present inventive matrix-anchored cyanovirins to produce pools of non-infectious virus. The present inventive method also can be used to remove gp120-presenting cells, infected cells that have gpl20 on their surfaces, from a sample.

The present invention, therefore, further provides a composition comprising naturally-occurring, non-infectious virus, such as a composition produced as described above. The composition can further comprise a carrier, such as a biologically or pharmaceutically acceptable carrier, and an immuno-adjuvant.

Preferably, the noninfectious virus is an immunodeficiency virus, such as HIV, 39 HIV-1l or HIV-2. Alternatively, and also preferably, the noninfectious virus is Fly. A composition comprising only naturally-occurring, non-infectious virus has many applications in research and the prophylactic treatment of a viral infection.

In terms of prophylactic treatment of a viral infection,rthe skilled artisan will appreciate the need to eliminate completely all infectious virus from the composition. If desired, fuirther treatment of the composition comprising noninfectious particles with virus-inactivating chemicals, such as imines or psoralens, and/or pressure or heat inactivation, will further the non-infectious nature of the composition. For examiple.- an immune response-inducing amount of the present inventive composition can be administered to an animal at risk for a viral infection in order to induce an immune response. The skilled artisan will appreciate that such a composition is a significant improvement over previously disclosed compositions in that the virus is non-infectious and naturally-occurring. Thus, there is no risk of inadvertent infection, greater doses can be administered in comparison to compositions comprising infectious viral particles, and the subsequent immune response will assuredly be directed to antigens present on naturally-occurring virus. The composition comprising naturally- occurring, noninfectious virus can be administered in any manner appropriate to induce an immune response. Preferably, the virus is administered, for example, intramuscularly, mucosally, intravenously, subcutaneously, or topically.

Preferably, the composition comprises naturally-oceuning, non-infectious human immunodeficiency virus comprising gp 120.

The composition comprising natural ly-occuring, non-infectious virus can be combined with various carriers, adjuvants, diluents or other anti-viral therapeutics, if desired. Appropriate carricrs include, for example, ovalbumin, albumin, globulins, hemocyanins, and the like. Adjuvants or immnuno-adjuvants are incorporated in most cases to stimulate further the immune system. Any physiologically appropriate adjuvant can be used. Suitable adjuvants for inclusion in the present inventive composition include, for example, aluminum hydroxide, beryllium sulfate, silica, kaolin, carbon, bacterial endotoxin, saponin, and the like.

Thus, the present invention also provides a method of inducing an immune response to a virus in an animal. The method comprises administering to the animal an immune response- inducing amount of a composition comprising natural ly-occurring, non-infectious virus as described above.

The appropriate dose of a composition comprising naturally-occuring, noninfectious virus required to induce an immune response to the virus in an animal is dependent on numerous factors, such as size of the animal and immune competency. The amount of composition administeted should be sufficient to induce a immnoral and/or -cellular immune response. The amount of non-infectious virus in a particular composition can be detennined using routine methods in the art, such as the Coulter HIV p 2 4 antigen assay (Coulter Corp., Hialeahi, FL). Any suitable dose of a composition comprising non-infectious virus is appropriate so long as an immune response is induced, desirably without the appearance of harmful side effects to the host. In this regard, compQsitions comprising from about 10' to about 10' particles, preferably from about 10' to about 10' particles, most preferably about 1O3 particles, are suitable for inducing an immune response.

One of ordinary skill can determine the effectiveness of the composition to induce an immune response using routine methods known in the art. Cellmed iated response can be determined by employing, for example, a virus antigenstimulated 1-cell proliferation assay. The presence of a humoral immune response can be detennined, for instance, with the Enzyme Linked Immunosorbent Assay (ELISA):' The skilled artisan will appreciate that there are numerous other suitable assays for evaluating induction of an immune response. To the extent that a dose is inadequate to induce an appropriate immune response, "booster" administrations can subsequently be administered in order to prompt a more effective immune response.

In terms of administration of the present inventive antiviral agents or conjugates thereof, the dosage can be in unit dosage form, such as a tablet or capsule. The tern "unit dosage foniv'. as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit 41 containing a predetermined quantity of a cyanovirin or conjugate thereof, alone or in combination with other antiviral agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.

The specifications for the unit dosage forms of the present invention depend on the particular cyanovirin, or conjugate or composition thereof, employed and the effect to be achieved, as well as the pharmacodynamics associated with each cyanovirin, or conjugate or composition thereof, in the host.

The dose administered should be an "antiviral effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending upon interindividual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level 0.1-1000 nM) desired in the patient that corresponds to a concentration of one or more cyanovirin or conjugate thereof, which inhibits a virus, such as HIV, in an assay known to predict for clinical antiviral activity of chemical compounds and biological agents. The "effective level" for agents of the present invention also can vary when the cyanovirin, or conjugate or composition thereof, is used in combination with AZT or other known antiviral compounds or combinations thereof.

One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective concentration" in the individual patient. One skilled in the art also can readily determine and use an appropriate indicator of the "effective concentration" of the compounds of the present invention by a direct analytical chemical analysis) or indirect with surrogate indicators such as p24 or RT) analysis of appropriate patient samples blood and/or tissues).

In the treatment of some virally infected individuals, it can be desirable to utilize a "mega-dosing" regimen, wherein a large dose of the cyanovirin or 42 conjugate thereof is administered, time is allowed for the drug to act, and then a suitable reagent is administered to the individual to inactivate the drug.

The pharmaceutical composition can contain other pharmaceuticals, in conjunction with the cyanovirin or conjugate thereof, when used to therapeuticaly treat a viral infection, such as that which results in AIDS. Representative examples of these additional pharmaceuticals include antiviral compounds, virucides, immunomodulators, immunostimulants, antibiotics and absorption enhancers. Exemplary antiviral compounds include AZT, ddl, ddC, gancylclovir, fluorinated dideoxynucleosides, nonnucleoside analog compounds, such as nevirapine (Shih et al., PNAS 88, 9878-9882, 1991), TIBO derivatives, such as R82913 (White et al., Antiviral Res. 16, 257-266, 1991), BI-RJ-70 (Merigan, Am.

J. Med. 90 (Suppl.4A), 8S-17S, 1991), michellamines (Boyd et al., J. Med. Chem.

37, 1740-1745, 1994) and calanolides (Kashman et al., J. Med. Chem. 35, 2735- 2743, 1992), nonoxynol-9, gossypol and derivatives, and gramicidin (Bourinbair et al., 1994, supra). Exemplary immunomodulators and immunostimulants include various interleukins, sCD4, cytokines, antibody preparations, blood transfusions, and cell transfusions. Exemplary antibiotics include antifungal agents, antibacterial agents, and anti-Pneumocystitis carnii agents. Exemplary absorption enhancers include bile salts and other surfactants, saponins, cyclodextrins, and phospholipids (Davis, 1992, supra).

Administration of a cyanovirin or conjugate thereof with other antiretroviral agents and particularly with known RT inhibitors, such as ddC, AZT, ddl, ddA, or other inhibitors that act against other HIV proteins, such as anti-TAT agents, is expected to inhibit most or all replicative stages of the viral life cycle.

The dosages of ddC and AZT used in AIDS or ARC patients have been published.

A virustatic range ofddC is generally between 0.05 pM to 1.0 pM. A range of about 0.005-0.25 mg/kg body weight is virustatic in most patients. The preliminary dose ranges for oral administration are somewhat broader, for example 0.001 to 0.25 nig/kg given in one or more doses at intervals of 2, 4, 6, 8, 12, etc.

hours. Currently, 0.01 mg/kg body weight ddC given every 8 hrs is preferred.

43 When given in combined therapy, the other antiviral compound, for example, can be given at the same time as the cyanovirin or conjugate thereof or the dosing can be staggered as desired. The two drugs also can be combined in a composition.

Doses of each can be less when used in combination than when either is used alone.

_It will also be appreciated by one skilled in the art that a DNA sequence of a cyanovirin or conjugate thereof of the present invention can be inserted ex vivo into mammalian cells previously removed from a given animal, in particular a human, host. Such cells can be employed to express the corresponding cyanovirin or conjugate in vivo after reintroduction into the host. Feasibility of such a therapeutic strategy to deliver a therapeutic amount of an agent in close proximity to the desired target cells and pathogens, virus, more particularly retrovirus, specifically HIV and its envelope glycoprotein gpl20, has been demonstrated in studies with cells engineered ex vivo to express sCD4 (Morgan et al., 1994, supra).

It is also possible that, as an alternative to ex vivo insertion of the DNA sequences of the present invention, such sequences can be inserted into cells directly in vivo, such as by use of an appropriate viral vector. Such cells transfected in vivo are expected to produce antiviral amounts of cyanovirin or a conjugate thereof directly in vivo.

Given the present disclosure, it will be additionally appreciated that a DNA sequence corresponding to a cyanovirin or conjugate thereof can be inserted into suitable nonmammalian host cells, and that such host cells will express therapeutic or prophylactic amounts of a cyanovirin or conjugate thereof directly in vivo within a. desired body compartment of an animal, in particular a human. Example 3 illustrates the transformation and expression of effective virucidal amounts of a cyanovirin in a non-mammalian cell, more specifically a bacterial cell. Example illustrates the transformation and expression of a cyanovirin in a nonmammalian cell, specifically a yeast cell.

In a preferred embodiment of the present invention, a method of femalecontrollable prophylaxis against HIV infection comprises the intravaginal administration and/or establishment of, in a female human, a persistent intravaginal population of lactobacilli that have been transformed with a coding sequence of the present invention to produce, over a prolonged time, effective virucidal levels of a cyanovirin or conjugate thereof, directly on or within the vaginal andor cervical and/or uterine mucosa. It is noteworthy that both the World Health Organization (WHO), as well as the U.S. National Institute of Allergy and Infectious Diseases, have pointed to the need for development of female-controlled topical microbicides, suitable for blocking the transmission of HIV, as an urgent global Priority (Lange et al., Lancet 341, 1356, 1993; Fauci, 1O NIALD News, April 27, 1995). A composition comprising a present inventive antiviral agent and a solid-support matrix is particularly useful in this regard, particularly when the solid-support matrix is a contraceptive device, such as a condom, a diaphragmn, a cervical cap, a vaginal ring, or a sponge.

The present invention also provides antibodies directed to the proteins of the present invention. The availability of antibodies to any given protein is highly advantageous, as it provides the basis for a wide variety of qualitative and quantitative analytical methods, separation and purification methods, and other useful applications directed to the subject proteins. Accordingly, given the present disclosure and the proteins of the present invention, it will be readily apparent to one skilled in the art that antibodies, in particular antibodies specifically binding to a protein of the present invention, can be prepared using well -established methodologies such as the methodologies described in detail by Harlow and Lane, in Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, pp. 1-725). Such antibodies can comprise both polyclonal and monoclonal. antibodies. Furthermore, such antibodies can be obtained and employed either in solution-phase or coupled to a desired solid-phase matrix, such as magnetic beads or a flow through matrix. Having in hand such antibodies as provided by the present invention, one skilled in the art will further appreciate that such antibodies;, in conjunction with well-established procedures such as described by Harlow and Lane (1988, sup~ra) comprise useful methods for the detection, quantification, or purification of a cyanovirin, conjugate thereof, or host cell transformed to produce a cyanovirin or conjugate thereof. Example 12 further illustrates an antibody specifically binding a cyanovirin.

Matrix-anchored anti-cyanovirin antibodies also can be used in a method to remove virus in a sample. Preferably, the antibodV binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2. Preferably, the matrix is a solid support matrix, such as a magnetic bead or a flow-through matrix. If the solid support matrix to which the anti-cyanovirin antibody is attached comprises magnetic beads, removal of the antibody-cyanovirin-virus complex can be readily accomplished using a magnet.

In view of the above, the present invention provides a method of removing virus from a sample. The method comprises contacting the sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, wherein the antiviral protein or the antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2, and (ii) the at least nine contiguous amino acids bind to the virus, and contacting the sample with an anti-cyanovirin antibody attached to a solid support matrix, whereupon the anti-cyanovirin antibody binds to the antiviral peptide, antiviral protein, antiviral peptide conjugate or antiviral protein conjugate to which is bound the virus, and separating the solid support matrix from the sample, whereupon the virus is removed from the sample. Preferably, the antiviral protein comprises SEQ ID NO: 2. Desirably, the virus that is removed is infectious, such as HIV. The sample can be blood, a component of blood, sperm, cells, tissue or an organ.

The antibody for use in the aforementioned method is an antibody that binds to a protein or a peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2, and, which protein or peptide can bind to and inactivate a virus.

The antibody can be coupled to the solid support matrix using similar methods and with similar considerations as described above for attaching a cyanovirin to a solid support matrix. For example, coupling methods and molecules employed to attach an anti-cyanovirin antibody to a solid support matrix, such as magnetic beads or a flow-through matrix, can employ biotin/streptavidin coupling or coupling through molecules, such as polyethylene glycol, albumin or dextran. For instance, essentially the same procedure as described in Example 7 for attaching a cyanovirin to a solid support matrix comprising magnetic beads can be used to attach an anti-cyanovirin antibody to magnetic beads. Also analogously, it can be shown that, after such coupling, the matrix-anchored anti-cyanovirin antibody retains its ability to bind to a protein or a peptide comprising at least nine contiguous amino acids of SEQ ID NO:2, which protein or peptide can bind to and inactivate a virus.

In view of the above, other embodiments of the present invention include: A. A method of removing virus from a sample, which method comprises: contacting said sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, wherein said antiviral protein or said 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, (iii) said at least nine contiguous amino acids bind to said virus, and (iv) said antiviral protein or antiviral peptide is biotinylated and through biotin is attached to streptavidin of streptavidin-coated magnetic beads, and separating said sample and said composition by contact with a magnet, whereupon virus is removed from said sample.

B. The method of A, wherein said antiviral protein comprises SEQ ID NO:2.

C. The method of claim A or B, wherein said sample is blood, a component of blood, sperm, cells, tissue or an organ.

D. 'The method of claim A or B, wherein said sample is a vaccine formulation a nd the virus that is removed is infectious.

E. The method of wherein said virus is IHV.

F. The method of claim A or B, wherein said virus is lilY.

G. The method of F, wherein said M-V is infectious.

H. A composition comprising streptavidin-coated magnetic beads to which is bound biotinylated, isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, in which said antiviral peptide or antiviral protein comprises at least nine contiguous amino acids of SEQ ID NO:2 that can bind to a virus.

I. The composition of H, wherein said antiviral protein comprises SEQ ID NO:2.

J. The composition of H or 1, wherein said at least nine contiguous amino acids bind gpl120 of HIV.

K_ The composition of J, wherein said HIV is infectious.

L. A composition comprising a naturally-occurring, non-infectious virus, wherein said composition is produced by: contacting a sample comprising infectious and non-infectious virus with an antiviral composition comprising an isolated and purified ntiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, wherein said antiviral protein or said antiviral peptide comprises at least nine 48 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 infectious virus, and separating said sample and said antiviral composition, whereupon infectious virus is removed from said sample.

M. The composition of L, wherein said solid support matrix is streptavidin-coated magnetic beads, said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate is biotinylated and through biotin is attached to streptavidin of the streptavidin-coated magnetic beads, and said separating is by contact with a magnet- N. A composition comprising a naturally-occurring, non-infectious virus, wherein said composition is produced by: contacting a sample comprising infectious and non-infectious virus with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, wherein said antiviral protein or said antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2, and (ii) said at least nine contiguous amino acids bind to said infectious virus, contacting said sample with an anti-cyanovirin antibody, wherein (i) said anti-cyanovirin antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 and (ii) said antibody is attached to a solid-support matrix, whereupon said anticyanovirin antibody binds to said antiviral peptide, antiviral protein, antiviral peptide conjugate or antiviral protein conjugate to which is bound said infectious virus, and separating said matrix-anchored anti-cyanovirin antibody and said sample, whereupon infectious virus is removed from said sample.

0. The composition of N, wherein said solid support matrix is a flowthrough matrix or a magnetic bead, in which case said separating is by contact with a magnet.

P. The composition of any of L-O, wherein said antiviral protein comprises SEQ ID NO: 2.

Q The compbsition of any of L-O, further comprising an immunoadjuvant.

R. The composition of any of L-O, wherein said virus is an immunodeficiency virus.

S. The composition of R, wherein said immnunodeficiency virus is a human immunodeficiency virus.

T. The composition of S, wherein said human immunodeficiency virus is HlV-1 orIHV-2.

U. A composition comprising naturally occurring, non-infectious human immunodeficiency virus comprising gpl2O.

V. A method of inducing an immune response to a virus in an animal, which method comprises administering to said animal an immune responseinducing amount of a composition of any of L-U.

The present inventive cyanovirins, conjugates, host cells, antibodies, compositions and methods are further described in the context of the following examples. These examples serve to illustrate further the ptresent invention and are not intended to limit the scope of the invention.

EXAMPLES

Example 1 This example shows details of anti-HIV bioassay-guided isolation and elucidation of pure cyanovirin from aqueous extracts of the cultured cyanobacterium, Nostoc ellipsosporum.

The method described in Weislow et al. (1989, supra) was used to monitor and direct the isolation and purification process. Cyanobacterial culture conditions, media and classification were as described previously (Patterson, J.

Phycol. 27, 530-536, 1991). Briefly, the cellular mass from a unialgal strain of Nostoc ellipsosporum (culture Q68D 170) was harvested by filtration, freeze-dried and extracted with MeOH-CH 2

CI

2 followed by H20. Bioassay indicated that only the H 2 0 extract contained HIV-inhibitory activity. A solution of the aqueous extract (30 mg/ml) was treated by addition of an equal volume of ethanol (EtOH). The resulting 1:1 H 2 0-EtOH solution was kept at -20°C for 15 hrs.

Then, the solution was centrifuged to remove precipitated materials (presumably, high molecular weight biopolymers). The resulting HIV-inhibitory supernatant was evaporated, then fractionated by reverse-phase vacuum-liquid chromatography (Coll et al., J. Nat. Prod. 49, 934-936, 1986; and Pelletier et al., JI Nat. Prod. 49, 892-900, 1986) on wide-pore C 4 packing (300, BakerBond WP-

C

4 and eluted with increasing concentrations of methanol (MeOH) in H 2 0. Anti- HIV activity was concentrated in the material eluted with MeOH-H 2 0 SDS- PAGE analysis of this fraction showed one main protein band, with a relative molecular mass (Mr) of approximately 10 kDa. Final purification was achieved by repeated reverse-phase HPLC on 1.9 x 15 cm pBondapak C 18 (Waters Associates) columns eluted with a gradient of increasing concentration of acetonitrile in H 2 0.

The mobile phase contained 0.05% TFA, pH=2. Eluted proteins and peptides were detected by UV absorption at 206, 280 and 294 nm with a rapid 51 spectral detector (Pharmacia LKB model 2140). Individual fractions were collected, pooled based on the UV chromatogram, and lyophilized. Pooled HPLC fractions were subjected to SDS-PAGE under reducing conditions (Laemmli, Nature 227, 680-685, 1970), conventional amino acid analysis, and testing for anti-HIV activity.

Figure IA is a graph of OD 206 nm versus time (min), which shows the pBondapak Cig HPLC chromatogram of nonreduced cyanovirin eluted with a linear CH 3

CN/H

2 0 gradient (buffered with 0.05% TFA) from 28-38% CH 3

CN.

Figure 1C is a graph of OD 206 nm versus time (min), which shows the chromatogram of cyanovirin that was first reduced with mercaptoethanol and then separated under identical HPLC conditions. HPLC fractions from the two runs were collected as indicated. 10% aliquots of each fraction were lyophilized, made up in 100 pl 3:1 H20/DMSO and assessed for anti-HIV activity in the XTT assay.

Figure IB is a bar graph of maximum dilution for 50% protection versus HPLC fraction, which illustrates the maximum dilution of each fraction that provided protection from the cytopathic effects of HIV infection for the nonreduced cyanovirin HPLC fractions. Corresponding anti-HIV results for the HPLC fractions from reduced cyanovirin are shown in Figure ID, which is a bar graph of maximum dilution for 50% protection versus HPLC fraction. 20% aliquots of selected HPLC fractions were analyzed by SDS-PAGE. In the initial HPLC separation, using a linear gradient from 30-50% CH 3 CN, the anti-HIV activity coeluted with the principal UV-absorbing peak at approximately 33% CH 3

CN.

Fractions corresponding to the active peak were pooled and split into two aliquots.

Reinjection of the first aliquot under similar HPLC conditions, but with a linear gradient from 28-38% CH3CN, resolved the active material into two closely eluting peaks at 33.4 and 34.0% CH 3 CN. The anti-HIV activity profile of the fractions collected during this HPLC run (as shown in Figure IB) corresponded with the two UV peaks (as shown in Figure 1 SDS-PAGE of fractions collected under the individual peaks showed only a single protein band.

52 The second aliquot from the original HPLC separation was reduced with mercaptoethanol prior to reinjection on the HPLC. Using an identical 28-38% gradient, the reduced material gave one principal peak (as shown in Figure 1C) that eluted later in the run with 36.8% CH 3 CN. Only a trace of anti-HIV activity was detected in the HPLC fractions from the reduced material (as shown in Figure ID) The two closely eluting HPLC peaks of the nonreduced material (Figure 1A) gave only one identical band on SDS-PAGE (run under reducing conditions), and reduction with mercaptoethanol resulted in an HPLC peak with a longer retention time than either of the nonreduced peaks. This indicated that disulfides were present in the native protein. Amino acid analysis of the two active peaks showed they had virtually identical compositions. It is possible that the two HPLC peaks resulted from cis/trans isomerism about a proline residue or from microheterogeneity in the protein sample that was not detected in either the amino acid analysis or during sequencing. The material collected as the two HIVinhibitory peaks was combined for further analyses and was given the name cyanovirin-N.

Example 2 This example illustrates synthesis of cyanovirin genes.

The chemically deduced amino acid sequence of cyanovirin-N was backtranslated to obtain a DNA coding sequence. In order to facilitate initial production and purification of recombinant cyanovirin-N, a commercial expression vector (pFLAG-1, from International Biotechnologies, Inc., New Haven, CT), for which reagents were available for affinity purification and detection, was selected. Appropriate restriction sites for ligation to pFLAG-1, and a stop codon, were included in the DNA sequence. Figure 2 is an example of a DNA sequence encoding a synthetic cyanovirin gene. This DNA sequence design couples the cyanovirin-N coding region to codons for a "FLAG" octapeptide at the N-terminal end of cyanovirin, providing for production of a FLAG-cyanovirin fusion protein.

A flowchart for synthesis of this DNA sequence is shown in Fig. 11. The DNA sequence was synthesized as 13 overlapping, complementary oligonucleotides and assembled to form the double-stranded codingsequence. Oligonucleotide elements of the synthetic DNA coding sequence were synthesized using a dual-column nucleic acid synthesizer (Model 392, Applied Biosystems Inc., Foster City, CA). Completed oligonucleotides were cleaved from-the columns and deprotected by incubation overnight at 56 0 C in concentrated ammonium hydroxide. Prior to treatment with T4 polynucleotide kinase, 33-66 mers were drop-dialyzed against distilled water. The 13 oligonucleotide preparations were individually purified by HPLC, and 10 nmole quantities of each were ligated with T4 DNA ligase into a 327 bp double-stranded DNA sequence. DNA was recovered and purified from the reaction buffer by phenol:chloroform extraction, ethanol precipitation, and further washing with ethanol. Individual oligonucleotide preparations were pooled and boiled for min to ensure denaturation. The temperature of the mixture was then reduced to 700 C for annealing of the complementary strands. After 20 min, the tube was cooled on ice and 2,000 units ofT4 DNA ligase were added together with additional ligase buffer. Ligation was performed overnight at 160 C. DNA was recovered and purified from the ligation reaction mixture by phenol:chloroform extraction and ethanol precipitation and washing.

The purified, double-stranded synthetic DNA was then used as a template in a polymerase chain reaction (PCR). One gl of the DNA solution obtained after purification of the ligation reaction mixture was used as a template. Thermal cycling was performed using a Perkin-Elmer instrument. "Vent" thermostable DNA polymerase, restriction enzymes, T4 DNA ligase and polynucleotide kinase were obtained from New England Biolabs, Beverly, MA. Vent polymerase was selected for this application because of its claimed superiority in fidelity compared to the usual Taq enzyme. The PCR reaction product was run on a 2% agarose gel in TBE buffer. The 327 bp construct was then cut from the gel and purified by electroelution. Because it was found to be relatively resistant to digestion with Hind III and Xho I restriction enzymes, it was initially cloned using the pCR- Script system (Stratagene). Digestion of a plasmid preparation from one of these clones yielded the coding sequence, which was then ligated into the multicloning site of the pFLAG-1 vector.

E. coli were transformed with the pFLAG-construct and recombinant clones were identified by analysis of restriction digests of plasmid DNA.

Sequence analysis of one of these selected clones indicated that four bases deviated from the intended coding sequence. This included deletion of three bases coding for one of four cysteine residues contained in the protein and an alteration of the third base in the preceding codon (indicated by the boxes in Figure In order to correct these "mutations," which presumably arose during the PCR amplification of the synthetic template, a double-stranded "patch" was synthesized, which could be ligated into restriction sites flanking the mutations (these Bst XI and Espl sites are also indicated in Figure The patch was applied and the repair was confirmed by DNA sequence analysis.

For preparation of a DNA sequence coding for native cyanovirin, the aforementioned FLAG-cyanovirin construct was subjected to site-directed mutagenesis to eliminate the codons for the FLAG octapeptide and, at the same time, to eliminate a unique Hind III restriction site. This procedure is illustrated in Figure 3, which 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. A mutagenic oligonucleotide primer was synthesized, which included portions of the codons for the Omp secretory peptide and cyanovirin, but lacking the codons for the FLAG peptide. Annealing of this mutagenic primer, with creation of a DNA hairpin in the template strand, and extension by DNA polymerase resulted in generation of new plasmid DNA lacking both the FLAG codon sequence and the Hind III site (refer to Figure 2 for details). Digestion of plasmid DNA with Hind III resulted in linearization of "wild-type" strands but not "mutant" strands. Since transformation of E. coli occurs more efficiently with circular DNA, clones could be readily selected which had the revised coding sequence which specified production of native cyanovirin-N directly behind the Omp secretory peptide. DNA sequencing verified the presence of the intended sequence. Site-directed mutagenesis reactions were carried out using materials (polymerase, buffers, etc.) obtained from Pharmacia Biotech, Inc., Piscataway,

NJ.

Example 3 This example illustrates expression of synthetic cyanovirin genes.

E. coli (strain DH5a) were transformed (by electroporation) with the pFLAG-1 vector containing the coding sequence for the FLAG-cyanovirin-N fusion protein (see Figure 2 for details of the DNA sequence). Selected clones were seeded into small-scale shake flasks containing (LB) growth medium with 100 pg/ml ampicillin and expanded by incubation at 37* C. Larger-scale Erlenmeyer flasks (0.5-3.0 liters) were then seeded and allowed to grow to a density of 0.5-0.7 OD 60 0 units. Expression of the FLAG-cyanovirin-N fusion protein was then induced by adding IPTG to a final concentration of 1.7 mM and continuing incubation at 30° C for 3-6 hrs. For harvesting ofperiplasmic proteins, bacteria were pelleted, washed, and then osmotically shocked by treatment with sucrose, followed by resuspension in distilled water. Periplasmic proteins were obtained by sedimenting the bacteria and then filtering the aqueous supematant through Whatman paper. Crude periplasmic extracts showed both anti-HIV activity and presence of a FLAG-cyanovirin-N fusion protein by Western or spotblotting. The construct for native cyanovirin-N described in Example 2 was used to transform bacteria in the same manner as described above for the FLAGcyanovirin-N fusion protein. Cloning, expansion, induction with IPTG, and harvesting were performed similarly. Crude periplasmic extracts showed strong anti-HIV activity on bioassay. A flowchart of the expression of synthetic cyanovirin genes is shown in Fig. 12.

Example 4 This example illustrates purification of recombinant cyanovirin proteins.

Using an affinity column based on an anti-FLAG monoclonal antibody (International Biotechnologies, Inc., New Haven, CT), FLAG-cyanovirin-N fusion protein could be purified as follows.

The respective periplasmic extract, prepared as described in Example 3, was loaded onto 2-20 ml gravity columns containing affinity matrix and washed extensively with PBS containing CA++ to remove contaminating proteins. Since the binding of the FLAG peptide to the antibody is Ca++-dependent, fusion protein could be eluted by passage of EDTA through the column. Column fractions and wash volumes were monitored by spot-blot analysis using the same anti-FLAG antibody. Fractions containing fusion protein were then pooled, dialyzed extensively against distilled water, and lyophilized.

For purification of recombinant native cyanovirin-N, the corresponding periplasmic extract from Example 3 was subjected to step-gradient

C

4 reversephase, vacuum-liquid chromatography to give three fractions: eluted with 100% H20, eluted with MeOH-H 2 0 and eluted with 100% MeOH.

The anti-HIV activity was concentrated in fraction Purification of the recombinant cyanovirin-N was performed by HPLC on a 1.9x15 cm gBondapak (Waters Associates) C g column eluted with a gradient of increasing concentration of CH 3 CN in H20 (0.05% TFA, v/v in the mobile phase). A chromatogram of the final HPLC purification on a lxl0 cm (Cohensive Technologies, Inc.) C 4 column monitored at 280 nm is shown in Figure 4, which is typical HPLC chromatogram during the purification of a recombinant native cyanovirin. Gradient elution, ml/min, from 100% H20 to H20-CH 3 CN was carried out over 23 min with 0.05% TFA in the mobile phase.

Example This example shows anti-HIV activities of natural and recombinant cyanovirin-N and FLAG-cyanovirin-N.

Pure proteins were initially evaluated for antiviral activity using an XTTtetrazolium anti-HIV assay described previously (Boyd, in AIDS. Etiologyv Diagnosis. Treatment and Prevention, 1988, supra; Gustafson et al., J. Med. Chem.

1978-1986, 1992; Weislow, 1989, sura; and Gulakowski, 1991, upra). The CEM-SS human lymphocytic target cell line used in all assays was maintained in RPMI 1650 medium (Gibco, Grand Island, NY), without phenol red, and was supplemented with 5% fetal bovine serum, 2 mM L-glutamine, and 50 tg/ml gentamicin (complete medium).

Exponentially growing cells were pelleted and resuspended at a concentration of2.0xl 0 5 cells/ml in complete medium. The Haitian variant of HIV, HTLV-IIIRF (3.54x106 SFU/ml), was used throughout. Frozen virus stock solutions were thawed immediately before use and resuspended in complete medium to yield 1.2x12 5 SFU/ml. The appropriate amounts of the pure proteins for anti-HIV evaluations were dissolved in H20-DMSO then diluted in complete medium to the desired initial concentration. All serial drug dilutions, reagent additions, and plate-to-plate transfers were carried out with an automated Biomek 1000 Workstation (Beckman Instruments, Palo Alto, CA).

Figures SA-5C are graphs of% control versus concentration which illustrate antiviral activities of native cyanovirin from Nostoc ellipsosporum recombinant native and recombinant FLAG-fusion cyanovirins. The graphs show the effects of a range of concentrations of the respective cyanovirins upon CEM-SS cells infected with HIV-1 as determined after 6 days in culture.

Data points represent the percent of the respective uninfected, nondrug-treated control values. All three cyanovirins showed potent anti-HIV activity, with an

EC

5 0 in the low nanomolar range and no significant eviderice of direct cytotoxicity to the host cells at the highest tested concentrations (up to 1.2 pM).

As an example of a further demonstration of the anti-HIV activity of pure cyanovirin-N, a battery of interrelated anti-HIV assays was performed in individual wells of 96-well microtiter plates, using methods described in detail elsewhere (Gulakowski, 1991, supra). Briefly, the procedure was as follows.

58 Cyanovirin solutions were serially diluted in complete medium and added to 96well test plates. Uninfected CEM-SS cells were plated at a density of lxi 04 cells in 50 pl of complete medium. Diluted HIV-1 was then added to appropriate wells in a volume of 50 jil to yield a multiplicity of infection of 0.6. Appropriate cell, virus, and drug controls were incorporated in each experiment. The final volume in each microtiter well was 200 pl. Quadruplicate wells were used for virusinfected cells. Plates were incubated at 37 C in an atmosphere containing 5% CO 2 for 4, 5, or 6 days.

Subsequently, aliquots of cell-free supernatant were removed from each well using the Biomek, and analyzed for reverse transcriptase activity, p24 antigen production, and synthesis of infectious virions as described (Gulakowski, 1991, supr). Cellular growth or viability then was estimated on the remaining contents of each well using the XTT (Weislow et al., 1989, sua), BCECF (Rink et al., J.

Cell Biol. 95, 189-196, 1982), and DAPI (McCaffrey et al., In Vitro Cell Develop.

Biol. 24, 247-252, 1988) assays as described (Gulakowski et al., 1991, To facilitate graphical displays and comparisons of data, the individual experimental assay results (of at least quadruplicate determinations of each) were averaged, and the mean values were used to calculate percentages in reference to the appropriate controls. Standard errors of the mean values used in these calculations typically averaged less than 10% of the respective mean values.

Figures 6A-6D are graphs of% control versus concentration (run), which illustrate anti-IV activity of a cyanovirin in a multiparameter assay format.

Graphs 6A, 6B, and 6C show the effects of a range of concentrations of cyanovirin upon uninfected CEM-SS cells and upon CEM-SS cells infected with HIV- 1 as determined after 6 days in culture. Graph 6A depicts the relative numbers of viable CEM-SS cells, as assessed by the BCECF assay. Graph 6B depicts the relative DNA contents of the respective cultures. Graph 6C depicts the relative numbers of viable CEM-SS cells, as assessed by the XTT assay. Graph 6D shows the effects of a range of concentrations of cyanovirin upon indices of infectious virus or viral replication as determined after 4 days in culture. These indices include viral reverse transcriptase viral core protein p 24 and syncytiumforming units In graphs 6A, 6B, and 6C, the data are represented as the percent of the uninfected, nondrug-treated control values. In graph 6D the data are represented as the percent of the infected, nondrug-treated control values.

As illustrated in Figure 6, cyanovirin-N was capable of complete inhibition of the eytopathic effects of HIV-1 upon CEM-SS human lymphoblastoid target cells in vitro; direct cytotoxicity of the protein upon the target cells was not observed at the highest tested concentrations. Cyanovirin-N also strikingly inhibited the production of RT, p24, and SFU in HIV-1-infected CEM-SS cells within these same inhibitory effective concentrations, indicating that the protein halted viral replication.

The anti-HIV activity of the cyanovirins is extremely resilient to harsh environmental challenges. For example, unbuffered cyanovirin-N solutions withstood repeated freeze-thaw cycles or dissolution in organic solvents (up to 100% DMSO, MeOH, or CH 3 CN) with no loss of activity. Cyanovirin-N tolerated detergent SDS), high salt (6 M guanidine HCI) and heat treatment (boiling, 10 min in H20) with no significant loss of HV inhibitory activity.

Reduction of the disulfides with mercaptoethanol, followed immediately by C 18 HPLC purification, drastically reduced the cytoprotective activity of cyanovirin-N.

However, solutions of reduced cyanovirin-N regained anti-HIV inhibitory activity during prolonged storage. When cyanovirin-N was reduced (P-mercaptoethanol, 6 M guanidine HCI, pH 8.0) but not put through C18 HPLC, and, instead, simply desalted, reconstituted and assayed, it retained virtually full activity.

Example 6 This example illustrates that the HIV viral envelope gpl20 is a principal molecular target of cyanovirin-N.

Initial experiments, employing the XTT-tetrazolium assay (Weislow et al., 1989, supra), revealed that host cells preincubated with cyanovirin (10 nM, I hr), then centrifuged free of cyanovirin-N, retained normal susceptibility to HIV infection; in contrast, the infectivity of concentrated virus similarly pretreated, then diluted to yield non-inhibitory concentrations of cyanovirin-N, was essentially abolished. This indicated that cyanovirin-N was acting directly upon the virus itself, acting as a direct "virucidal" agent to prevent viral infectivity even before it could enter the host cells. This was further confirmed in time-ofaddition experiments, likewise employing the XTT-tetrazolium assay (Weislow, 1989, supra), which showed that, to afford maximum antiviral activity, cyanovirin- N had to be added to cells before or as soon as possible after addition of virus as shown in Figure 7, which is a graph of uninfected control versus time of addition (hrs), which shows results of time-of-addition studies of a cyanovirin, showing anti-HIV activity in CEM-SS cells infected with HIV-lRF. Introduction of cyanovirin or ddC (10 nM and 5 pM concentrations, respectively) was Sdelayed by various times after initial incubation, followed by 6 days incubation, then assay of cellular viability (linegraphs) and RT (open bars, inset). Points represent averages of at least triplicate determinations. In marked contrast to the reverse transcriptase inhibitor ddC, delay of addition of cyanovirin-N by only 3 hrs resulted in little or no antiviral activity (Figure The aforementioned results suggested that cyanovirin-N inhibited HIV-infectivity by interruption of the initial interaction of the virus with the cell; this would, therefore, likely involve a direct interaction ofcyanovirin-N with the viral gpl20. This was confirmed by ultrafiltration experiments and dot-blot assays.

Ultrafiltration experiments were performed to determine if soluble gp120 and cyanovirin-N could bind directly, as assessed by inhibition of passage of cyanovirin-N through a 50 kDa-cutoffultrafilter. Solutions of cyanovirin (30 pg) in PBS were treated with various concentrations of gp 20 for 1 hr at 370 C, then filtered through a 50 kDa-cutoffcentrifugal ultrafilter (Amicon). After washing 3 times with PBS, filtrates were desalted with 3 kDa ultrafilters; retentates were lyophilized, reconstituted in 100 pl H 2 0 and assayed for anti-HIV activity.

Figure 8 is a graph of OD (450 nm) versus cyanovirin concentration (g/ml), which illustrates cyanovirin/gp 120 interactions defining gp 120 as a principal molecular target of cyanovirin. Free cyanovirin-N was readily eluted, as evidenced by complete recovery of cyanovirin-N bioactivity in the filtrate. In contrast, filtrates from cyanovirin-N solutions treated with gpl20 revealed a concentration-dependent loss of filtrate bioactivity; moreover, the 50kDa filter retentates were all inactive, indicating that cyanovirin-N and soluble interacted directly to form a complex incapable of binding to gp 120 of intact virus.

There was further evidence of a direct interaction of cyanovirin-N and in a PVDF membrane dot-blot assay. A PVDF membrane was spotted with pg CD4 10 pg aprotinin 10 pg bovine globulin and decreasing amounts of cyanovirin; 6 pg 3 pg 1.5 pg 3 0.75 pg 0.38 pg 0.19 pg [61, 0.09 pg and 0.05 ig then washed with PBST and visualized per manufacturer's recommendations. A dot blot of binding of cyanovirin and a gp 120-HRP conjugate (Invitrogen) showed that cyanovirin-N specifically bound a horseradish peroxidase conjugate of gp 120 (gpl20-HRP) in a concentration-dependent manner.

Example 7.

This example illustrates the preparation and biological activity of a composition of the present invention comprising a functional cyanovirin coupled to a solid support matrix.

Recombinant CV-N (rCV-N) produced in E. coli was compared with biotinylated CV-N (bCV-N) at 0.05 pg or 0.5 pg for in vitro inactivation of 100

TCID

50 of a primary isolate (HIV-1/UG/021/92) spiked into fetal bovine serum.

Aliquots of 104 streptavidin-coated magnetic glass beads were reacted at room temperature with 0.5 pg bCV-N for 60 minutes and washed to remove free bCV- N. The binding of bCV-N to the beads was detected with polyclonal rabbit antibodies against CV-N, using FITC-labeled goat anti-rabbit IgG in flow cytometric analysis of the beads. The bead-bound sessile CV-N (sCV-N) and unbound control beads were tested for antiviral activity against 100 TCID 50 of HIV. Following incubation at 370 C for 90 minutes, the viral supernates were

I

62 cultured for 7 days in PHA-stimulated human PMBC, and the synthesis of P24 antigen was assessed.

At 0.5 Pg, both rCV-N and bCV-N were equally effective in inactivating 100 TCID 5 0 of HIV; however, at 0.05 pg, they were only 40% effective in inactivating 100 TCID 50 The bead-bound sCV-N completely inactivated 100

TCID

5 0 of HIV, though a fraction of non-infectious HIV appeared to remain adherent to sCV-N as accessed by RT-PCR. Thus, matrix-anchored cyanovirin provides a practical and effective means to remove infectious virus from noninfectious virus in a sample.

Example g This example further illustrates the extraordinarily broad range of antiretroviral activity against diverse lab-adapted and clinical strains of human and nonhuman primate immunodeficiency retroviruses. Table 1 below shows the comparative ranges of anti-immunodeficiency virus activities of cyanovirin-N and sCD4 tested against a wide range of virus strains in diverse host cells. Particularly noteworthy is the similar potency of cyanovirin-N against both lab-adapted strains as well as clinical isolates of HIV. This was in sharp contrast to the lack of activity of sCD4 against the clinical isolates.

The EC50 values (Table 1) were determined from concentration-response curves from eight dilutions of the test agents (averages from triplicate wells per concentration); G910-6 is an AZT-resistant strain; A17 is a pyridinone-resistant strain; HIV-1 Ba-L was tested in human peripheral blood macrophage (PBM) cultures by supernatant reverse transcriptase activity; all other assays employed XTT-tetrazolium (Gulakowski et al., 1991, supra). Further details of virus strains, cell lines, clinical isolates, and assay procedures are published (Buckheit et al., AIDS Res. Hum. Retrovir. 10, 1497-1506, 1994; Buckheit et al., Antiviral Res. 43-56, 1994; and references contained therein). In Table 1, N.D.=not determined 63 TABLE 1. Comparative Ranges of Antiviral Activity of CV-N and sCD4 EC50(nM) a Virus Target Cells Cyanovirin-N s CD4 HIV-1 Laboratory Strains RF CEM-SS 0.5 0.8 RF U937 0.5 0.1 IIIB CEM-SS 0.4 1.6 IIIB MT-2 0.4 13 MN MT-2 2.3 N.D.

G-910-6 MT-2 5.8 N.D.

A17 MT-2 0.8 13 HIV-1 Promonocytotropic Isolates 214 CEM-SS 0.4

N.D.

SKI CEM-SS 4.8 N.D.

HIV-1 Lymphotropic Isolates 205 CEM-SS 0.8 N.D.

GI CEM-SS 0.9 N.D.

HIV-1 Clinical Isolates WEJO PBL 6.7 >100 VIHU PBL 5.5 >100 BAKI PBL 1.5 >100 WOME PBL 4.3 >100 HIV-2 ROD CEM-SS 7.6 >200 MS CEM-SS 2.3 N.D.

SIV

DeltaB670 174xCEM 11 Example 9 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 cells. More specifically, this example illustrates construction and expression of a conjugate DNA coding sequence for a cyanovirin/Pseudomonas-exotoxin which selectively kills viral host cells.

A DNA sequence (SEQ ID NO:3) coding for FLAG-cyanovirin-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 position of the FLAG-cyanovirin-N coding sequence using standard recombinant DNA procedures. This construct is illustrated schematically in Fig. 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). Fig. 10 illustrates the results of this testing. As anticipated, the infected H9/IIIB cells expressing cell-surface were dramatically more sensitive to the toxic effects of PPE than were the uninfected H9 or CEM-SS cells. The ICo 5 values determined from the concentration-effect curves were 0.014 nM for H9/IIIB compared to 0.48 and 0.42 nM for H9 and CEM-SS, respectively.

Example 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-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 Western blot analysis. 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 11, which follows, indicated that this increased molecular weight was due to post-translational modification (N-linked oligosaccharides) of the FLAG-cyanovirin-N.

Example 11 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 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 anticyanovirin-N polyclonal antibodies (see, Example 12).

Similar to the observations with the mammalian forms described in Example 10, the elevated apparent molecular weight of the yeast-derived product on PAGE and Western blot 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. This treatment reduced the apparent molecular weight of the product to that equivalent to native recombinant 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 consistent with that of native recombinant FLAGcyanovirin-N.

Example 12 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 pg of cyanovirin-N was dissolved in 100 pl 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 pg of cyanovirin-N per rabbit was dissolved in 1000 gl of a 1:1 suspension of PBS and Freunds incomplete adjuvant and administered by intraperitoneal injection; on days 42, 70, 98 and 122, 10 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 western-blot analysis using a 1:1000 to 1:5000 dilution of the rabbit IgG fractions.

The antibody prepared according to the aforementioned procedure specifically bound to a protein of the present invention. SDS-PAGE of a wholecell lysate, from E. coli strain DH5a engineered to produce cyanovirin-N, was carried 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.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.

One skilled in the art additionally will appreciate that, likewise by wellestablished, routine procedures see Harlow and Lane, 1988, supra), 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.

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 compounds and methods may 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.

In the specification and claims the term 'comprising' shall be understood to have a broad meaning similar to the term 'including' and will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term 'comprising' such as 'comprise' and 'comprises'.

Claims (11)

1. A method of inhibiting prophylactically or therapeutically a viral infection of a host, which method comprises administering to said host an antiviral effective amount of a conjugate comprising an isolated and purified antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2, wherein said at least nine contiguous amino acids bind to a virus, chemically coupled or fused to an anti-cyanovirin antibody, whereupon administration of said antiviral effective amount of said conjugate, said viral inhibition is inhibited.

2. The method of claim 1, wherein said antiviral protein comprises SEQ ID NO: 2.

3. The method of claim 1 or claim 2, wherein said antiviral protein or antiviral peptide is further coupled to at least one effector component, which can be the same or different, selected from the group consisting of an immunological reagent and a toxin.

4. A method of inhibiting prophylactically or therapeutically a viral infection of a host, which method comprises topically administering to a host an antiviral effective amount of a composition comprising an isolated and purified antiviral agent selected from the group consisting of an antiviral protein, an antiviral peptide, an antiviral protein conjugate, and an antiviral peptide conjugate, wherein said antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2, (ii) said at least nine contiguous amino acids of SEQ ID NO: 2 have antiviral activity, and (iii) said antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate is attached to a solid support matrix, whereupon administration of said antiviral effective amount of said composition, said viral infection is inhibited.

The method of claim 4, wherein said solid support matrix comprises a contraceptive device.

6. The method of claim 5, wherein said contraceptive device is a condom, a diaphragm, a cervical cap, a vaginal ring or a sponge.

7. A matrix-anchored anti-cyanoviran antibody, wherein said antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 and wherein said antibody is attached to a magnetic bead or a flow-through matrix.

8. A composition comprising the matrix-anchored anti-cyanovirin antibody of claim 7.

9. A matrix-anchored anti-cyanoviran antibody, wherein said antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 and wherein said antibody is attached to a solid support matrix via streptavidin/biotin coupling or coupling through polyethylene glycol, albumin or dextran.

The matrix-anchored anti-cyanoviran antibody of claim 9, wherein said solid support matrix is a magnetic bead or a flow-through matrix.

11. A composition comprising the matrix-anchored anti-cyanoviran antibody according to claim 9 or claim DATED THIS THIRD DAY OF OCTOBER 2003 THE UNITED STATES OF AMERICA REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH HUMAN SERVICES BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS