WO2012109314A2 - Potent hiv inhibitors targeting ccr5 and gp41 - Google Patents

Abstract

The present disclosure provides chimeric polypeptides comprising a CCR5-binding protein and a gp41-binding protein and methods of using these chimeric polypeptides or related compositions to prevent or treat HIV infection.

Description

POTENT HIV INHIBITORS TARGETING CCR5 AND GP41

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of United States Provisional Patent Application No. 61/441 ,190, filed February 9, 201 1 the contents of which is incorporated by reference into the present application in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. AI079777 awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF INVENTION

[0003] The present disclosure generally relates to compositions and methods for preventing or treating HIV infections.

BACKGROUND OF THE DISCLOSURE

[0004] Approximately 33 million people are currently infected with HIV, and millions more are infected each year. There is currently no vaccine, and treatments usually involve inhibiting viral activity post-infection, by inhibiting the HIV protease or reverse transcriptase. More recently, therapies that target other parts of the viral life cycle have been approved, including an HIV integrase inhibitor.

[0005] One of the most promising areas in the fight against HIV/AIDS has been the development of entry inhibitors, which generally bind to either the viral surface or the human cell surface to stop HIV before it can enter a cell. FIG. 1 shows a diagram of HIV entry: the HIV surface protein gp120 first makes contact with human cell surface protein CD4, which causes a conformational rearrangement in gp120, allowing the protein to then bind its co-receptor on the cell surface (either the chemokine receptor CCR5 or CXCR4). During this process, the HIV protein gp41 is exposed and its fusion peptide enters the cell surface. Toward the end of the infection process, the C-terminal helical trimer folds over to contact the N-terminal trimer of gp41 , forming a 6-helix bundle that likely pulls the membranes of the two entities in closer proximity to assist fusion of the virus to the cell. Recently, it has been reported that some of these events may occur in the endosome.

[0006] Inhibition of HIV entry can be achieved by blocking one or more of the events that lead to infection. Proteins, particularly lectins that bind to gp120, have been shown to be effective inhibitors, as are peptides that bind to gp41 to stop 6- helix bundle formation. In particular, so-called C-peptides that are derived from the C-terminus of gp41 effectively bind to the N-terminus of gp41 to inhibit infection. One of these peptides, T-20, has been approved for clinical use. Another strategy to inhibit HIV infection involves binding the co-receptor on the human cell surface, particularly CCR5. Natural ligands for CCR5, namely chemokines MIP-1 b, MIP-1 a and RANTES, were found to be able to block HIV infection. It was later shown that variants of these chemokines, particularly RANTES, could lead to even more highly potent inhibition.

[0007] Despite the effectiveness of entry inhibition strategies, many of them have serious drawbacks. While the recent RANTES variants are potent, they work by binding CCR5, so are only effective against R5 tropic virus, not against X4 tropic virus. While the C-peptides are effective against most strains of HIV, their potency is limited to nanomolar levels, and the virus can evolve so that the peptides bind less well.

[0008] Considering the stepwise nature of the HIV entry process, there are certain time windows in which multiple targets are simultaneously susceptible to inhibition. Binding of co-receptor inhibitors and fusion peptides to their targets can both be achieved after the exposure of gp41 and before gp120 interacts with its co-receptor. Evidence suggests that co-receptor binding is a key factor in the kinetic properties of fusion, and that lowered co-receptor density or weakened co- receptor-gp120 binding slows down gp41 mediated cell fusion and prolongs the time window of the intermediate states of gp41 for fusion peptides to bind. SUMMARY OF THE DISCLOSURE

[0009] It is discovered herein that protein inhibitors that target both CCR5 and gp41 are potent inhibitors of the HIV fusion process and thus are effective in inhibiting HIV infection. A non-limiting example of such a protein inhibitor is a chimeric polypeptide composed of a CCR5- binding protein, such as RANTES variants 5P12-RANTES and 5P14-RANTES, and a gp41 -binding protein, such as the C-peptide, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed high anti-viral potency at low-picomolar levels, and are active against both R5 and X4 tropic virus. This inhibition was was up to 100 fold better than the RANTES variant alone or in combination with unlinked C37. Furthermore, such chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides.

[0010] Thus, in one aspect, the disclosure provides an isolated chimeric polypeptide comprising, or alternatively consisting essentially of, or alternatively consisting of, a first portion comprising a CCR5-binding protein and a second portion comprising a gp41 -binding protein.

[0011] In one aspect, the chimeric polypeptide does not comprise an lgG1 antibody that specifically recognizes CCR5. In another aspect, the chimeric polypeptide does not comprise an antibody that specifically recognizes CCR5. In yet another aspect, the chimeric polypeptide does not comprise an antibody. In yet still another aspect, the chimeric polypeptide does not comprise an antibody or an antibody fragment. In some aspects, the chimeric polypeptide is monomeric or includes a single polypeptide chain. Compositions are also provided, comprising the chimeric polypeptide and a carrier.

[0012] In one aspect, the gp41 -binding protein is one or more of a C-peptide; a N-peptide; C37; C-37ac; C37(Q652L); N-acetylated, C-term amidated C37; N- acetylated, C-term amidated C37(Q652L); C34; C52L; T-2635; T20; N-peptides; N17; N23; N36 or a substantial homologue thereof.

[0013] In one aspect, the CCR5-binding protein is selected from RANTES, P2-RANTES, PSC-RANTES, 5P12-RANTES, 5P14-RANTES, 6P4-RANTES, ΜΙΡ-1 α, ΜΙΡ-1 β, U83A or a substantial homologue of any one thereof. Biological equivalents of P2-RANTES include, for example, 5P12-RANTES, 5P14-RANTES or 6P4-RANTES.

[0014] In another aspect, the chimeric polypeptide further comprises a peptide linker between the first portion and the second portion.

[0015] Also provided is a polynucleotide encoding for any of the above chimeric polypeptides, a DNA construct comprising an expression vector and the polynucleotide, or an isolated host cell transformed with the polynucleotide.

[0016] The disclosure further provides a method for preventing or inhibiting HIV entry into a cell or HIV replication in a cell capable of hosting HIV infection, comprising, or alternatively consisting essentially of, or yet further consisting of, contacting the cell with an effective amount of any of the above chimeric polypeptides or compositions. In one aspect, the chimeric polypeptides or compositions inhibit entry into the cell and in addition or alternatively, they act as fusion inhibitors. In some embodiments, the cell is an animal cell, such as a mammalian cell, e.g., a human cell. In one aspect of the disclosure, the cell is a human cell.

[0017] Further provided is a method for treating a subject in need thereof, comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of any of the above chimeric polypeptides or compositions. The subject can be a subject infected with HIV or a subject at risk of HIV infection. In some embodiments, the subject is an animal, a mammal, or a human. In some embodiments, administration of the chimeric polypeptides or composition is by injection or topical application.

[0018] Another aspect of the disclosure provides a peptide conjugate comprising, or alternatively consisting essentially of, or alternatively consisting of, a carrier covalently or non-covalently linked to an isolated chimeric polypeptide of the disclosure. In some embodiments, the carrier comprises a liposome, or alternatively a micelle, or alternatively a pharmaceutically acceptable polymer, or a carrier, e.g. a pharmaceutically acceptable carrier. [0019] Yet another aspect of the disclosure provides an isolated polynucleotide encoding for an isolated chimeric polypeptide of the disclosure. Also provided is a DNA construct comprising an expression vector and a polynucleotide. In one aspect of the DNA construct, the vector is a plasmid vector, a yeast artificial chromosome, or a viral vector. In one aspect, the vector of the DNA construct comprises a protein tag. Protein tags can be selected from a His-tag, a SUMO-tag, a GST-tag, a myc-tag, or a FLAG-tag provided in expression constructs commercially available from, e.g., Invitrogen, Carlsbad, CA. Compositions comprising, or alternatively consisting essentially of, or yet further consisting of the isolated polynucleotides as described above and host cells as described are further provide by this disclosure.

[0020] The disclosure, in another aspect, provides an antibody that binds an isolated chimeric polypeptide of the disclosure. The antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a derivative or fragment thereof as defined below. In one aspect, the fragment comprises, or alternatively consists essentially of, or yet further consists of the CDR of the antibody. In one aspect, the antibody is detectably labeled or further comprises a detectable label conjugated to it. Also provided is a hybridoma cell line that produces a monoclonal antibody of this disclosure. Compositions comprising or alternatively consisting essentially of or yet further, consisting of one or more of the above embodiments are further provided herein.

[0021] The disclosure, in one aspect, provides a method for preventing or inhibiting HIV entry into a cell, comprising contacting the cell with an effective amount of an isolated chimeric polypeptide or an effective amount of a polynucleotide encoding the chimeric polypeptide of the disclosure. The contacting can be in vitro or in vivo. The cell can be an animal cell, a mammalian cell, or a human cell. In a particular aspect, the cell is a human cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a diagram of the HIV cell entry mechanism: HIV gp120 (light gray ovals) makes contact with the human cell surface receptor CD4 (orange), which causes structural change in gp120, allowing its binding to the co-receptor (dark green) CCR5 or CXCR4 on the cell surface. During this process, the HIV protein gp41 is exposed and its fusion peptide enters the cell surface. At the end of the entry process, the N-terminal trimer of hairpins (dark gray) folds over to contact the C-terminal trimer (gray) which leads to the formation of a 6-helix bundle that likely pulls the two membranes in closer proximity to assist fusion.

[0023] FIGS. 2A-2B include ¹H-¹⁵N 2D HSQC spectra of the chimeric proteins and the parent RANTES variants to verify the structural integrity of the proteins. FIG. 2A shows overlay of 5P12-linker-C37 (gray peaks) with 5P12-RANTES (black peaks); FIG. 2B shows overlay of 5P14-linker-C37 (gray peaks) with 5P14- RANTES (black peaks).

[0024] FIGS. 3A-3F show antiviral activities of the chimeric inhibitors against R5 tropic virus. Chimeric inhibitors showed higher anti-HIV potency than the control groups (RANTES variants alone, C37 alone, or 1 :1 mixture of the RANTES variants and C37). FIGS. 3A and 3B show dell-cell fusion assay using effector cells expressing ADA viral envelopes. FIGS. 3C and 3D show single- cycle viral infection assay (Ba-L strain pseudoviral particles infecting TZM-bl cells). FIGS. 3E and 3F show replication-competent viral assay (Ba-L strain virus infecting TZM-bl cells). Data shown are typical results of single assays done in triplicate. Error bars represent standard deviations of the data.

[0025] FIGS. 4A-4F show antiviral activities of the chimeric inhibitors against X4 tropic virus. The chimeric inhibitors retain the anti-viral activity of C37 in X4 assays: FIG. 4A: Magi-X4 cell (expresses only CXCR4, but not CCR5, on the surface) based cell-cell fusion assays; FIG. 4B. Magi-X4 cell based single-cycle viral infection assays. The anti-viral activity of chimeric inhibitors against X4 virus is greatly enhanced if the cells co-express CCR5 receptors: FIG 4C. TZM-bl cell (expresses both CCR5 and CXCR4 on the surface) based fusion assays; FIG. 4D. TZM-bl based single-cycle viral infection assays. This enhancement of the chimeric protein requires binding to CCR5: When the CCR5 receptors on the cell surface are occupied by pre-incubation with CCR5-binding protein, the chimeric inhibitors showed no enhancement over C37: FIG 4E. TZM-bl cell based fusion assays; FIG. 4F. TZM-bl cell based single-cycle viral infection assays. The cells were pre-incubated with 100 nM 5P12 or 5P14. Data shown are typical results of single assays done in triplicate.

[0026] FIG. 5A-5F show data that reveal the mechanism of action of the chimeric inhibitors. FIG 5A. Mutations on the C37 segment of 5P12-linker-C37 cause reduced or loss of activity against X4-tropic virus, while substitution of 5P12 with a different N-terminus (that of P2-RANTES) has no effect against X4-tropic virus. The X4- tropic antiviral potency was determined on Magi-X4 cells (which express only CXCR4, but not CCR5, on the surface) against HXB2 strain pseudotyped virus particles. FIG. 5C. Mutations on either the RANTES variant segment or the C37 segment of 5P12-linker- C37 cause reduced activity against R5-tropic virus. The R5-tropic antiviral potency was determined using TZM-bl cells (which express both CCR5 and CXCR4 on the surface) against Ba-L strain pseudotyped virus particles. FIG. 5E. When the cells co-express CCR5, mutations on either the RANTES variant segment or the C37 segment of 5P12- linker-C37 cause reduced activity against X4-tropic virus. The X4-tropic antiviral potency was determined on TZM cells (which express both CCR5 and CXCR4 on the surface) against HXB2 strain pseudotyped virus particles. FIG. 5B. Changing the original 10- amino-acid linker to a shorter 3-amino-acid linker or a longer 20-amino-acid linker does not affect the native activity of C37 against X4 tropic virus. The X4-tropic antiviral potency was determined on Magi-X4 cells (which express only CXCR4, but not CCR5, on the surface) against HXB2 strain pseudotyped virus particles. FIGS. 5D, 5F. 5P12- 3AA-C37 shows reduced anti-viral activity in both R5-tropic single-cycle viral assays and TZM-bl cell based X4-tropic single-cycle viral assays, while 5P12-20AA-C37 shows very similar activity to 5P12-linker-C37. The R5-tropic antiviral potency was determined using TZM-bl cells (which express both CCR5 and CXCR4 on the surface) against Ba-L strain pseudotyped virus particles (D). The TZM-bl cell based X4-tropic antiviral potency was determined on TZM cells against HXB2 strain pseudotyped virus particles (F). Data shown are typical results of single assays done in triplicate. Error bars represent standard deviations of the data.

[0027] FIG. 6A-6C illustrate a model of action of the chimeric inhibitors: FIG. 6A. during the process of R5 tropic viral entry, the chimeric inhibitor can bind to the CCR5 receptor and block the co-receptor-gp120 interaction, and at the same time delivers the C37 fusion peptide to the nearby gp41 targets. In this way, the chimeric inhibitors block R5-tropic HIV entry at both steps more effectively. FIG. 6B. during the process of X4-tropic viral entry, only the C37 part of the chimeric inhibitor is active, and the chimeric inhibitor functions exactly as a fusion peptide by binding to the N-terminal trimer-of-hairpins of gp41 . FIG. 6C. When the target cells of the X4-tropic virus co-express both CCR5 and CXCR4 receptors, the chimeric inhibitors can inhibit viral entry more efficiently. Since CCR5 and CXCR4 form hetero-oligomers on the cell surface, the chimeric inhibitors can bind to CCR5 and deliver the C37 peptide to the nearby X4 infection site. By specific delivery of C37 to its target and possibly increasing the local concentration of C37 on the cell surface, the chimeric inhibitors block HIV more efficiently than C37 alone.

[0028] FIG. 7 shows extent of potency enhancement compared to sensitivity to C37. For virus strains that are particularly sensitive to C37 such as Ba-L, 5P12- linker-C37 and 5P14-linker-C37 showed 70 - and 23 - fold potency enhancement over 5P12 and 5P14, respectively. But for virus strains that are less sensitive to C37 such as 6535, 5P12-linker-C37 was only 2.5 fold better than 5P12, while 5P14-linker-C37 showed no enhancement over 5P14. These data indicate that the linked C-peptide is critical for the enhancement of the inhibition of the RANTES variant in the chimera, and suggest that effect of the fusion peptide on the specific viral strain determines the magnitude of the relative potency of the enhancement over the RANTES variant alone.

[0029] FIG. 8 shows CCR5 receptor density comparison by flow cytometry. The CCR5 receptor expression levels on HeLa-TZM-bl cells and Hel_a-P5L cells were compared using flow cytometry. The cells were incubated with FITC conjugated anti-CCR5 antibody (clone 2D7, BD Biosciences), and the fluorescence values were determined using a FACSAria cytometer (BD Biosciences). The results showed that the lower the CCR5 density, the more potent the chimeric inhibitor

[0030] FIG. 9A-9B list the amino acid sequences (SEQ ID NO: 1 and 2) of the chimeric inhibitors as examples.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001 ) Molecular Cloning: A Laboratory Manual, 3^rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991 ) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;

Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5^th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of

Experimental Immunology.

[0032] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1 .0 or 0.1 , as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0033] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a pharmaceutically acceptable carrier" includes a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

[0034] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0035] A "subject" of diagnosis or treatment is a cell or an animal such as a mammal, or a human. Non-human animals subject to diagnosis or treatment are those subject to HIV or similar virus (e.g., Simian Immunodeficiency Virus (SIV)) that include, for example, simians, murine, such as, rats, mice, canine, such as dogs, cats, leporids, such as rabbits, livestock, sport animals, and pets.

[0036] The term "protein", "peptide" and "polypeptide" are used

interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. Single letter and three letter abbreviations of the naturally occurring amino acids are known in the art and "X" is used herein to indicate an unnatural or unidentified amino acid.

[0037] A "chimeric polypeptide", "chimeric protein" or "fusion protein" refers to a protein, peptide or polypeptide created through the joining of two or more amino acid sequences or alternatively created by expression of a joint nucleotide sequence comprising two or more nucleotide sequences which originally code for separate proteins, peptides, polypeptides. Translation of joined nucleotide sequence, also known as a fusion gene, results in a single polypeptide, the "chimeric polypeptide", with functional properties derived from each of the original proteins.

[0038] The term "monomeric", as used herein, refers to a polypeptide that has a single peptide chain and therefore has one N-terminus and one C-terminus. In some aspects, a monomeric polypeptide excludes protein or protein complexes that include multiple peptide chains.

[0039] A "linker" or "peptide linker" refers to a peptide sequence linked to a polypeptide sequence at both ends of the linker peptide sequence. In one aspect, the linker is from about 1 to about 50 amino acid residues long or alternatively 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 2 to about 40, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 5, about 4 to about 40, about 4 to about 30, about 4 to about 20, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 5 to about 40, about 5 to about 30, about 5 to about 20, about or 5 to about 10 amino acid residues long. In a particular aspect, the linker is from about 1 to about 20 amino acid residues long. In another particular aspect, the linker is from about 3 to 10 amino acid residues long.

[0040] The terms "polynucleotide" and "oligonucleotide" are used

interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the

polynucleotide. The sequence of nucleotides can be interrupted by

non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide

encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0041] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0042] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. [0043] As used herein, the term "biological equivalent thereof when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology or alternatively a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement, and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid while still maintaining desired structure or functionality . Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.

[0044] "Hybridization" refers to hybridization reactions can be performed under conditions of different "stringency". Conditions that increase the stringency of a hybridization reaction are widely known and published in the art: see, for example, Sambrook, et al., infra. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C, 37°C, 50°C, and 68 °C; buffer concentrations of 10 X SSC, 6 X SSC, 1 X SSC, 0.1 X SSC (where SSC is 0.15 M NaCI and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours and washes of increasing duration, increasing frequency, or decreasing buffer concentrations.

[0045] A biological equivalent of P2-RANTES includes without limitation 5P12-RANTES and 5P14-RANTES as well as those described in Gaertner et al. (2008) PNAS 105:17706-1771 1 . PSC-RANTES is another biological equivalent of RANTES designed by Lederman et al. (2004) Science 306:485-7.

[0046] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1 . Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50

sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0047] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0048] A "substantial homologue" of a polynucleotide or polypeptide refers to a polynucleotide or a polypeptide having a substantial homology or sequence identity to the polynucleotide or polypeptide. In one aspect, a "substantial homology" is greater than about 80% homology, or alternatively greater than about 80% homology, or alternatively greater than about 90% homology or alternatively greater than about 95% homology, or alternatively greater than about 98% homology.

[0049] The term "a homolog of a nucleic acid" refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.

[0050] As used herein, "expression" refers to the process by which

polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

[0051] The term "encode" as it is applied to polynucleotides refers to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0052] A "composition" is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0053] A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0054] "An effective amount" refers to the amount of an active chimeric polypeptide or a pharmaceutical composition sufficient to induce a desired biological and/or therapeutic result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present disclosure, the result will typically involve prevention or inhibition of HIV infection (or SIV if appropriate) or alleviation of signs or symptoms of HIV or SIV infection. The effective amount will vary depending upon the health condition or disease stage of the subject being treated, timing of administration of the chimeric polypeptide, the manner of administration and the like, all of which can be determined readily by one of ordinary skill in the art. [0055] As used herein, the terms "treating," "treatment" and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.

[0056] "Treating" also covers any treatment of a disorder in a mammal, and includes: (a) preventing a disorder from occurring in a subject that may be predisposed to a disorder, but may have not yet been diagnosed as having it, e.g., prevent HIV or SIV infection to a subject at risk of HIV or SIV infection or prevent HIV or SIV infection to a healthy cell in a subject; (b) inhibiting a disorder, i.e., arresting its development, e.g., inhibiting HIV infection; or (c) relieving or ameliorating the disorder, e.g., reducing HIV or SIV infection.

[0057] As used herein, to "treat" further includes systemic amelioration of the symptoms associated with the pathology and/or a delay in onset of symptoms. Clinical and sub-clinical evidence of "treatment" will vary with the pathology, the subject and the treatment.

[0058] "Administration" can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of

administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non- limiting examples of route of administration include oral administration, nasal administration, injection, and topical application. [0059] The agents and compositions of the present disclosure can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.

[0060] An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0061] The term "conjugated moiety" refers to a moiety that can be added to an isolated chimeric polypeptide by forming a covalent bond with a residue of chimeric polypeptide. The moiety may bond directly to a residue of the chimeric polypeptide or may form a covalent bond with a linker which in turn forms a covalent bond with a residue of the chimeric polypeptide.

[0062] A "peptide conjugate" refers to the association by covalent or non- covalent bonding of one or more polypeptides and another chemical or biological compound. In a non-limiting example, the "conjugation" of a polypeptide with a chemical compound results in improved stability or efficacy of the polypeptide for its intended purpose. In one embodiment, a peptide is conjugated to a carrier, wherein the carrier is a liposome, a micelle, or a pharmaceutically acceptable polymer.

[0063] "Liposomes" are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE),

phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin,

phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol- amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan- olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC),

palmitoyloleoylphosphatidylethanolamine (POPE) and

dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1 -carb- oxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Choi). Negatively charged lipids include

phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG),

dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles

(LUVs).

[0064] A "micelle" is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle). Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue. [0065] The phrase "pharnnaceutically acceptable polymer" refers to the group of compounds which can be conjugated to one or more polypeptides described here. It is contemplated that the conjugation of a polymer to the polypeptide is capable of extending the half-life of the polypeptide in vivo and in vitro. Non- limiting examples include polyethylene glycols, polyvinylpyrrolidones,

polyvinylalcohols, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.

[0066] "Pharmaceutically acceptable carriers" refers to any diluents, excipients, or carriers that may be used in the compositions of the disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0067] A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides;

lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0068] A polynucleotide of this disclosure can be delivered to a cell or tissue using a gene delivery vehicle. "Gene delivery," "gene transfer," "transducing," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

[0069] A "plasmid" is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the

chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.

[0070] "Plasmids" used in genetic engineering are called "plasmic vectors". Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.

[0071] A "yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearised by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. Yeast expression vectors, such as YACs, Yips (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than BACs, producing chimeric effects.

[0072] A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus- based vectors, have also been developed for use in gene therapy and

immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. [0073] As used herein, "retroviral mediated gene transfer" or "retroviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0074] Retroviruses carry their genetic information in the form of RNA;

however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0075] In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071 . Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/1 1984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0076] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.

[0077] Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this disclosure. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this disclosure are other non-limiting techniques.

[0078] An example of a solid phase support include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation..

[0079] "Eukaryotic cells" comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. A eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non- limiting examples include simian, bovine, porcine, murine, rats, avian, reptilian and human. [0080] "Prokaryotic cells" that usually lack a nucleus or any other membrane- bound organelles and are divided into two domains, bacteria and archaea.

Additionally, instead of having chromosomal DNA, these cells' genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1 -2 μιτι in diameter and 10 μιτι long).

Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0081] A "host cell", as used herein, refers to a living cell in which a virus, a plasmid or any other nucleic acid reproduces. Non-limiting examples of host cells include eukaryotic cells such as plant cells, yeast, algae and mammalian cells, and prokaryotic cells such as E. coli cells.

[0082] As used herein, an "antibody" includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0083] The antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, e.g., murine, rat, sheep and canine.

[0084] The term "human antibody" as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., Cm, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. Similarly, antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, and the like) and other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies. Further, chimeric antibodies include any combination of the above. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans or other species relative to non-modified antibodies. Thus, a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non- human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.

[0085] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody that is "derived from" a human germline

immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins. A selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0086] A "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term also intends recombinant human antibodies. Methods to making these antibodies are described herein.

[0087] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by

recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. Methods to making these antibodies are described herein.

[0088] As used herein, "isotype" refers to the antibody class (e.g., IgM or lgG1 ) that is encoded by heavy chain constant region genes. [0089] The terms "polyclonal antibody" or "polyclonal antibody composition" as used herein refer to a preparation of antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.

[0090] The terms "monoclonal antibody" or "monoclonal antibody

composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0091] A "biologically active fragment" of an antibody refers to a peptide fragment that recognizes the target of the antibody. In some aspects, a biologically active fragment of an antibody is a fragment antigen-binding (Fab fragment) region of the antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain.

[0092] As used herein, the term "label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histadine tags (N-His), magnetically active isotopes, e.g., ¹¹⁵Sn, ¹¹⁷Sn and ¹¹⁹Sn, a non-radioactive isotopes such as ¹³C and ¹⁵N, polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, nonradioactive isotopes, radioisotopes, fluorochromes, chemiluminescent

compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0093] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable

luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0094] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the

Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^th ed.).

[0095] In another aspect, the fluorescent label is functional ized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0096] "Fusion inhibitors" or "entry inhibitors" as used herein interchangeably and refer to a class of antiretroviral drugs, used in combination therapy for the treatment of HIV infection and the like. This class of drugs interferes with one or more of the binding, fusion and entry of an HIV virion to a human cell. By blocking these steps in HIV's replication cycle, such agents inhibit or slow HIV infection. There are several key proteins involved in the HIV entry process: CD4, a protein receptor found on the surface of helper T cells in the human immune system, also called CD4+ T cells; gp120, a protein on HIV surface that binds to the CD4 receptor; CCR5, a second receptor found on the surface of CD4+ cells, called a chemokine co-receptor; CXCR4, another chemokine co-receptor found on CD4+ cells; and gp41 , a HIV protein, closely associated with gp120, that penetrates the cell membrane.

[0097] A "gp41 -binding protein" or "gp41 -binding peptide" refers to a protein that binds to the gp41 protein. HIV binds to the host CD4+ cell receptor via the viral protein GP120; GP41 , a viral transmembrane protein, then undergoes a conformational change that assists in the fusion of the viral membrane to the host cell membrane. Non-limiting examples of gp41 -binding proteins include C- peptides, C37, C34, C52L, T-2635 and T20.

[0098] "C-peptides", or "synthetic C peptides" refer to peptides that are derived from the C-terminal heptad repaeat of the HIV type 1 (HIV-1 ) gp41 envelope protein. These C-peptides bind to the N-terminal heptad repeat of gp41 and inhibit HIV fusion. One C-peptide (T-20, also called Fuzeon or enfuvirtide from Trimeris/Roche) is currently in clinical use. The drawbacks of these peptides are that they generally require relatively high doses when in clinical use. And even in in vitro studies, they have been surpassed in terms of low

concentrations needed for effectiveness by both griffithsin alone and variants of CC chemokines. But they are still considered to be a very clinically useful class of drugs.

[0099] "CC chemokines" or "β-chemokines" are chemokines that have two adjacent cysteines near their amino terminus. There have been at least 27 distinct members of this subgroup reported for mammals, called CC chemokine ligands (CCL)-1 to -28; CCL10 is the same as CCL9. Chemokines of this subfamily usually contain four cysteines (C4-CC chemokines), but a small number of CC chemokines possess six cysteines (C6-CC chemokines). An example of a CC chemokine is monocyte chemoattractant protein-1 (MCP-1 or CCL2) which induces monocytes to leave the bloodstream and enter the surrounding tissue to become tissue macrophages. CC chemokines induce cellular migration by binding to and activating CC chemokine receptors, ten of which have been discovered to date and called CCR1 -10. These receptors are expressed on the surface of different cell types allowing their specific attraction by the chemokines.

[0100] "C37" is a peptide that is derived from C34 which in turn is a sequence in gp41 that binds to the N-terminal region of gp41 to stop the 6 helix bundle formation. It has the sequence

HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL that is derived from HIV- 1 . HIV-1 - HXB2 residues 625 to 661 contains the entire C34 sequence (W628 to L661 ) as reported by Root et al. (2001 ) Science 291 :884-888.

[0101] "C37-ac" is C-terminally acetylated, N-terminally amidated C37 peptide and is intended to be included in the use of the term "C37". That is, the two ends are "capped" so that they are not charged.

[0102] "T20" or "Enfuvirtude" is an HIV fusion inhibitor, marketed under the trade name Fuzeon®. Without being bound by theory, T20 is believed to work by disrupting the HIV-1 molecular machinery at the final stage of fusion with the target cell, preventing uninfected cells from becoming infected. A biomimetic peptide, enfuvirtide was rationally designed to mimic components of the HIV-1 fusion machinery and displace them, preventing normal fusion. Enfuvirtide binds to GP41 preventing the creation of an entry pore for the capsid of the virus, keeping it out of the cell. Generally, C-peptides can inhibit HIV-1 membrane fusion by binding to the amino-terminal trimeric coiled coil of the same protein. T- 20 contains an additional tryptophan-rich sequence motif whose binding site extends beyond the gp41 coiled-coil region yet provides the key determinant of inhibitory activity in T-20.

[0103] "C34" is a synthetic C-peptide composed of a peptide sequence that overlaps with T-20 but contains the gp41 coiled-coil cavity binding residues, ⁶²⁸WMEW⁶³¹. C34 is known to compete with the CHR of gp41 for the

hydrophobic grooves of the NHR yet is incapable of functioning at a post-lipid mixing stage (Stoddart et al. (2008) J. Biol. Chem. 283(49):34045-52). [0104] "C52L" is a recombinant peptide inhibitor that includes both the C- peptide and tryptophan-rich regions of T-20 (Deng (2007) Biochemistry

46(14):4360-9). The C52L peptide potently inhibits in vitro infection of human T cells. C52L can be expressed in bacteria so it might be more economical to manufacture on a large scale than T-20-like peptides produced by chemical synthesis.

[0105] "T-2635" is a helix-stabilized second generation fusion inhibitor with antiviral activity against virus strains resistant to enfuvirtide. It was designed by Dwyer et al. (Dwyer et al. (2007) Proc. Natl. Acad. Sci. USA 104(31 ):12772-7).

[0106] A "CCR5-binding protein" refers to a protein that binds to CCR5.

CCR5, short for chemokine (C-C motif) receptor 5, is a protein which in humans is encoded by the CCR5 gene which is located on chromosome 3 on the short (p) arm at position 21 . CCR5 has also recently been designated CD195 (cluster of differentiation 195). The CCR5 protein functions as a chemokine receptor in the CC chemokine group. The natural chemokine ligands that bind to this receptor are RANTES, MIP-1 a and MIP-1 β. CCR5 is predominantly expressed on T cells, macrophages, dendritic cells and microglia. It is likely that CCR5 plays a role in inflammatory responses to infection, though its exact role in normal immune function is unclear. Non-limiting examples of CCR5-binding proteins include RANTES, P2-RANTES, MIP-1 a, MIP-1 β, U83A and CCR5 antibodies.

[0107] "RANTES", "Chemokine (C-C motif) ligand 5" or simply "CCL5" refers to a protein which in humans is encoded by the CCL5 gene. CCL5 is an 8kDa protein classified as a chemotactic cytokine or chemokine. CCL5 is chemotactic for T cells, eosinophils, and basophils, and plays an active role in recruiting leukocytes into inflammatory sites. With the help of particular cytokines (i.e., IL-2 and IFN-γ) that are released by T cells, CCL5 also induces the proliferation and activation of certain natural-killer (NK) cells to form CHAK (CC-Chemokine- activated killer) cells. It is also an HIV-suppressive factor released from CD8+ T cells. This chemokine has been localized to chromosome 17 in humans.

RANTES was first identified in a search for genes expressed "late" (3-5 days) after T cell activation. It was subsequently determined to be a CC chemokine and expressed in more than 100 human diseases. RANTES expression is regulated in T lymphocytes by Kruppel like factor 13 (KLF13). RANTES was earlier called Regulated upon Activation, Normal T-cell Expressed, and Secreted, abbreviated RANTES. There have been many variants of the CC chemokine RANTES (and some of other CC chemokines) that have been shown to have strong anti-HIV activity. Several of these have been patented including P2- RANTES (Hartley et al. (2003) J. Virol. 77:637-644) and PSC-RANTES. Most recently, next generation RANTES variants have been isolated by random mutagenesis, and these have the combination of high effectiveness (at low (pM) concentration) as well as being able to be produced from E. coli. The publication which introduced P2-RANTES is Hartley et al. (2003) J. Virol. 77:637-644. Some other RANTES variants are shown in Gaertner et al. (2008) PNAS 105:17706- 1771 1 . "PSC-RANTES" is another RANTES variant designed by Lederman et al. (Lederman et al. (2004) Science 306:485-7).

[0108] "MIP-1 a" or "macrophage inflammatory protein-1 a", and "ΜΙΡ-1 β" or "macrophage inflammatory protein-1 β" are from the CC (cysteine-cysteine) chemokine subfamily. These soluble factors are chemotactic for specific types of leukocyte populations and are involved in the regulation of cell-mediated immunity. MIP-1 a and ΜΙΡ-1 β are produced by monocytes, macrophages, lymphocytes, and other cell types (see generally, e.g., Matsukawa et al. (2000) Chemokines and innate immunity. Rev. Immunogenet. 2:339-358). MIP-1 a and MIP-1 β have been shown to inhibit HIV entry by binding to the co-receptor CCR5.

[0109] "U83A" is a distant chemokine homolog encoded by a human herpesvirus-6 variant (Dewin & Gompels (2006) J. Immunol. 176(1 ):544-56). U83A can efficiently and potently induce calcium mobilization in cells expressing single human CCR1 , CCR4, CCR6, or CCR8. U83A can also induce chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8.

[0110] A "gp120-binding protein" refers to a protein that binds gp120. gp120 is a glycoprotein exposed on the surface of the HIV envelope that binds to the CD4 receptor. gp120 is essential for virus entry into cells as it plays a vital role in seeking out specific cell surface receptors for entry. The crystal structure of gp120 complexed to D1 D2 CD4 and a neutralizing antibody Fab was solved in 1998. It is organized with an outer domain, an inner domain with respect to its termini and a bridging sheet. The gp120 gene is around 1 .5 kb long and codes for around 500 amino acids. Three gp120s, bound as heterodimers to a transmembrane glycoprotein, gp41 , are thought to combine in a trimer to form the envelope spike, which is involved in virus-cell attachment. Non-limiting examples of gp120-binding proteins include Griffinthsin, cyanovirin-N (CVN), 12p1 ,

CD4M33 and CD4M47. In certain embodiments of the present disclosure, the chimeric polypeptide further comprises a portion that includes a gp120-binding protein. In some embodiments, the composition of the present disclosure further comprises a gp120-binding protein.

Descriptive Embodiments

[0111] A microbicide is a composition that can be used to reduce the infectivity of microbes such as HIV. It can be formulated into a cream or gel and used to prevent sexual spread of HIV. For example, for use in developing countries, the microbicide needs to be inexpensive to produce, stable under high temperature, and active at the lower pH's in the urogenital tract. This disclosure satisfies these needs and provides related advantages as well.

[0112] It is discovered herein that chimeric polypeptides comprised of a gp41 - binding protein and a CCR5-binding protein, are potent inhibitors to HIV infection.

Polypeptides and Compositions

[0113] One aspect of the present disclosure provides an isolated chimeric polypeptide comprising, or alternatively consisting essentially of, or alternatively consisting of, a first portion comprising a CCR5- binding protein and a second portion comprising a gp41 -binding protein. In some aspects, the chimeric polypeptide does not comprise an lgG1 antibody that specifically recognizes CCR5. In some aspects, the chimeric polypeptide is monomeric.

[0114] In some embodiments, the first portion is N-terminal to the second portion. [0115] Also provided is an isolated chimeric polypeptide comprising, or alternatively consisting essentially of or yet further consisting of a first portion comprising a CCR5- binding protein and a second portion comprising a gp41 binding peptide, wherein the chimeric polypeptide does not comprise an lgG1 antibody that specifically recognizes CCR5. In one aspect, the gp41 -binding peptide comprises, or alternatively consists essentially of, or yet further consists of, an amino acid sequence of one or more of a C-peptide; a N-peptide; C37; C- 37ac; C37(Q652L); N-acetylated, C-term amidated C37; N-acetylated, C-term amidated C37(Q652L); C34; C52L; T-2635; T20; N-peptides; N17; N23; N36 or a substantial homologue thereof. In a further aspect, gp41 -binding peptide comprises, or alternatively consists essentially of, or yet further consists of, an amino acid of one or more of C37; C37-ac; C37(Q652L); N-acetylated, C-term amidated C37; N-acetylated, C-term amidated C37(Q652L) or a substantial homologue thereof. The substantial homologue is an amino acid sequence having greater than about 80% homology, or alternatively greater than about 80% homology, or alternatively greater than about 90% homology or alternatively greater than about 95% homology, or alternatively greater than about 98% homology, to the amino acid sequence of the respective gp41 -binding peptide.

[0116] In a further aspect, the isolated chimeric polypeptide as described above comprises a CCR5-binding protein that comprises, or alternatively consists essentially of, or yet further consist of, RANTES, P2-RANTES, PSC-RANTES, 5P12-RANTES, 5P14-RANTES, 6P4-RANTES, ΜΙΡ-1 α, ΜΙΡ-1 β, U83A or a substantial homologue of any one thereof. In a further aspect, the CCR5- binding protein comprises, or alternatively consists essentially of, or yet further consist of, an amino acid sequence of 5P12-RANTES or 5P14-RANTES or a substantial homologue thereof.

[0117] In each of these embodiments, the substantial homologue is an amino acid sequence having greater than about 80% homology, or alternatively greater than about 80% homology, or alternatively greater than about 90% homology or alternatively greater than about 95% homology, or alternatively greater than about 98% homology to the respective gp41 -binding peptide or CCR5- binding protein. [0118] The first portion may be N-terminal to the second portion.

[0119] In some embodiments, the isolated chimeric polypeptide further comprises a peptide linker between the first portion and the second portion. In one aspect, the linker is from about 1 to about 50 amino acid residues long or alternatively about 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 2 to about 40, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 5, about 4 to about 40, about 4 to about 30, about 4 to about 20, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 5 to about 40, about 5 to about 30, about 5 to about 20, about or 5 to about 10 amino acid residues long. In a particular aspect, the linker is from about 1 to about 20 amino acid residues long. In another particular aspect, the linker is from about 3 to about 10 amino acid residues long. In one aspect, the peptide linker is a polypeptide that comprises one or more amino acids, wherein at least one or more is selected from alanine, glycine or serine.

[0120] In some embodiments, the isolated chimeric polypeptide further comprises at least one of a protein start site, a polyhistidine tag, and/or a protease cleavage site, each operatively linked to the isolated chimeric

polypeptide. Methods of designing, selecting and using protein start,

polyhistidine tags, protease cleavage site in a protein expression system and operatively linking them to an expression target are known in the art. See, generally, Baneyx (2004) Protein Expression Technologies: Current Status and Future Trends, Taylor & Francis, 1 ^st Ed.

[0121] For any chimeric polypeptide of the disclosure, the gp41 -binding protein can be selected from C37, C34, C52L, T-2635, T20 or a substantial homologue or biological equivalent of any one thereof. In one aspect, the gp41 - binding protein is C37 or a substantial homologue or biological equivalent thereof.

[0122] For any chimeric polypeptide of the disclosure, the CCR5-binding protein can be selected from RANTES, P2-RANTES, PSC-RANTES, MIP-1 a, ΜΙΡ-1 β, U83A, a CCR5 antibody or a substantial homologue or biological equivalent of any one thereof. In one aspect, the CCR5-binding protein is 5P12- RANTES, 5P14-RANTES or a substantial homologue or biological equivalent thereof.

[0123] Polypeptides comprising the amino acid sequences of the disclosure can be prepared by expressing polynucleotides encoding the polypeptide sequences of this disclosure in an appropriate host cell. This can be

accomplished by methods of recombinant DNA technology known to those skilled in the art. Accordingly, this disclosure also provides methods for recombinantly producing the polypeptides of this disclosure in a eukaryotic or prokaryotic host cells, as well as the isolated host cells used to produce the proteins. The proteins and polypeptides of this disclosure also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431 A, Foster City, CA, USA. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this disclosure also provides a process for chemically synthesizing the proteins of this disclosure by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.

[0124] It is known to those skilled in the art that modifications can be made to any peptide to provide it with altered properties. Polypeptides of the disclosure can be modified to include unnatural amino acids. Thus, the peptides may comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids {e.g., β-methyl amino acids, C-a-methyl amino acids, and N-a-methyl amino acids, etc.) to convey special properties to peptides.

Additionally, by assigning specific amino acids at specific coupling steps, peptides with a-helices, β turns, β sheets, a-turns, and cyclic peptides can be generated. Generally, it is believed that a-helical secondary structure or random secondary structure is preferred.

[0125] In a further embodiment, subunits of polypeptides that confer useful chemical and structural properties will be chosen. For example, peptides comprising D-amino acids may be resistant to L-amino acid-specific proteases in vivo. Modified compounds with D-amino acids may be synthesized with the amino acids aligned in reverse order to produce the peptides of the disclosure as retro-inverso peptides. In addition, the present disclosure envisions preparing peptides that have better defined structural properties, and the use of

peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. In another embodiment, a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri-CH₂NH-R₂, where R-i, and R₂ are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a molecule would be resistant to peptide bond hydrolysis, e.g., protease activity. Such molecules would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown, or protease activity. Furthermore, it is well known that in certain systems constrained peptides show enhanced functional activity (Hruby (1982) Life Sciences 31 :189-199 and Hruby et al. (1990) Biochem J. 268:249-262); the present disclosure provides a method to produce a constrained peptide that incorporates random sequences at all other positions.

[0126] Non-classical amino acids may be incorporated in the peptides of the disclosure in order to introduce particular conformational motifs, examples of which include without limitation: 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazrnierski et al. (1991 ) J. Am. Chem. Soc. 1 13:2275-2283); (2S,3S)-methyl- phenylalanine, (2S,3R)- methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazrnierski & Hruby (1991 ) Tetrahedron Lett. 32(41 ):5769-5772); 2-aminotetrahydronaphthalene-2- carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylate (Miyake et al. (1989) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylic acid (Zechel et al. (1991 ) Int. J. Pep. Protein Res. 38(2):131 -138); and HIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep. Protein Res. 42(1 ):68-77) and (Dharanipragada et al. (1992) Acta. Crystallogr. C. 48:1239-1241 ).

[0127] The following amino acid analogs and peptidomimetics may be incorporated into a peptide to induce or favor specific secondary structures: LL- Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); β-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081 -5082); β-turn inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5057-5060); a-helix inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:4935-4938); a-turn inducing analogs (Kemp et al. (1989) J. Org. Chem. 54:109:1 15); analogs provided by the following references: Nagai & Sato (1985) Tetrahedron Lett. 26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p. 1687; a Gly-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bond isostere (Clones et al. (1988) Tetrahedron Lett. 29:3853-3856); tetrazole (Zabrocki et al. (1988) J. Am. Chem. Soc. 1 10:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501 :509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci. 1 12:323-333 and Garvey et al. (1990) J. Org. Chem. 56:436.

Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Patent No. 5,440,013, issued August 8, 1995 to Kahn.

[0128] It is known to those skilled in the art that modifications can be made to any peptide by substituting one or more amino acids with one or more functionally equivalent amino acids that does not alter the biological function of the peptide. In one aspect, the amino acid that is substituted by an amino acid that possesses similar intrinsic properties including, but not limited to, hydrophobicity, size, or charge. Methods used to determine the appropriate amino acid to be substituted and for which amino acid are know to one of skill in the art. Non-limiting examples include empirical substitution models as described by Dahoff et al. (1978) In Atlas of Protein Sequence and Structure Vol. 5 suppl. 2 (ed. M.O.

Dayhoff), pp. 345-352. National Biomedical Research Foundation, Washington DC; PAM matrices including Dayhoff matrices (Dahoff et al. (1978), supra, or JTT matrices as described by Jones et al. (1992) Comput. Appl. Biosci. 8:275-282 and Gonnet et al. (1992) Science 256:1443-1 145; the empirical model described by Adach & Hasegawa (1996) J. Mol. Evol. 42:459-468; the block substitution matrices (BLOSUM) as described by Henikoff & Henikoff (1992) Proc. Natl.

Acad. Sci. USA 89:1 -1 ; Poisson models as described by Nei (1987) Molecular Evolutionary Genetics. Columbia University Press, New York.; and the Maximum Likelihood (ML) Method as described by Muller et al. (2002) Mol. Biol. Evol. 19:8- 13.

[0129] Another aspect of the disclosure provides a composition comprising, or alternatively consisting essentially of, or alternatively consisting of, an isolated chimeric polypeptide comprising a first portion comprising a CCR5- binding protein and a second portion comprising a gp41 -binding protein, wherein the chimeric polypeptide does not comprise an lgG1 antibody that specifically recognizes CCR5.

[0130] In one aspect of the above composition, the chimeric polypeptide is 5P12-RANTES-linker-C37 or a substantial homologue or biological equivalent thereof.

[0131] In another aspect of the above composition, the chimeric polypeptide is 5P14-RANTES-linker-C37 or a substantial homologue or biological equivalent thereof.

[0132] The mole ratio between the first polypeptide and the second

polypeptide can be from about 1 : 10 to about 10:1 , or alternatively from about 1 :9 to about 9:1 , about 1 :8 to about 8:1 , about 1 :7 to about 7:1 , about 1 :6 to about 6:1 , about 1 :5 to about 5:1 , about 1 :4 to about 4:1 , about 1 :3 to about 3:1 , about 1 :2 to about 2:1 . In another aspect, the mole ratio between the first polypeptide and the second polypeptide is about 1 :1 .

[0133] For any of the above composition, the gp41 -binding protein can be selected from C37, C34, C52L, T-2635, T20 or a substantial homologue or biological equivalent of any one thereof. In one aspect, the gp41 -binding protein is C37 or a substantial homologue or biological equivalent thereof. [0134] For any of the above connpositions, the CCR5-binding protein can be selected from RANTES, P2-RANTES, PSC-RANTES, ΜΙΡ-1 α, ΜΙΡ-1 β, U83A, a CCR5 antibody or a substantial homologue or biological equivalent of any one thereof. In one aspect, the CCR5-binding protein is 5P12-RANTES or a substantial homologue or biological equivalent thereof. In one aspect, the CCR5- binding protein is 5P14-RANTES or a substantial homologue or biological equivalent thereof.

[0135] In another aspect, any of the above compositions further comprises a carrier. The carrier can be a solid phase carrier, a gel, an aqueous liquid carrier, a paste, a liposome, a micelle, albumin, polyethylene glycol, a pharmaceutically acceptable polymer, or a pharmaceutically acceptable carrier, such a phosphate buffered saline.

[0136] The compositions of the disclosure can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others.

Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Compositions may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

[0137] Compositions may be prepared as liquid suspensions or solutions using a sterile liquid, such as oil, water, alcohol, and combinations thereof.

Pharmaceutically suitable surfactants, suspending agents or emulsifying agents, may be added for oral or parenteral administration. Suspensions may include oils, such as peanut oil, sesame oil, cottonseed oil, corn oil and olive oil.

Suspension preparation may also contain esters of fatty acids, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension compositions may include alcohols, such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil and petrolatum, and water may also be used in suspension compositions. [0138] The compositions of this disclosure are formulated for pharmaceutical administration to a mammal, preferably a human being. Such compositions of the disclosure may be administered in a variety of ways, preferably topically or by injection.

[0139] Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. Compounds may be formulated for parenteral administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection may be in ampoules or in multi-dose containers.

[0140] In addition to dosage forms described above, pharmaceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the disclosure. It should be understood that a specific dosage and treatment regimen for any particular subject will depend upon a variety of factors, including the activity of the specific antidote employed, the age, body weight, general health, sex and diet, renal and hepatic function of the subject, and the time of administration, rate of excretion, drug combination, judgment of the treating physician or veterinarian and severity of the particular disease being treated.

Polypeptide Conjugates

[0141] Another aspect of the disclosure provides a peptide conjugate comprising, or alternatively consisting essentially of, or alternatively consisting of, a carrier covalently or non-covalently linked to an isolated chimeric polypeptide of the disclosure. In some embodiments, the carrier comprises a liposome, or alternatively a micelle, or alternatively a pharmaceutically acceptable polymer, or a pharmaceutically acceptable carrier.

[0142] The polypeptides and polypeptide conjugates of the disclosure can be used in a variety of formulations, which may vary depending on the intended use. For example, one or more can be covalently or non-covalently linked (complexed) to various other molecules, the nature of which may vary depending on the particular purpose. For example, a peptide of the disclosure can be covalently or non-covalently complexed to a macromolecular carrier, including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. A peptide can be conjugated to a fatty acid, for introduction into a liposome, see U.S. Patent No. 5,837,249. A peptide of the disclosure can be complexed covalently or non- covalently with a solid support, a variety of which are known in the art and described herein. An antigenic peptide epitope of the disclosure can be associated with an antigen-presenting matrix such as an MHC complex with or without co-stimulatory molecules.

[0143] Examples of protein carriers include, but are not limited to,

superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin, myoglobulin, and immunoglobulin.

[0144] Peptide-protein carrier polymers may be formed using conventional cross-linking agents such as carbodimides. Examples of carbodimides are 1 - cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1 -ethyl-3-(3- dimethyaminopropyl) carbodiimide (EDC) and 1 -ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide.

[0145] Examples of other suitable cross-linking agents are cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo- bifunctional epoxide, a homo-bifunctional imido-ester, a homo-bifunctional N- hydroxysuccinimide ester, a homo-bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound may be used. Also included are hetero-bifunctional compounds, for example, compounds having an amine- reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl- reactive group.

[0146] Specific examples of such homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters

dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartrate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1 ,4-di- [3'-(2'-pyridyldithio) propionamido]butane, bismaleimidohexane, and bis-N- maleimido-1 , 8-octane; the bifunctional aryl halides 1 ,5-difluoro-2,4- dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional

photoreactive agents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1 ,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide,

carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o- tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides N1 N'-ethylene-bis(iodoacetamide), N1 N'-hexamethylene-bis(iodoacetamide), N1 N'-undecamethylene-bis(iodoacetamide), as well as benzylhalides and halomustards, such as a1 a'-diiodo-p-xylene sulfonic acid and tri(2- chloroethyl)amine, respectively. [0147] Examples of common hetero-bifunctional cross-linking agents that may be used to effect the conjugation of proteins to peptides include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 - carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N- succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-(y-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N- maleimidomethyl) cyclohexane-1 -carboxyl-hydrazide), SMPT

(succinimidyloxycarbonyl-a-methyl- a -(2-pyridyldithio)toluene), and SPDP (N- succinimidyl 3-(2-pyridyldithio)propionate).

[0148] Cross-linking may be accomplished by coupling a carbonyl group to an amine group or to a hydrazide group by reductive amination.

[0149] The chimeric polypeptides or polypeptides of the compositions of the disclosure also may be formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules may be done through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created using charged polymers such as poly-(L- glutamic acid) or poly-(L-lysine) which contain numerous negative and positive charges, respectively. Adsorption of peptides may be done to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non- covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein. Finally, peptides may be non- covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. These biotin-binding proteins contain four binding sites that can interact with biotin in solution or be covalently attached to another molecule. (See Wilchek (1988) Anal. Biochem. 171 :1 -32). Peptides can be modified to possess biotin groups using common biotinylation reagents such as the N- hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts with available amine groups on the protein. Biotinylated peptides then can be incubated with avidin or streptavidin to create large complexes. The molecular mass of such polymers can be regulated through careful control of the molar ratio of

biotinylated peptide to avidin or streptavidin.

[0150] Also provided by this application are the peptides and polypeptides described herein conjugated to a label, e.g., a tag (His-tag), label e.g., a fluorescent or bioluminescent label, for use in the diagnostic methods. For example, detectably labeled peptides and polypeptides can be bound to a column and used for the detection and purification of antibodies. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine,

tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Molecular Probes Handbook.

[0151] The chimeric polypeptides or polypeptides of the compositions of the disclosure also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare antibodies, the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one. However, for the purpose of illustration only, suitable adjuvants include, but are not limited to, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant and mineral salts.

Isolated Polynucleotides, Host Cells and Compositions

[0152] Yet another aspect of the disclosure provides an isolated

polynucleotide encoding for an isolated chimeric polypeptide, an antibody, or a biologically active fragment of the antibody of the disclosure. Also provided is a DNA construct comprising an expression vector and a polynucleotide. In one aspect of the DNA construct, the vector is a plasmid vector, a yeast artificial chromosome, or a viral vector. In one aspect, the vector of the DNA construct comprises a protein tag. Protein tags can be selected from a His-tag, a SUMO- tag, a GST-tag, a myc-tag, or a FLAG-tag provided in expression constructs commercially available from, e.g., Invitrogen, Carlbad, CA.

[0153] Another aspect of the disclosure provides an isolated host cell transformed with a polynucleotide or a DNA construct of the disclosure. The isolated host cells can be a prokaryotic or a eukaryotic cell. Yet another aspect of the disclosure provides an isolated transformed host cell expressing an isolated chimeric polypeptide, an antibody or a biologically active fragment of the antibody of the disclosure. The isolated host cells can be a prokaryotic or a eukaryotic cell.

[0154] Also provided are polynucleotides encoding substantially homologous and biologically equivalent polypeptides to the inventive polypeptides and polypeptide complexes. Substantially homologous and biologically equivalent intends those having varying degrees of homology, such as at least 80 %, or alternatively, at least 85 %, or alternatively at least 90 %, or alternatively, at least 95 %, or alternatively at least 98 % homologous as defined above and which encode polypeptides having the biological activity as described herein. It should be understood although not always explicitly stated that embodiments to substantially homologous polypeptides and polynucleotides are intended for each aspect of this disclosure, e.g., polypeptides, polynucleotides and antibodies.

[0155] The polynucleotides of this disclosure can be replicated using conventional recombinant techniques. Alternatively, the polynucleotides can be replicated using PCR technology. PCR is the subject matter of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds, Birkhauser Press, Boston (1994)) and references cited therein. Yet further, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA. Accordingly, this disclosure also provides a process for obtaining the polynucleotides of this disclosure by providing the linear sequence of the polynucleotide, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the

nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can operatively link the polynucleotides to regulatory sequences for their expression in a host cell, described below. The polynucleotides and regulatory sequences are inserted into the host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods well known to those of skill in the art. A process for obtaining

polynucleotides by this method is further provided herein as well as the

polynucleotides so obtained.

[0156] Also provided are host cells comprising one or more of the

polypeptides or polynucleotides of this disclosure. In one aspect, the

polypeptides are expressed and can be isolated from the host cells. In another aspect, the polypeptides are expressed and secreted. In yet another aspect, the polypeptides are expressed and present on the cell surface (extracellularly).

Suitable cells containing the inventive polypeptides include prokaryotic and eukaryotic cells, which include, but are not limited to bacterial cells, algae cells, yeast cells, insect cells, plant cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells. A non-limiting example of algae cells is red alga Griffithsia sp. from which Griffithsin was isolated

(Toshiyuki et al. (2005) J. Biol. Chem. 280(10):9345-53). A non-limiting example of plant cells is a Nicotiana benthamiana leaf cell from which Griffithsin can be produced in a large scale (O'Keefe (2009) Proc. Nat. Acad. Sci. USA

106(15):6099-6104). Examples of bacterial cells include Escherichia coli

(Giomarelli et al. (2006), supra), Salmonella enteric, Streptococcus gordonii and lactobacillus (Liu et al. (2007) Cellular Microbiology 9:120-130; Rao et al. (2005) PNAS 102:1 1993-1 1998; Chang et al. (2003) PNAS 100(20):1 1672-1 1677; Liu et al. (2006) Antimicrob. Agents & Chemotherapy 50(10):3250-3259). The cells can be purchased from a commercial vendor such as the American Type Culture Collection (ATCC, Rockville Maryland, USA) or cultured from an isolate using methods known in the art. Examples of suitable eukaryotic cells include, but are not limited to 293T HEK cells, as well as the hamster cell line CHO, BHK-21 ; the murine cell lines designated NIH3T3, NSO, C127, the simian cell lines COS, Vero; and the human cell lines HeLa, PER.C6 (commercially available from Crucell) U- 937 and Hep G2. A non-limiting example of insect cells include Spodoptera frugiperda. Examples of yeast useful for expression include, but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Patent Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.

Antibody Compositions

[0157] The disclosure, in another aspect, provides an antibody that binds an isolated chimeric polypeptide of the disclosure. The antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a derivative or fragment thereof as defined below. In one aspect, the antibody is detectably labeled or further comprises a detectable label conjugated to it.

[0158] Also provided is a composition comprising the antibody and a carrier. Further provided is a biologically active fragment of the antibody, or a

composition comprising the antibody fragment. Suitable carriers are defined supra.

[0159] Further provided is an antibody-peptide complex comprising, or alternatively consisting essentially of, or yet alternatively consisting of, the antibody and a polypeptide specifically bound to the antibody. In one aspect, the polypeptide is the chimeric polypeptide against which the antibody is raised.

[0160] This disclosure also provides an antibody capable of specifically forming a complex with a protein or polypeptide of this disclosure, which are useful in the therapeutic methods of this disclosure. The term "antibody" includes polyclonal antibodies and monoclonal antibodies, antibody fragments, as well as derivatives thereof (described above). The antibodies include, but are not limited to mouse, rat, and rabbit or human antibodies. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc. The antibodies are also useful to identify and purify therapeutic polypeptides. Methods of the Disclosure

[0161] The disclosure, in one aspect, provides a method for preventing or inhibiting HIV entry into a cell, comprising contacting the cell with an effective amount of an isolated chimeric polypeptide or an effective amount of a

composition of the disclosure. The cell can be an animal cell, a mammalian cell, or a human cell. In a particular aspect, the cell is a human cell.

[0162] Also provided is a method for treating a subject in need thereof, comprising administering to the subject an effective amount of an isolated chimeric polypeptide or an effective amount of a composition of the disclosure. In one aspect, the subject is an HIV patient. In another aspect, the subject is a subject at risk of HIV infection. In one aspect, the subject is an animal, a mammal, or a human. In a particular aspect, the subject is a human.

[0163] The methods of the present disclosure are useful in inhibiting entry of HIV into a cell, preventing or inhibiting HIV infection, and/or treating HIV infection. The effectiveness of the methods, accordingly, can be measured by methods known in the art. In one aspect, the methods inhibit HIV infection by at least about 10%, or alternatively at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or at least 98%. Methods of determining inhibition of HIV entry/infection are known in the art, such as the cell fusion assay used in the experimental examples.

[0164] The disclosure further provides a method for identifying an agent useful for prevention or treatment of HIV infection, comprising a) contacting an HIV virus with a cell under suitable condition, the cell being in contact with a candidate agent and assaying the ability of the agent to inhibit HIV entry into the cell; and b) comparing that to the ability of any one or more agent of: a chimeric

polypeptide, a peptide conjugate, an isolated polynucleotide or a DNA construct of the disclosure, to inhibit HIV infection, wherein the ability of the agent in step a) that is greater than or substantially equal to the ability of the agent of step b) identifies the agent as useful for the prevention or treatment of HIV infection; and wherein the ability of the agent in step a) that is substantially less than the ability of the agent of step b) identifies the agent as not useful for the prevention or treatment of HIV infection. In one aspect, "substantially less" refers to at least about 10% less, or alternatively at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least 98%. Methods of determining an agent's ability to prevent, inhibit, or treat HIV infection are known in the art and further exemplified in the experimental examples.

[0165] Route of administration for the methods can be any methods disclosed herein, including but not limited to injection or topical application.

[0166] Also provided is a method for preparing an isolated chimeric

polypeptide of the disclosure, comprising expressing a polynucleotide encoding the chimeric polypeptide in an isolated prokaryotic or an isolated eukaryotic host cell. Non-limiting examples of host cells include an E. coli cell, lactobacillus, a plant cell, an algae, or a mammalian cell. In one aspect, the method further comprises isolating the polypeptide produced by the isolated host cell.

[0167] Accordingly, also provided is an isolated prokaryotic or eukaryotic host cell comprising a polynucleotide of the disclosure, and a composition comprising a carrier and a prokaryotic or eukaryotic host cell as described herein.

[0168] The current disclosure, in yet another aspect, provides a method for identifying an agent useful for prevention or treatment of HIV infection, comprising contacting an HIV virus with a cell capable being infected with HIV under suitable conditions, the cell being in contact with a candidate agent and an isolated chimeric polypeptide or a composition of the disclosure, wherein a decrease in infection compared to a cell being in contact with the chimeric polypeptide or the composition only identifies the agent as an agent useful for prevention or treatment of HIV infection.

[0169] HIV virus exclusively infects and causes disease in humans therefore so far there are no ideal model exists that can imitate the natural history and pathogenesis of HIV infection and AIDS in the human body. However, the data from animal models provides conceptual insights into immune responses elicited by investigational vaccines, and reassurance of safety, guiding preclinical development and the decision to enter into clinical trials in humans. Non-human primate studies play a leading role in efforts to develop an HIV vaccine.

[0170] Macaque monkeys infected with simian immunodeficiency virus (SIV), a virus closely related to HIV can be a good HIV animal model. This model is useful because SIV in macaques follows a similar disease course to HIV. A hybrid virus created by replacing SIV envelope with HIV envelope but retaining the inner core of SIV virus (called SHIVs), replicates acute HIV infection in macaques, and causes rapid disease progression leading to death.

[0171] A number of other animal models have been used to obtain

information that can have application to HIV. Feline immunodeficiency virus (FIV), transgenic mice that contain part of the HIV genome or co-receptors for viral entry, and severe combined immune deficiency (SCID) mice reconstituted with human immune system cells or tissues are some of the animal models being used to study pathogenesis.

Kits

[0172] An aspect of the disclosure provides a kit for use in preventing or inhibiting HIV entry into a cell, comprising, or alternatively consisting essentially of, or alternatively consisting of, an isolated chimeric polypeptide of the

disclosure, and instructions to use.

[0173] Also provided is a kit for use in treating a subject in need thereof, comprising, or alternatively consisting essentially of, or alternatively consisting of, an isolated chimeric polypeptide or a composition of the disclosure, and instructions to use.

[0174] Kits may further comprise suitable packaging and/or instructions for use of the compositions. The compositions can be in a dry or lyophilized form, in a solution, particularly a sterile solution, or in a gel or cream. The kit may contain a device for administration or for dispensing the compositions, including, but not limited to, syringe, pepitte, transdermal patch and/or microneedle. [0175] The kits may include other therapeutic compounds for use in conjunction with the compounds described herein. These compounds can be provided in a separate form or mixed with the compounds of the present disclosure.

[0176] The kits will include appropriate instructions for preparation and administration of the composition, side effects of the compositions, and any other relevant information. The instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc.

[0177] In another aspect of the disclosure, kits for treating a subject who suffers from or is susceptible to the conditions described herein are provided, comprising a container comprising a dosage amount of a composition as disclosed herein, and instructions for use. The container can be any of those known in the art and appropriate for storage and delivery.

[0178] Kits may also be provided that contain sufficient dosages of the effective composition or compound to provide effective treatment for a subject for an extended period, such as a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, or 8 weeks or more.

EXAMPLES

[0179] The disclosure is further understood by reference to the following examples, which are intended to be purely exemplary of the disclosure. The present disclosure is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the disclosure only. Any methods that are functionally equivalent are within the scope of the disclosure. Various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims. Example 1

[0180] This example shows that, RANTES variants linked to the C-peptide C37 are even more effective against R5 virus than the parent RANTES variants, leading to low-picomolar HIV inhibition, and the chimeric proteins also exhibited nano-molar anti-X4 activity, which can be further enhanced by co-expression of CCR5 receptor on the target cell surface.

Materials and Methods

Protein Production and Purification

[0181] The genes for 5P12, 5P12-linker-C37, 5P14, 5P14-linker-C37 and P2- linker-C37 were synthesized using overlapping oligonucleotides from Bioneer Corp (Alameda, CA). Mutations to 5P12-linker-C37 were made using the

QuikChange Site-Directed Mutagenesis method (Stratagene, La Jolla, CA).

These genes were expressed in the pET-28b expression vector (Novagen, Madison, Wl) along with an N-terminal SUMO tag. The vectors were transformed into BL21 (DE3), and grown in 1 liter ¹⁵N minimal medium using ¹⁵NH CI as the only nitrogen source. Protein production was induced with 1 mM IPTG when the absorbance at 600 nM reached 0.7. The cells were incubated with shaking for 20 hours at 22°C after induction and then harvested by centrifugation. The cell pellet was resuspended in cracking buffer (500 mM NaCI, 20 mM Tris, pH 8.0) with 10 mM benzamidine, and French pressed twice at 16,000 psi. The solution was centrifuged at 20,000 *g for 30 minutes. The supernatant was discarded and the pellet was resuspended in 10 ml refolding buffer (5 M Guanidinium/HCI, 3 mM EDTA, 50 mM Tris, 50 mM NaCI, pH 8.0) with 10 mM β-mercaptoethanol. The resuspended solution was incubated at room temperature for 2 hours with stirring followed by a centrifugation at 20,000 xg for 60 minutes. The supernatant containing the denatured protein was added dropwise into 90 ml dripping buffer (50 mM NaCI, 20 mM Tris, pH 8.0) with 10 mM β-mercaptoethanol. The solution was incubated overnight at 4°C and then precipitants were removed by

centrifugation at 20,000 xg for 60 minutes. The supernatant was dialyzed in 4 L dialysis buffer (50 mM NaCI, 20 mM Tris, pH 8.0) with slow stirring, and the buffer was changed after 6 hours. After dialysis, the solution was centrifuged again, and the supernatant, containing refolded protein, was passed through a Ni chelating column and eluted with imidazole in 500 mM NaCI, 50 mM Tris (pH 8.0) buffer. The purified proteins were dialyzed in 4 L 50 mM NaCI, 20 mM Tris buffer (pH 8.0) to remove imidazole. To cleave the SUMO tag, recombinant ULP1 protease was added and the solution was incubated overnight at 4°C. ULP1 protease was added and the solution was incubated overnight at 4°. (ULP1 protease was produced and purified in our lab as briefly described: ULP1 was expressed in LB medium using a pET-28b vector and the cells were collected and French pressed. The ULP1 protease from the supernatant was purified using a Ni chelating column). Precipitated matter was removed by centrifugation at 20,000 xg for 30 minutes and the product was separated from the SUMO tag using an acetonitrile gradient on a C4 reversed phase chromatography column (Vydac, Hesperia, CA) on an Akta purification system (GE Healthcare), and then lyophilized by the Labconco freeze dry system (Labconco Corporation, Kansas City, MO). Applicants were able to obtain a yield of 5 mg pure protein from 1 liter E. coli prep. For proteins containing a 20-amino-acid linker, the protocol was modified to include an extra step of centrifugation to remove unfolded protein before adding TFA and acetonitrile for the final C4 column purification step. C37 peptide: The N-acetylated and C-amidated fusion peptide C37 was purchased from Genescript (Piscataway, NJ).

[0182] The amino acid sequence of the chimeric inhibitors are shown FIG. 9. FIG. 9A shows the amino acid sequence of 5P12-linker-C37. FIG. 9B shows the amino acid sequence of 5P14-linker-C37.

NMR Spectroscopy

[0183] All NMR data were acquired at 25 °C on a four-channel 600 MHz Bruker Avance III spectrometer equipped with a GRASP II gradient accessory and a TCI cryoprobe, which has an actively-shielded Z-gradient coil. NMR samples were prepared by adding ¹⁵N-labelled lyophilized proteins into 20 mM sodium phosphate buffer (pH = 2.5) with 5% D₂O. The chemical shift was referenced relative to internal DSS (2,2-dimethyl-2- silapentane-5-sulfonic acid) ( Wishart, D. S.et al., (1995) J. Biomol. NMR 6, 135-140). The data were

processed using NmrPipe ( Delaglio, F. et al., (1995) J. Biomol. NMR 6, 277-293) and analyzed using PIPP ( Garrett, D. S. et al., (1991 ) J. Magn. Reson. 95, 214- 220). For 2D HSQC spectra, SW=6982.631 Hz (1 H) and 1700.030 Hz (15N), with 512^* points in ¹H and 128^* points in ¹⁵N.

Cell Lines and Viruses

[0184] HeLa-ADA, and Hel_a-P5L cells were kindly provided by Dr. M. Alizon and Dr. Anne Brelot (Cochin Institute, Paris, France) ( Pleskoff, O. et al., (1997) Science 276, 1874-1878). HeLa-TZM-bl, HL2/3 and Magi-CXCR4 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Hela-TZM-bl cell line from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tanzyme Inc ( Takeuchi, Y.et al., (2008) J Virol 82, 12585- 12588; Wei, X.et al., (2002) Antimicrob Agents Chemother 46, 1896-1905;

Derdeyn, C. A. et al., (2000) J Virol 74, 8358-8367; Piatt, E. J., Wehrly, K.et al., (1998) J Virol 72, 2855-2864); HL2/3 from Dr. Barbara K. Felber and Dr. George N. Pavlakis ( Ciminale, V. et al., (1990) AIDS Res Hum Retroviruses 6, 1281 - 1287); Magi-CXCR4 was donated by Dr. Michael Emerman (Vodicka, M. A. et al., (1997) Virology 233, 193-198). 293FT cells were kindly provided by Dr. Jennifer Manilay and were originally obtained from Invitrogen (Carlsbad, CA). Viruses used in replication-competent viral assays and PBMC assays including HIV-1 Ba- L, ADA and 1MB were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 Ba-L from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo (Gartner, S. et al., (1986) Science 233, 215-219); HIV-1 ADA-M from Dr. Howard Gendelman (Gendelman, H. E. et al., (1992) J Immunol 148, 422-429; Westervelt, P. et al., (1991 ) Proc Natl Acad Sci U S A SS, 3097-3101 ; Gendelman, H. E. et al., (1989) AIDS 3, 475- 495; Gendelman, H. E. et al. (1988) J Exp Med 167, 1428-1441 ); HIV-1 1MB from Dr, Robert C. Gallo (Popovic, M. et al., (1984) Lancet 2, 1472-1473; Popovic, M.et al., (1984) Science 224, 497-500; Ratner, L. et al., (1985) Nature 313, 277- 284).

Cell-Cell Fusion Assay

[0185] HIV-1 cell-cell fusion assays were carried out as previously described (Pleskoff, O. et al., (1997) Science 276, 1874-1878). Briefly, 10⁴/well target cells (Hel_a-P5L for R5-tropic fusion assay, HeLa-TZM-bl and Magi-CXCR4 for X4- tropic fusion assay) were plated in a 96 well plate. After 12 hours incubation, the medium was replaced with 50 μΙ_ per well fresh RPMI 1640 (DMEM for the X4 assay). Different concentrations of inhibitors were added and mixed well. 10⁴/well effector cells (HeLa-ADA for R5 assay, HL2/3 for X4 assay) in 50 μΙ_ medium were then added to each well. The cells were incubated at 37 °C for 24 hours to allow fusion. Cells were lysed with 0.5% NP-40 (US Biological) in PBS (pH 7.4) for 30 min, then 30 μΙ_ PBS with 8 mM substrate CPRG (chlorophenol red- -D- galactopyranoside, Calbiochem), 20 mM KCI and 10 mM β-mercaptoethanol (Sigma) was added to each well. The absorbance signal at wavelength 570 nm and 630 nm were measured. Data was analyzed using KaleidaGraph (synergy Software, Reading PA).

Single-round viral infection assay

[0186] Plasmids used to generate the pseudotyped viral particles were all obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Plasmid pNL4-3.Luc.R^"E^" (with deletion of the envelope and vpr genes), and envelope plasmids pSV-ADA, pSV-JRFL from Dr. Nathaniel Landau (Connor, R. Let al., (1995) Virology 206, 935-944); pHEF- VSVG from Lung-Ji Chang (Chang, L. J. et al., (1999) Gene Ther 6, 715-728). pCAGGS-SF162-gp160 from Leonidas Stamatatos and Dr. Cecilia Cheng-Mayer (Cheng-Mayer, C. et al., (1997) J Viron\ , 1657-1661 ; Stamatatos, L. et al., (1998) AIDS Res Hum Retroviruses 14, 1 129-1 139; Stamatatos, L., Lim, M., and Cheng-Mayer, C. (2000) AIDS Res Hum Retroviruses 16, 981 -994); HXB2-env from Dr. Kathleen Page and Dr. Dan Littman (Page, K. A. et al., (1990) J Virol 64, 5270-5276). HIV-1 clone Ba-L.01 from Dr. John Mascola (Li, Y. et al., (2006) J Virol 80, 1414-1426); pSVIII-91 US005.1 1 from Dr. Beatrice Hahn (Gao, F. et al., (1996) J Virol 70, 1651 -1667); 6535, clone 3 (SVPB5) from Dr. David Montefiori, Dr. Feng Gao and Dr. Ming Li (Li, M. et al., (2005) J Virol 79, 10108-10125). To make the pseudo-typed viral particles, 293FT cells were co-transfected with the pNL4-3.Luc.R-E-plasmid and an envelope plasmid using the ProFection

Mammalian Transfection System (Promega). 48 hours post-transfection, the supernatant were collected, centrifuged and filtered with a 0.45 μΜ syringe filter. The viral stocks were stored at -80 °C. For the infection assay, 10⁴ per well cells (HeLa-TZM-bl cell for both the R5 and X4 tropic assays, Magi-CXCR4 cells for the control "X4-only" assays) were plated in a 96 well plate. The next day, after changing the medium, different concentration of inhibitors were added to the wells and mixed, and then the virus particles were added. The final volume was adjusted to 100 μΙ_ per well. After incubation for 3 days (the medium was changed once), the cells were lysed and the substrate CPRG was added (as described above). The absorbance signal at wavelength 570 nm and 630 nm were measured and the 570/630 ratio for each well was calculated. The data were plotted using Microsoft Excel, and the IC50 was determined using a linear equation fitted between two data points surrounding 50% inhibition. For presentation purposes, data shown in the figures were plotted and fitted as curves using a four-parameter logistic equation in Kaleidagraph (Synergy

Software, Reading, PA).

Replication competent viral assay and PBMC assay

[0187] The replication competent viral assays were performed as previously described et al., (2008) Antimicrob Agents Chemother 52, 1768-1781 ). TZM-bl cells were used for both the CCR5 and CXCR4 tropic assays and the HeLa- CXCR4 cells were used for the control X4 only assays. The PBMCs were freshly isolated and used in viral assays as previously described (Lackman-Smith, C. et al., (2008) Antimicrob Agents Chemother 52, 1768-1781 ; Ptak, R. G., Gallay, P. A., Jochmans, D. et al., (2008) Antimicrob Agents Chemother 52, 1302-1317). The viruses used to infect the target cells were HIV-1 Ba-L, ADA (CCR5 tropic), and 1MB (CXCR4 tropic) strains. Each assay was conducted in parallel with control compounds AMD3100 (CXCR4 inhibitor; positive control inhibitor for 1MB, and negative control inhibitor for Ba-L and ADA) and TAK779 (CCR5 inhibitor; positive control inhibitor for Ba-L and ADA, and negative control inhibitor for 1MB) (data not shown). Cytotoxicity of the inhibitors was assayed using the CellTiter 96 AQueous One Solution cell proliferation assay (Promega) (Lackman-Smith, C. et al., (2008) Antimicrob Agents Chemother 52, 1768-1781 ). All tested compounds showed no toxicity at the highest tested concentration (100 nM). Data were plotted and presented as described for the single-round viral assays.

Receptor density comparison using flow cytometry [0188] The CCR5 receptor expression levels on HeLa-TZM-bl cells and Hel_a-P5L cells were compared using flow cytometry. The cells were incubated with FITC conjugated anti-CCR5 antibody (clone 2D7, BD Biosciences), and the fluorescence values were determined using a FACSAria cytometer (BD

Biosciences). Flow cytometric data was analyzed using FlowJo software

(TreeStar).

Results

Design of the chimeric proteins

[0189] 5P12-RANTES and 5P14-RANTES are variants of the chemokine RANTES developed by Gaertner et al., each with 10 different amino-acid mutations at the N terminus (Gaertner, H. et al., (2008) Proc Natl Acad Sci U S A 105, 17706-1771 1 ). Unlike natural RANTES, which is an agonist to CCR5, 5P12 triggers neither receptor sequestration nor cell signaling, while 5P14 causes receptor sequestration but not signaling. Lack of receptor-signaling activity is a valuable property in an anti-HIV agent since immunologic activation could lead to more susceptibility to infection. The flexible N-terminus of both 5P12 and 5P14 are important for their receptor related anti-HIV function, but the C-terminal amino acids, which typically form an α-helix in the chemokine structure, are likely not functionally important. So it is chosen to link the C37 peptide to the C-terminus of 5P12 and 5P14, leaving the N-terminus intact. The C-termini of 5P12 and 5P14 were covalently linked to the N-terminus of C37 peptide using a 10-amino-acid flexible glycine/serine linker, GGGGSGGGGS (SEQ ID NO: 3). The engineered chimeric proteins are denoted as 5P12-linker-C37 (SEQ ID NO: 1 , FIG. 9A) and 5P14-linker-C37 (SEQ ID NO: 2, FIG. 9B). The sequences of 5P12-linker-C37 and 5P14-linker-C37 are also provided below.

A: 5P12-linker-C37 (SEQ ID NO: 1)

QGPPLMATQSCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQV CANPEKKWVREYINSLEMSGGGGSGGGGSHTTWMEWDREINNYTSLIHSL IEESQNQQEKNEQELL

B: 5P14-linker-C37 (SEQ ID NO: 2)

QGPPLMSLQVCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQV CANPEKKWVREYINSLEMSGGGGSGGGGSHTTWMEWDREINNYTSLIHSL IEESQNQQEKNEQELL [0190] All the individual RANTES variants and chimeric inhibitors were expressed in E. coli, and purified to no less than 95% purity as determined by SDS-PAGE. Proteins were tested by NMR to determine the structural integrity. ¹H-¹⁵N correlation spectra revealed that all proteins are nicely folded (FIG. 2). 5P12-linker-C37 and 5P14-linker-C37 exhibited the same peak placement as free 5P12 and 5P14, respectively, except for the extra peaks in the unstructured region of the spectrum, which are likely caused by the linker and the C37 peptide, which is known to be unstructured in the absence of its binding partner. These data indicate that linking C37 to a RANTES variant does not compromise the native structure of the RANTES variant.

Anti-viral potencies of 5P12-linker-C37 and 5P14-linker-C37 against R5-tropic HIV viruses

[0191] In vitro assays including the cell-cell fusion assay, single-cycle viral infection assay, replication-competent viral assay and PBMC assay were conducted to evaluate the anti-viral potencies of the chimeric inhibitors (FIG. 3). Control compounds including RANTES variants alone, C37 alone, and a mixture of RANTES variants with C37 (1 :1 ratio) were tested in parallel with the chimeric inhibitors.

[0192] In R5-tropic cell-cell fusion assays (Table 1 ), consistent with previously published data, 5P12-RANTES and 5P14-RANTES showed anti-HIV IC₅o values of 50 pM and 30 pM respectively, while the IC₅₀ of C37 was in the low-nano-molar range, 2 orders of magnitude higher. As expected, because of the large differences of the RANTES variants and C37 in anti-viral potencies, simply mixing 5P12 or 5P14 with C37 in 1 :1 ratio exhibited similar potency as 5P12 or 5P14 alone. But the chimeric inhibitors exhibited anti-viral potencies stronger than either of the components alone, or the unlinked combination of the two (Table 1 , FIG. 3A, B). These findings suggest the enhancement is not due to simply adding 2 inhibitors together, but rather an intra-molecular mechanism. Table 1 : Anti-HIV activity of linker compounds in R5 cell-cell fusion assays

Inhibitors 5P12 C37 5P12+C37 5P12-linker-C37

IC50 (nM) 0.051 ± 0.01 9.1 ± 4.1 0.044 ± 0.005 0.009 ± 0.003

Inhibitors 5P14 C37 5P14+C37 5P14-linker-C37

IC50 (nM) 0.03 ± 0.002 9.1 ± 4.1 0.03 ± 0.01 0.006 ± 0.001

Results are average IC50 ± SD from 4 or more independent experiments in triplicate. R5 fusion stands for P5L (R5) cells fusion with Hela-ADA cells.

[0193] Extensive testing with multiple strains of R5 virus in single-cycle viral infection assays in TZM-bl cells revealed similar results (Table 2, FIG. 3 C, D).

Table 2: Anti-HIV activity of linker compounds in Single-cycle viral assay

5P12-linker-

HIV virus Tropism 5P12 C37 5P14-linker-C37 5P14

C37

Ba-L R5 0.004 ± 0.001 0.29 ± 0.09 15 ± 0.6 0.007 ± 0.001 0.16 ± 0.01

SF162 R5 0.006 ± 0.001 0.59 ± 0.09 38 ± 13 0.017 ± 0.005 0.22 ± 0.06

ADA R5 0.025 ± 0.004 0.47 ± 0.09 44 ± 14 0.037 ± 0.002 0.18 ± 0.03

JRFL R5 0.015 ± 0.001 0.51 ± 0.01 49 ± 7.4 0.02 ± 0.006 0.14 ± 0.03

US005 R5 0.03 ± 0.006 0.20 ± 0.02 59 ± 16 0.025 ± 0.006 0.09 ± 0.03

6535 R5 0.22 ± 0.05 0.55 ± 0.07 261 ± 57 0.08 ± 0.02 0.10 ± 0.03

HXB2 (Magi-

X4 18 ± 7.4 9.6 ± 1.3 14 ± 5.0 >500 X4)

HXB2 (TZM) X4 0.001 ± 0.0003 6.1 ± 0.3 0.001 ± 0.0001 >500

VSV-G Ctrl >500 >500 >500 >500 >500

Results are average IC50 ± SD (nM) from 4 or more independent experiments in triplicate. [0194] For all 6 strains tested, 5P12-linker-C37 and 5P14-linker-C37 exhibited up to 100 fold greater potency compared to 5P12 and 5P14 alone, or 1 :1 mixtures of them with C37 (Table 3).

Table 3: Anti-HIV activity of 5P12 + C37 and 5P14+ C37 in Single-round viral assays

HIV virus Tropism 5P12+C37 5P14+C37

BaL R5 0.30 ± 0.05 0.16 ± 0.03

SF162 R5 0.61 ± 0.11 0.13 ± 0.08

ADA R5 0.54 ± 0.06 0.13 ± 0.009

JRFL R5 0.56 ± 0.05 0.19 ± 0.09

US005 R5 0.28 ± 0.08 0.10 ± 0.01

6535 R5 0.7 ± 0.2 0.08 ± 0.008

HXB2 X4 9.6 ± 1.3 8.5 ± 1.5

(Magi-X4)

HXB2 X4 3.6 ± 0.1 4.6 ± 0.3

(TZM)

VSV-G > 500 > 500 > 500

Results are average IC50 ± SD (nM) from 4 or more independent experiments in triplicate. 5P12+C37 and 5P14+C37 groups showed similar R5 anti-viral activity to 5P12 and 5P14, and similar X4 anti-viral activity to C37.

[0195] It was also found that the potency enhancement is strain dependent: for virus strains that are particularly sensitive to C37 such as Ba-L, 5P12-linker- C37 and 5P14-linker-C37 showed 70- and 23- fold potency enhancement over 5P12 and 5P14, respectively. But for virus strains that are less sensitive to C37 such as 6535, 5P12-linker-C37 was only 2.5 fold better than 5P12, while 5P14- linker-C37 showed no enhancement over 5P14. These data indicate that the linked C-peptide is critical for the enhancement of the inhibition of the RANTES variant in the chimera, and suggest that effect of the fusion peptide on the specific viral strain determines the magnitude of the relative potency of the enhancement over the RANTES variant alone (FIG. 7).

[0196] A series of assays with replication-competent HIV was also carried out. CCR5 tropic HIV-1 ADA and HIV-1 Ba-L strains were used to infect TZM-bl cells at different concentrations of inhibitors. Compared to the results of the single-round viral infection assay, all inhibitors were less effective in inhibiting viral replication. For example, 5P12 and 5P14 had nanomolar rather than mid- picomolar inhibition against tested strains. But the chimeric inhibitors consistently showed stronger inhibition, with 5P12-linker-C37 showing up to 157 fold enhancement over 5P12 alone, and with 5P14-linker-C37 showing up to 56 fold better inhibition than 5P14 alone (Table 9, FIG. 3 E, F).

Table 9: Anti-HIV activity of chimeric inhibitors in replication-competent viral assay

Tropis 5P12-linker- 5P14-linker-

HIV virus 5P12 C37 5P14 m C37 C37

Ba-L R5 0.08 ± 0 12.61 ± 8.19 >100 0.08 ± 0.01 4.52 ± 0.64

ADA R5 0.65 ± 0.08 28.5 ± 5.9 >100 0.63 ± 0.06 21.8 ± 0.1

IIIB (HeLa-

X4 10.1 ± 0.1 > 500 10.4 ± 0.2 9.23 ± 1.77 > 500 X4)

IIIB (TZM) X4 0.05 ± 0 > 500 77.1 ± 21.1 0.04 ± 0 > 500

Results are average IC₅₀ ± the uncertainty of the average (half the difference) (nM) from 2 independent experiments in triplicate. ± 0 indicates the two experiments yielded identical IC₅₀s.

[0197] As described above, the chimeric inhibitors consistently showed better inhibition than the parent compounds against HIV in engineered cell lines, which may be different than natural human cells in properties such as receptor expression level. To determine the success of these chimeric inhibitors on primary human cells and to get an estimation of their real potency against HIV on its natural targets, the inhibitors were tested on human peripheral blood mononuclear cells (PBMC). The PBMC results confirmed the previous findings, with 5P12-linker-C37 being 45 fold better than 5P12 alone, and 5P14-linker-C37 being 26 fold better than 5P14 alone (Table 4).

Table 4. Anti-HIV activities of the chimeric inhibitors in PBMC assay

HIV virus Ba-L 91US001 91US004 IIIB (X4)¹ CMU02 92UG001 92UG001

(R5)¹ (R5)² (R5)² (X4)² (X4)² (X4R5)²

5P12-linker- 0.015 ± 0.02 0.04 0.44 ± 0.07 0.57 18.1 C37 0.005 0.02

5P12 0.675 ± 0.18 0.21 > 100 > 100 > 100 > 100

0.5

C37 13.85 ± 28.6 49.4 7.0 ± 2.5 18.2 39 58.7

8.3

5P14-linker- 0.015 ± 0.09 0.27 3.1 ± 2.3 > 100 > 100 > 100 C37 0.005

5P14 0.395 ± 0.07 0.12 > 100 0.04 42.4 9.47

0.26

¹Results are average IC50 ± the uncertainty of the average (half the difference) nM) from 2 independent experiments in triplicate.

Results are IC50 (nM) from one independent assay in triplicate.

Anti-viral potencies of 5P12-linker-C37 and 5P14-linker-C37 against X4-tropic HIV viruses

[0198] Since 5P12 and 5P14 work by binding CCR5 and are therefore only effective against R5-tropic virus, they showed no inhibition against X4 envelopes, either in cell-cell fusion assays or in viral assays using X4-tropic virus against Magi-X4 cells, as expected (Table 1 , Table 5, FIG. 4 A, B).

Table 5: Anti-HIV activity of linker compounds in X4 cell-cell fusion assays

Inhibitors C37 5P12-linker-C37 5P12 + C37 5P14-linker-C37 5P14 + C37

X4 (nM) 1 .5 ± 0.3 4.7 ± 0.9 2.2 ± 0.2 4.3 ± 0.5 2.4 ± 0.7

R5/X4 (nM) 2.1 ± 0.8 0.006 ± 0.003 2.1 ± 0.2 0.005 ± 0.002 2.6 ± 1 .0

Results are average IC50 ± SD from 4 or more independent experiments in triplicate. X4 fusion stands for Magi-X4 cells fusion with HL2/3 cells. R5/X4 fusion stands for TZM (R5/X4) cells fusion with HL2/3 cells.

[0199] In contrast, the peptide C37 is active against both R5 and X4 tropic virus due to its ability to bind gp41 , and this peptide exhibits nanomolar-level inhibition potency in X4 fusion and viral assays. The designed chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 also show anti-X4 activity due to the action of the C37 portion of the molecule, as shown in FIG. 4 A and B. These chimeric inhibitors exhibit IC₅₀ values nearly identical to C37, demonstrating their effectiveness even when one portion (the RANTES variant) is not utilized for the inhibition.

[0200] More striking results are observed when performing assays with X4- tropic virus on the TZM-bl cell line, which expresses both CXCR4 and CCR5 receptors. While the IC₅₀ of C37 alone remains the same as on the Magi-X4 cell line, the anti-viral potency of the chimeric inhibitors increased 400- and 6,000- fold compared to C37 in cell fusion and in single-cycle viral assays, respectively (Table 2, 5, FIG. 4 C, D). Parent protein controls 5P12-RANTES and 5P14- RANTES showed no inhibition as expected, indicating that CCR5 binding alone by the RANTES variants does not inhibit X4-tropic viral entry, as expected.

Furthermore, potency enhancement was not seen when 5P12 or 5P14 were mixed with C37 in 1 : 1 ratio, which led to the same activity as C37 alone. The high potency of the chimeric proteins against X4 virus under these conditions suggests a strong enhancement by the intramolecular action of both components of the chimeric protein. This enhancement likely involves the RANTES variants being bound to CCR5 receptors, placing the C37 part of the chimera in the proper position to bind its target gp41 .

[0201] Given the fact that CCR5 and CXCR4 receptors form hetero-oligomers on the cell surface, it is very likely that by binding to the CCR5 receptors, the RANTES variants in the chimeric protein could specifically deliver C37 to its nearby virus target, which is presumably using the nearby CXCR4 as a co- receptor, to achieve the strong enhancement of X4 inhibition potency. To provide evidence for this, experiments were carried out in which the CCR5 receptors were blocked prior to adding X4 tropic virus and chimeric inhibitor. In both fusion and single-cycle viral assays, target cells were pre-incubated with 100 nM 5P12 or 5P14 before 5P12-linker-C37 or 5P14-linker-C37 were added. Since the CCR5 receptors were blocked by excessive amount of RANTES variants and the chimeric inhibitors could not bind to CCR5 receptors, their X4 inhibition potency reverted to that of C37 alone (Table 2, 5, FIG. 4 E, F).

[0202] Replication-competent viral assays were also conducted to confirm the anti-viral activities of chimeric inhibitors against X4-tropic virus replication.

Similar results were observed as from the single cycle assays: while the chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed exactly the same IC₅₀ values as C37 on Hel_a-X4 cells (which expresses only CXCR4 receptors on the surface), they exhibited 1500-1900 fold increased potency against X4 tropic HIV- 1 1MB strain on TZM-bl cells which co-expressed CCR5 and CXCR4 receptors.

[0203] Altogether, the data show that chimeric inhibitors fully retain the anti- X4 activity of C37, and that this anti-X4 potency can be further enhanced when the target cells co-express CCR5 on the surface.

[0204] Viral assays against PBMC, some of which also express both CCR5 and CXCR4 receptors on the surface, were then carried out to evaluate the X4 tropic anti-viral potencies of these chimeric inhibitors on natural human cells. As expected, 5P12 and 5P14 alone did not show any inhibition against the X4-tropic HIV-1 1MB strain since these proteins are only able to bind the CCR5 coreceptor and not CXCR4. The peptide C37 alone showed an l_C5o of 7 nM. The chimeric inhibitors, however, showed inhibition that was up to 16 fold better than C37 alone as judged by IC50, most likely due to the coexpression of CCR5 receptors on some of the cells (Table 5). This result showed that the chimeric inhibitors were extremely potent against X4-tropic viruses even on natural human cells. Further testing on clinical X4-tropic strains CMU02 and 92UG029 exhibited 260- fold and 68-fold improvement by 5P12-linker-C37 over C37. 5P14-linker-C37 also showed greatly enhanced activity over C37 for strain CMU02, although not for 92UG029 (Table 4). The chimeric inhibitors were also tested against a clinical dual-tropic strain, 92UG001 . In this case, the RANTES variants 5P12 and 5P14 alone showed no activity, while C37 had an IC50 of 59 nM. Both 5P12-linker-C37 and 5P14-linker-C37 showed several fold increased potency over C37 alone (Table 4).

Receptor density comparison using flow cytometry

[0205] The CCR5 receptor expression levels on HeLa-TZM-bl cells and Hel_a-P5L cells were compared using flow cytometry. The cells were incubated with FITC conjugated anti-CCR5 antibody (clone 2D7, BD Biosciences), and the fluorescence values were determined using a FACSAria cytometer (BD Biosciences). Flow cytometric data was analyzed using FlowJo software

(TreeStar) (Table 6).

Table 6: Anti-HIV activity in R5 fusion assays with P5L and TZM-bl cell lines

Cell lines Hela-P5L TZM-bl

CCR5 density Low High

5P12 0.051 ± 0.01 14 ± 1 .4

5P12+C37 0.044 ± 0.005 7.0 ± 1 .3

5P12-linker-C37 0.009 ± 0.003 0.8 ± 0.2

C37 9.1 ± 4.1 340 ± 80

5P14 0.03 ± 0.002 1 1 ± 2.4

5P14+C37 0.03 ± 0.01 4.8 ± 0.2

5P14-linker-C37 0.006 ± 0.001 0.6 ± 0.02

Results are average IC₅₀ ± SD (nM) from 4 or more independent experiments in triplicate. Fusion with Hela-ADA cells.

Mechanism of the chimeric inhibitors

[0206] Several assays, including R5 and X4 tropic cell fusion assays, single- cycle viral infection assays, replication-competent viral assays, and PBMC assays indicate the success of the strategy to covalently link RANTES variants with a C-peptide. These experiments also provide evidence suggesting the potency is probably due to the excellent inhibition of the RANTES variant, along with its ability to specifically deliver the C37 to its gp41 target. Therefore, experiments were designed to further characterize the mechanism of action of the chimeric inhibitors, first focusing on the relative importance of the C37 portion.

[0207] A series of mutations were made to 5P12-linker-C37 (FIG. 5, Table 7, Table 8), and the corresponding effects were evaluated with cell fusion and single-cycle viral assays. It has previously been shown that that the mutation of lie to Asp in position 642 in the C-peptide causes a 10,000 fold drop of anti-HIV activity, almost completely abolishing its function, while the lie mutation to Asp at position 656 causes a moderate, 80 fold decrease of activity. Therefore, mutations to Asp were made on the 642th and 656th positions of C37 in 5P12- linker-C37. To test whether mutations reduce the activity of the chimeric protein, the mutants were tested with Magi-X4 cell based fusion and viral assays. 5P12- Iinker-C37I642D completely lost its ability to inhibit X4 at lower than 500 nM concentration, while 5P12-Iinker-C37I656D showed 10 - and 3 - fold decrease in activity in fusion and viral assays, respectively compared to the wild type chimera (FIG. 5A). These mutants that weaken the potency of the C-peptide also reduced the overall effectiveness of the chimeric protein against R5-tropic viruses. The anti-viral potency of both 5P12-Iinker-C37I642D and 5P12-Iinker-C37I656D were much lower compared to 5P12-linker-C37, inhibiting similarly to free 5P12 (FIG. 5C, Table 7, Table 8). Similar results were observed in X4 assays with TZM-bl cells that contain both CCR5 andCXCR4 on the surface.

Table 7. Anti-HIV activities of 5P12-linker-C37 mutations in single-cycle viral assay

Mutation in

Mutation in C37 Change of linker length RANTES

5P12-

HIV 5P12- 5P12- 5P12-

Tropism linker- P2- 5P12- virus linker- linker- (GGGGS)₄- C37 RANTES- GGS- C371642 C37I656 C37 linker-C37 C37

D D

0.004 ± 0.24 ± 0.20 ± 0.06 ± 0.002 ±

Ba-L R5 0.82 ± 0.09

0.001 0.04 0.02 0.001 0.001

0.006 ± 0.75 ± 0.27 ± 0.1 ± 0.004 ±

SF162 R5 0.80 ± 0.02

0.001 0.36 0.07 0.02 0.0005

0.025 ± 0.29 ± 0.15 ± 0.1 ± 0.016 ±

ADA R5 0.61 ± 0.05

0.004 0.02 0.02 0.003 0.004

HXB2

(Magi- X4 18 ± 7.4 17 ± 2.4 > 500 60 ± 20 27 ± 3.1 11 ± 6.4

X4)

HXB2 0.001 ± 0.10 ± 0.28 ± 0.002 ±

X4 0.28 ± 0.07 > 500

(TZM) 0.0003 0.02 0.05 0.001

VSV-

Ctrl > 500 > 500 > 500 > 500 > 500 > 500 G

Results are average IC₅₀ ± SD (nM) from 4 or more independent experiments in triplicate.

[0208] The RANTES portion of 5P12-linker-C37 was also mutated to determine the effect of the RANTES portion on the overall chimeric protein. In particular, the potent 5P12 N-terminus was changed into that of another RANTES variant, P2-RANTES, which differs from 5P12 by the first 10 amino-acids. P2-RANTES is also an R5 ligand, but with lower anti-viral potency against R5 virus (nanomolar- level inhibition of cell fusion and pseudotyped viral infection). Substitution of the RANTES part of the linker protein did not affect the C-peptide portion of the chimera, as evidenced by the similar activity as 5P12-linker-C37 and C37 in X4- tropic assays under conditions where only the C37 portion would be expected to be active (Table 7, Table 8, FIG. 5A). But this variant did show decreased activity in R5 and TZM-bl X4 assays (FIG. 5C, E).

Table 8: Activities of 5P12-linker-C37 mutations in cell-cell fusion assays

Mutation in

Mutation in C37 Change of linker length

RANTES

5P12-

Tropism P2- 5P12- 5P12- linker-C37 5P12-linker- 5P12-GGS- RANTES- linker- (GGGGS)₄- C37I656D C37

linker-C37 C37I642D C37

0.009 ± 0.06 ± 0.07 ±

1.2 ± 0.09 0.03 ± 0.01 0.01 ± 0.002

0.003 0.005 0.003

X4 (Magi- 4.7 ± 0.9

3.9 ± 0.5 59.4 ± 15.4 4.1 ± 0.9 6.1 ± 1.7 X4)

0.006 ± 0.003 ±

X4 (TZM) 0.2 ± 0.09 0.05 ± 0.01 0.008 ± 0.003

0.003 0.001

Results are average IC50 ± SD (nM) from 4 or more independent experiments in triplicate.

[0209] These data indicate that both parts of the linker protein are necessary and that they have to be functioning on the same molecule to show an

enhancement of potency.

[0210] Having demonstrated the necessity of both portions of the chimera, it is hypothesized that the observed enhancement of the chimeric inhibitors was likely due to the specific delivery of C37 to the nearby gp41 target by the RANTES variant as it binds to the CCR5 co-receptor. To probe this possibility, the effects of different linker length on the overall activity of the linker protein were tested. Mutant chimeric inhibitors with 3-amino-acid linker "GGS" and 20-amino-acid linker "(GGGGS)₄" were made to compare with the 5P12-linker-C37 which has a flexible 10-amino-acid linker (GGGGS)2. NMR experiments were done to confirm the structural integrity of the mutants. The spectra showed that the 20-amino-acid long linker led to a significant portion of unfolded protein using our regular purification method, so we modified the protocol to obtain pure and fully folded chimeric protein with the 20-amino-acid long linker. Finally, 2D HSQC spectra verified the structural integrity of all mutant chimeric proteins and showed that the change of linker length did not affect the structure of 5P12 (data not shown). As a control, X4-tropic single round virus assays using MAGI-X4 cells (containing no CCR5) confirmed that different linker length did not affect the anti-viral function of the C37 portion of these chimeras (FIG. 5B). This was expected, since it has already been shown that under these conditions the C-peptide is the only component involved in inhibition while the RANTES variant is not active against X4 tropic strains. In contrast, R5 tropic viral assays with 3 different strains showed that the shorter linker 5P12-GGS-C37 has the lowest anti-viral potency, 15-fold lower compared to that 5P12-linker-C37. The moiety with the longer linker, 5P12-(GGGGS)₄-C37, showed almost the same activity as 5P12-linker- C37. Similar results were observed in X4-tropic assays using TZM-bl (containing both surface R5 and X4) as target cells, where 5P12-GGS-C37 showed the lowest ability to inhibit, and where 5P12-linker-C37 had similar activity to 5P12- (GGGGS)₄-C37, and (Table 7).

[0211] Since the individual activities of 5P12 and C37 were not affected, the change of anti-viral activity in these altered chimeras was likely caused by the change in linker length. The data suggest that the linker has to be long enough to allow both parts of the linker protein to function at the same time, and support the hypothesis that the enhancement of potency in the chimeric inhibitor is due to the specific delivery of C37 to the nearby gp41 target by the RANTES variant as it binds to CCR5.

[0212] The chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 exhibited anti-viral potency higher than either of the individual components alone or in combination. They were able to inhibit R5 tropic HIV at low picomolar level in all the in vitro assays, and therefore are among the most potent entry inhibitors yet reported. The chimeric inhibitors also fully retained the anti-X4 activity of C37, and this anti-X4 potency can be further enhanced when the cells co-express CCR5 on the surface. The chimeras therefore overcome the major drawbacks of the parent co-receptor inhibitors 5P12 and 5P14 which lack activity against X4 tropic virus. Another advantage is that by blocking HIV entry at two steps, the chimeric inhibitors are less likely to be evaded by virus through mutations.

[0213] The chimeric inhibitors of the present invention are straightforward and inexpensive to produce in E. coli, highly active against R5 viruses, active against X4-tropic viruses regardless of the presence of a particular co-receptor on the surface of the target cell, and are very potent on PBMCs. Single-chain

monomeric chimeric proteins are particularly easy to generate and more effective than larger molecules such as antibodies.

[0214] The overall effectiveness of anti-HIV chimeric inhibitors relies on two major factors aside from the components' innate effectiveness: viral susceptibility to the C37 peptide, and CCR5 receptor density. As shown in Table 2, the effectiveness of the chimeric inhibitors on 6 different singe-round R5 viruses was tested. The viruses showed variable sensitivity to C37, with C37 inhibition IC₅₀ ranging from 15 nM to 261 nM, while showing quite similar sensitivity to 5P12 and 5P14. The relative potency enhancement of the chimeric inhibitors over the parent RANTES variants also varied from 1 to 100 fold, and was largely in proportion to the virus' susceptibility to C37: the more sensitive the virus to C37, the more potency enhancement of the chimeric inhibitor over the RANTES variants alone against that virus (FIG. 7).

[0215] The effectiveness of the chimeras also depend on receptor density on the target cells, which is also true for other inhibitors: Lower receptor density leads to more sensitivity to inhibition. In the present invention, R5-tropic fusion assays were carried out on 2 cell lines with differing amounts of CCR5 on the surface, and the results showed that the lower the CCR5 density, the more potent the chimeric inhibitor (FIG. 8, Table 6). [0216] Extensive in vitro viral assays and mutagenesis studies were carried out to investigate the mechanism of the chimeric inhibitors. In R5 tropic viral assays, the chimeric inhibitors showed up to 100 fold potency enhancement over the parent RANTES variants while a simple mixture of the RANTES variants and C37 showed no enhancement, indicating that C37 enhanced the R5 anti-viral potency of the RANTES variants, and the mechanism involves both components being covalently linked. Similar conclusions can be drawn from the results of an X4 tropic viral assay on TZM-bl cells, which contain both CCR5 and CXCR4 on their surface. While the only active part of the chimeric inhibitor against X4 tropic virus is C37, as much as 6000 fold enhancement of potency was observed. This effect disappeared when the CCR5 receptors were blocked, suggesting that the

RANTES variant is binding CCR5 and specifically delivering the C37 portion of the chimera to gp41 . Mutagenesis on either part of the chimeric protein showed that both parts are essential and they are functioning at the same time. Change of linker length also provided valuable information about the space requirements of this intra-molecular mechanism.

[0217] Based on these findings the following model is proposed to explain the mechanism of the chimeric inhibitors on both R5 and X4 tropic viruses. The inhibitors likely inhibit R5 tropic virus by binding to both the CCR5 co-receptor and the gp41 N-terminal trimer of hairpin simultaneously or near-simultaneously. By binding to the co-receptor, the chimeric inhibitors could specifically delivery C37 near to its target on gp41 , and potentially increase the local concentration of C37 on the cell surface (FIG. 6A). When inhibiting X4 virus on cells containing only CXCR4 receptors, the chimeric inhibitors behave essentially as C37 alone by binding only to gp41 (FIG. 6B). When the cells express both CCR5 and CXCR4, the chimeric inhibitors can deliver C37 to its target by binding to a CCR5 receptor that is presumably in proximity to a CXCR4 that is being used as a co- receptor for infection due to the known hetero-oligomerization of CCR5 with CXCR4. By specific delivery of C37 to its target and possibly increasing the local concentration of C37 on the cell surface, the chimeric inhibitors block HIV more efficiently than C37 alone (FIG. 6C). [0218] It is reported here the success of a strategy to covalently link potent CCR5 binding proteins with a gp41 -binding C peptide. The chimeric inhibitors exhibited extremely high anti-viral potency, and were able to inhibit both R5 and X4 tropic viruses. Since the inhibitors block HIV at two steps, they are likely more resistant to viral mutations. Also, as fully recombinant inhibitors, they are inexpensive and relatively easy to produce. Overall, these inhibitors are excellent candidates for HIV microbicides. This work could also provide insight for a general approach for optimizing existing HIV entry inhibitors or designing new inhibitors.

[0219] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure.

Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

CLAIMS:

1 . An isolated chimeric polypeptide comprising a first portion comprising a CCR5- binding protein and a second portion comprising a gp41 -binding protein, wherein the chimeric polypeptide does not comprise an lgG1 antibody that specifically recognizes CCR5.

2. The isolated chimeric polypeptide of claim 1 , wherein the gp41 -binding protein comprises one or more of a C-peptide; a N-peptide; C37; C37-ac;

C37(Q652L); N-acetylated, C-term amidated C37; N-acetylated, C-term amidated C37(Q652L); C34; C52L; T-2635; T20; N-peptides; N17; N23; N36 or a

substantial homologue thereof.

3. The isolated chimeric polypeptide of claim 2, wherein the gp41 -binding protein comprises one or more of C37; C37-ac; C37(Q652L); N-acetylated, C- term amidated C37; N-acetylated, C-term amidated C37(Q652L) or a substantial homologue thereof.

4. The isolated chimeric polypeptide of claim 3, wherein the gp41 -binding protein comprises C37 or a substantial homologue thereof.

5. The isolated chimeric polypeptide of any one of claims 1 to 4, wherein the CCR5-binding protein comprises RANTES, P2-RANTES, PSC-RANTES, 5P12-RANTES, 5P14-RANTES, 6P4-RANTES, ΜΙΡ-1 α, ΜΙΡ-1 β, U83A or a substantial homologue of any one thereof.

6. The isolated chimeric polypeptide of claim 5, wherein the CCR5- binding protein comprises 5P12-RANTES or 5P14-RANTES or a substantial homologue thereof.

7. The isolated chimeric polypeptide of any one of claims 1 to 6, wherein the first portion is N-terminal to the second portion.

8. The isolated chimeric polypeptide of any of claims 1 to 7, further comprising a peptide linker between the first portion and the second portion.

9. The isolated chimeric polypeptide of claim 8, wherein the peptide linker comprises from about 1 to about 20 amino acids.

10. The isolated chimeric polypeptide of claim 8 or 9, wherein the peptide linker comprises one or more amino acids selected from alanine, glycine or serine.

1 1 . The isolated chimeric polypeptide of any one of claims 1 to 10, further comprising at least one of a protein start site, a polyhistidine tag, and/or a protease cleavage site, each operatively linked to the chimeric polypeptide.

12. The isolated chimeric polypeptide of any one of claims 1 to 1 1 , wherein the cheimeric polypeptide is a monomeric.

13. The isolated chimeric polypeptide of any one of claims 1 to 12, further comprising an anti-HIV small molecule or compound bound to the isolated chimeric polypeptide.

14. A peptide conjugate comprising a carrier covalently or non-covalently linked to an isolated chimeric polypeptide of any one of claims 1 to 13.

15. An antibody that binds an isolated chimeric polypeptide of any one of claims 1 to 13.

16. The antibody of claim 15, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized antibody.

17. An antibody fragment that binds an isolated chimeric polypeptide of any one of claims 1 to 13.

18. The antibody of any one of claims 15 to 17, further comprising a detectable label conjugated to the antibody.

19. A biologically active fragment of the antibody of claim 15 or 16.

20. An antibody-peptide complex comprising the antibody of any one of claims 16 to 19 and a polypeptide that the antibody specifically recognizes.

21 . An isolated polynucleotide encoding the chimeric polypeptide of any one of claims 1 to 12 or the antibody of any one of claims 15 to 19.

22. A DNA construct comprising an expression vector and a

polynucleotide of claim 21 .

23. The DNA construct of claim 22, wherein the vector is a plasmid vector, a yeast artificial chromosome, or a viral vector.

24. The DNA construct of claim 22 or 23, wherein the vector further comprises a protein tag.

25. The DNA construct of claim 24, wherein the protein tag is selected from a His-tag, a SUMO-tag, a GST-tag, a myc-tag or a FLAG-tag.

26. A chimeric polypeptide of any one of claims 1 to 13 or a peptide conjugate of claim 14 or an antibody of 15 to 19 or an isolated polynucleotide of claim 21 or the DNA construct of any one of claims 22 to 25, further comprising a detectable label.

27. A composition comprising a carrier and one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an antibody of 15 to 19; d) an isolated polynucleotide of claim 21 ; or e) the DNA construct of any one of claims 22 to 25.

28. The composition of claim 27, wherein the carrier is selected from a gel, an aqueous liquid carrier, a paste, a liposome, a micelle, albumin,

polyethylene glycol, a pharmaceutically acceptable polymer, or a

pharmaceutically acceptable carrier.

29. An isolated host cell transformed with an isolated polynucleotide of claim 21 or a DNA construct of any one of claims 22 to 25.

30. The isolated host cell of claim 29, wherein the host cell is selected from a prokaryotic cell or a eukaryotic cell.

31 . A composition comprising an isolated host cell of claims 29 or 30.

32. The composition of claim 31 , wherein the carrier is selected from a gel, an aqueous liquid carrier, a paste, a liposome, a micelle, albumin,

polyethylene glycol, a pharmaceutically acceptable polymer, or a

pharmaceutically acceptable carrier.

33. A method for preventing or inhibiting human immunodeficiency virus (HIV) entry into a cell, comprising contacting the cell with an effective amount of any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an antibody of 15 to 19; d) an isolated

polynucleotide of claim 21 ; or e) the DNA construct of any one of claims 22 to 25.

34. The method of claim 33, wherein the contacting is in vitro or in vivo.

35. A method for inhibiting HIV infection in a subject, comprising

administering to the subject an effective amount of any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25.

36. The method of claim 35, wherein the subject is infected with HIV.

37. The method of claim 35, wherein the subject is at risk of HIV infection.

38. The method of any of claims 35 to 37, wherein the subject is a mammal or a human.

39. The method of any one of claims 32 to 38, wherein the effective amount is administered by injection or topical application.

40. A method for preparing an isolated chimeric polypeptide of one of claims 1 to 13 comprising expressing a polynucleotide encoding the isolated chimeric polypeptide of one of claims 1 to 13 in an isolated host cell.

41 . The method of claim 40, wherein the host cell is a prokaryotic cell or a enkaryotic cell.

42. The method of claim 40 or 41 , further comprising isolating the chimeric polypeptide from the isolated host cell.

43. A therapy comprising any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25 for the treatment or prevention of HIV infection.

44. Use of any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25, in the

manufacture of a medicament for treatment or prevention of HIV infection.

45. A kit for use in preventing or inhibiting HIV entry into a cell, comprising of any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25, and instructions to use.

46. A kit for use in treating a subject in need thereof, comprising of any one or more of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25, and instructions for use.

47. A method for identifying an agent useful for prevention or treatment of HIV infection, comprising:

a) contacting an HIV virus with a cell under suitable condition, the cell being in contact with a candidate agent and assaying the ability of the agent to inhibit HIV entry into the cell;

b) comparing that to the ability of any one or more agent of: a) a chimeric polypeptide of any one of claims 1 to 13; b) a peptide conjugate of claim 14; c) an isolated polynucleotide of claim 21 ; or d) the DNA construct of any one of claims 22 to 25, to inhibit HIV infection,

c) wherein the ability of the agent in step a) that is greater than or substantially equal to the ability of the agent of step b) identifies the agent as useful for the prevention or treatment of HIV infection; and

d) and wherein the ability of the agent in step a) that is substantially less than the ability of the agent of step b) identifies the agent as not useful for the prevention or treatment of HIV infection.