WO2013023166A2 - Hapivirins and diprovirins - Google Patents

Abstract

Hapivirin or diprovirin peptides are small polypeptide agents with potent activity against viruses. A pharmaceutical composition comprising hapivirin or diprovirin as an active agent is administered therapeutically to a patient.

Description

HAPIVIRINS AND DIPROVIRINS

GOVERNMENT RIGHTS

This invention was made with Government support of Grant No. AI067327, awarded by the National Institutes of Health. The Government has certain rights in this invention.

INTRODUCTION

Background

[01] Natural polycationic antimicrobial peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. Nearly all of these peptides have membrane affinity, and can permeate and permeabilize bacterial membranes, resulting in injury, lysis, and/or death to the microbes. In particular, the human peptides known as defensins are produced by mammalian and avian leukocytes (e.g. neutrophils, some macrophages) and epithelial cells.

[02] Influenza virus is a continuing threat to global health, causing approximately 36,000 deaths annually in the USA alone. In non-pandemic years the virus tends to be seasonal and to affect children and the elderly primarily. The pandemic of 1918, caused by an H1 N1 subtype, resulted in millions of deaths including those of healthy, young adults. The first 21 ^st century pandemic occurred in 2009, and again involved an H1 N1 variant that caused mortality in young individuals. Since influenza A viruses have unstable segmented RNA genomes and multiple wild-animal reservoirs that facilitate genetic reassortment, the threat of future pandemics is likely to remain indefinitely.

[03] Influenza viral infections are exemplars of the importance of innate immunity because the mutability of the virus allows it to evade prior adaptive immune responses. After initial exposure to a novel influenza viral strain, it takes 5 to 7 days before specific antibodies and T cells arrive in the lung to clear the virus. This delay defines the critical time window in which innate immunity operates to confine influenza viruses to the upper respiratory tract.

[04] Defensins are cysteine-rich cationic and amphipathic peptides found in plants, insects, mammals and birds. Human neutrophils contain large stores of oc-defensins ⁽HNPs) 1-3 in their primary (azurophilic) granules, and several β-defensins are expressed in human pulmonary (and other) epithelial cells. Collectively, the activity of these a- and β-defensins encompasses Gram-positive and Gram-negative bacteria, fungi, and viruses, θ-defensins are cyclic defensins found in certain non-human primates but not in humans, chimpanzees or gorillas. Their disappearance from the Hominidae is attributable to a premature stop codon within the signal sequence. Retrocyclins 1 and 2 can be considered to be "humanized" θ-defensins because their sequences are encoded within the human genome

l and because recent experiments showed that human epithelial cells can produce retrocyclins peptides when exposed to an aminoglycoside that allows the stop codon to be bypassed.

[05] We have shown that synthetic retrocyclins have stronger IAV neutralizing activity than a- and β-defensins and that they also induce viral aggregation and promote the uptake of IAV by neutrophils. Pulmonary surfactant protein D (SP-D) is an important innate inhibitor of seasonal IAV strains. In contrast to oc-defensins HNP1 and 2, which bind strongly to SP-D and interfere with its antiviral activity retrocyclins and SP-D have additive antiviral activity when combined.

[06] There is a clinical need for novel antiviral agents that have low toxicity against mammalian cells. The present invention addresses this need.

SUMMARY OF THE INVENTION

[07] Methods and compositions are provided for the use of hapivirin or diprovirin peptides.

Hapivirin or diprovirin peptides are small antiviral and anti-plaque agents. Their structures are somewhat similar to those of retrocyclins, theta-defensin peptides with potent anti-viral, antitoxic and antibacterial properties. However, compared to retrocyclins, the new peptides are smaller, have simpler structures, and will be easier and less expensive to manufacture.

[08] A pharmaceutical composition comprising hapivirin or diprovirin as an active agent is administered to a patient suffering from a viral infection. Alternatively, a pharmaceutical composition comprising hapivirin or diprovirin is administered as a protective agent to a normal individual facing potential exposure to virus or to pathogenic microbes. Hapivirin or diprovirin may be administered alone, or in combination with other agents, e.g. antibiotics and/or other antiviral agents, and antiviral agents as a cocktail of effective peptides, etc. The peptides are shown to be effective when administered in combination with surfactant protein D. Hapivirin or diprovirin-mediated killing is also useful for modeling and screening novel antibiotics.

BRIEF DESCRI PTION OF THE DRAWINGS

[09] Figure 1 . Schematic representation of synthesized HpV and DpV peptides - All peptides were synthesized as a C-terminal amides as described in Methods.

[10] Figure 2. Comparison of some amino acids used as substituents in position 7 of HpVs.

[11] Figure 3. IAV neutralizing activity of Hapivirins - Aliquots of the Phil82 strain of IAV were pre-incubated with 1 or 2μg/ml of the indicated Hapivirins. Results are mean±SEM of 3 experiments using the fluorescent focus assay on MDCK cells. All of the tested HpVs significantly reduced infectious foci as compared to virus alone (p<0.05).

[12] Figure 4. Dose response for viral neutralization by HpV1 1 , HpV17, HpV18, and HpV19 - Neutralizing activity of the most potent Hapivirins was compared using A549 and MDCK cells. Panels A-C used the Phil82 viral strains and panel D used the PR-8 strain. Mean±SEM of 4 experiments

[13] Figure 5. Dose response curves for neutralization of IAV by selected Diprovirins - The neutralizing activity of different doses of DpVs that had high (panels A and B) or intermediate (panels C and D) activity on screening assays were tested. Mean±SEM of 4 experiments

[14] Figure 6. Effect of delaying addition of defensins on neutralizing activity - All the peptides were used at concentrations of 1 μg/ml. The defensins were either pre-incubated with IAV (Phil82 strain) as in figure 5 (Group 1 ), or added to the MDCK or A549 cells 15 (Group 1 ) or 45 (Group 3) minutes after viral infection of the cells. In Group 1 all defensins significantly inhibited viral nucleoprotein expression 7 hours after infection (with the single exception of DpV21 in A549 cells). In contrast, no defensin inhibited IAV in groups 2 or 3.

[15] FIGURE 7. Viral-aggregating activity of selected HpVs, DpVs, and RCs. (A-C) Show results of viral aggregation assays using light scattering, in which control buffer (lAValone) or HpVs, DpVs, or RCs were added at time 0. The results are expressed as percentage of light absorption at time 0 and are mean + SEM of three or more experiments in each case. In (A) and (B), 8 mg/ml peptides were used, and in (C) 10 mg/ml was used. Aggregation induced by HpV 1 1 and 17 was highly significant compared with control (p , 0.001 ) and was significantly greater than aggregation induced by RC2 by ANOVA. Aggregation induced by HpV 15 and RC2 was also significant compared with IAV alone (p , 0.05). Aggregation induced by DpV 13 was significantly greater than that induced by RC1 , as assessed by ANOVA (C). The lower panels show results of electron micrographs of untreated virus, or virus treated with the indicated HpVs or DpVs demonstrating virus aggregation. The electron microscopy results were representative of at least three similar experiments using 40 mg/ml peptides.

[16] Figure 8. Aggregation of S. aureus and zymosan particles by HpV1 1 and DpV1607 - Fluorescently labeled S. aureus and zymosan particles were obtained from Molecular Probes and incubated with various HpVs and DpVs, followed by examination using a fluorescent microscope. Representative pictures (from 3 or more experiments) showing aggregation of S. aureus by HpV1 1 (24μg/ml) and DpV1607 (60 μg ml) are shown.

[17] Figure 9. Comparison the ability of HpVs or DpVs to increase viral uptake by neutrophils or RAW cells - FITC labeled Phil 82 virus was used to detect viral uptake by flow cytometry. Panel A shows uptake of virus that was treated with various concentrations of HpV 17 and 18. Panel B shows similar experiments using RAW cells. Results are mean±SEM of 7 experiments for panels A and B using separate neutrophil donors or RAW cells harvested on different days. Panel C shows neutrophil uptake of virus after pre-incubation with the indicated DpVs. Results are mean±SEM of 3 experiments using separate neutrophil donors. All of the tested DpVs caused significant increases in viral uptake by neutrophils at the 3C^g/ml concentration (p<0.03), but only DpV1632 caused a significant increase in uptake at 15 μg/ml. All of the tested peptides caused significant increases in neutrophil or RAW cell uptake of IAV (p<0.05).

[18] Figure 10. Ability of HpVs or DpVs to inhibit IAV -induced TNF generation by human monocytes - Human peripheral blood monocytes were infected with IAV (Phil82 strain) followed by culture in vitro for 18 hrs. TNF was measured by ELISA on culture supernatants as described. Pre-treatment of IAV with the indicated peptides reduced cytokine production as compared to virus alone (^* indicates significant reduction at p<0.05). Results are mean±SEM of 4-5 experiments using separate monocyte donors.

DESCRI PTION OF THE SPECIFIC EMBODIMENTS

[19] Novel compositions and methods are provided for the use of hapivirin or diprovirins and hapivirin or diprovirin analogs as therapeutic and/or prophylactic agents. Hapivirin or diprovirin(s) are administered alone or in combination with other active agents to a patient suffering from an infection, in a dose and for a period of time sufficient to reduce the patient population of pathogenic viruses. Alternatively, a pharmaceutical composition comprising hapivirin or diprovirin or other circular mini-defensins or is administered as a protective agent to a normal individual.

[20] Specific treatments of interest include, without limitation: using hapivirin or diprovirin or a hapivirin or diprovirin analog to prevent or treat infection, for example by influenza virus; aerosol administration to the lungs of patients with cystic fibrosis to combat infection or forestall the emergence of resistance to other inhaled antibiotics; instillation into the urinary bladder of patients with indwelling catheters to prevent infection; application to the skin of patients with serious burns; opthalmic instillation, directly or in ophthalmic solutions, to treat or prevent infection; intravaginal application to treat bacterial vaginosis and/or prevent sexually transmitted viral infection. The hapivirin or diprovirins may be administered alone or in conjunction with other antiviral therapy.

[21 ] The peptide form of hapivirin or diprovirins provides a basis for further therapeutic development, by modification of the polypeptide structure to yield modified forms having altered biological and chemical properties. The native or modified forms are formulated in a physiologically acceptable carrier for therapeutic uses, or are otherwise used as an antimicrobial agent. HAPIVIRIN OR DIPROVIRIN COMPOSITIONS

[22] For use in the subject methods, an hapivirin or diprovirin peptide or peptide analog may be used. Hapivirin or diprovirin peptides are generally of from about 10 to 20 amino acids in length, usually from about 12 to 18 amino acids in length, and may be about 13, about 14, about 15, about 16, about 17 residues in length. By the term "amino acid", various analogs of naturally occurring amino acids are intended, including d-amino acids and analogs that include, without limitation, Cha-(L)-cyclohexyl-Alanine, Ctb-(L)-S-t-butyl- Cysteine, Cts-(L)-S-t-butylthio-Cysteine, Cam-(L)-Sacetamidomethyl-Cysteine, Oic-(L)- Octahydroindole-2-carboxylic acid, Tle-(L) tert-Leucine, r-(D)-Arginine, k-(D)-Lysine, PEG3- 1 1 -Amino-3,6,9-trioxaundecanoic acid.

[23] Hapivirin or diprovirins include the peptides set forth in Figure 1 , and include specifically the hapivirins of SEQ ID NO:1 -19, which are designated as HpV1 -17, respectively; diprovirins as set forth in SEQ ID NO:20-41 ; and diprovirins second generation as set forth in SEQ ID NO:42-73. The sequence of the polypeptides may be altered in various ways known in the art to generate targeted changes in sequence. The polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by one amino acid, and may differ by two amino acids. The sequence changes may be substitutions, insertions or deletions.

Table of Peptides

DpV1620 Tyr Cys SEQ ID NO:60

DpV1621 Lys Cys SEQ ID N0:61

DpV1622 Aer Cys SEQ ID NO:62

DpV1623 lie Cys^SH SEQ ID NO:63

DpV1624 lie Ser SEQ ID NO:64

DpV1625 lie Thr SEQ ID NO:65

DpV1626 lie Aib SEQ ID NO:66

DpV1627 lie Arg SEQ ID NO:67

DpV1628 lie Glu SEQ ID NO:68

DpV1629 lie Cys^suaH SEQ ID NO:69

DpV1630 lie Cys Mes SEQ ID NO:70

DpV1631 lie Cys PEG₅ SEQ ID N0:71

DpV1632 lie Cys Aoa-PEG₅ SEQ ID NO:72

Abbreviations: Abu-4-Aminobutyric acid, Ach-1 -Aminocyclohexanoic acid, Ahp-7-Aminoheptanoic acid, Ahx-6-Aminohexanoic acid, Aib-Aminoisobutyric acid, Aoc-8-Aminooctanoic acid, Aoa- Aminooxyacetic acid, Ava-5-Aminopentanoic acid, β Ala^-Alanine, Chg-Cyclohexylglycine, Cha- Cyclohexylalanine, Cpg-Cyclopentylglycine, C^S03H-Cysteic acid, Cys^SH-Cysteine (reduced), hSer^Me- Homoserine 0 methyl ether, ldc-(S)-lndoline-2-carboxylic acid, Mes-Methanesulfonyl group, Nal-1 - Naphthylalanine, Nle-Norleucine, Nva-Norvaline, Oic-(S)-Octahydroindole-2- carboxylic acid, Ser^Me- Serine O-methyl ether, Thr^Me-Threonine O-methyl ether, Tic-(3S)-1 ,2,3,4-Tetrahydroisoquinoline-3- carboxylic acid, Tle-tert-Leucine, DGK- Asp-Gly-Lys triad, PEG₅-21 -amino-4,7,10,13,16,19- hexaoxaheneicosanoic acid.

[24] The protein may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post- translational modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation. In this situation, the peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.

[25] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[26] Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non- naturally occurring synthetic amino acids.

[27] The subject peptides may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[28] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[29] The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

[30] In one embodiment of the invention, the antimicrobial peptide consists essentially of a polypeptide sequence set forth above. By "consisting essentially of" in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the sequence set forth in the seqlist, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) of the polypeptide.

HAPIVI RIN OR DI PROVIRIN CODING SEQUENCES

[31 ] The invention includes nucleic acid sequences encoding hapivirin or diprovirins; and fragments and derivatives thereof. Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to one of ordinary skill in the art when provided with the disclosure here.

[32] Hapivirin or diprovirin coding sequences can be generated by methods known in the art, e.g. by in vitro synthesis, recombinant methods, etc. to provide a coding sequence to corresponds to a linear hapivirin or diprovirin polypeptide that could serve as an intermediate in the production of the cyclic hapivirin or diprovirin molecule. Using the known genetic code, one can produce a suitable coding sequence.

[33] Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50 'C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55 °C in 1 XSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50 'C or higher and 0.1 XSSC (9 mM saline/0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, e.g., U.S. patent no. 5,707,829. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g. primate species, particularly human; rodents, such as rats and mice; canines, felines, bovines, ovines, equines, fish, yeast, nematodes, etc.

[34] Nucleic acids of the invention also include naturally occurring variants of the nucleotide sequences (e.g., degenerate variants, allelic variants, etc.). Variants of the nucleic acids of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions. For example, by using appropriate wash conditions, variants of the nucleic acids of the invention can be identified where the allelic variant exhibits at most about 25-30% base pair (bp) mismatches relative to the selected nucleic acid probe. In general, allelic variants contain 15-25% bp mismatches, and can contain as little as even 5-15%, or 2-5%, or 1 -2% bp mismatches, as well as a single bp mismatch.

[35] The invention also encompasses homologs corresponding to the nucleic acids of SEQ ID NO:5, where the source of homologous genes can be any mammalian species, e.g., primate species, particularly human; rodents, such as rats; canines, felines, bovines, ovines, equines, fish, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs generally have substantial sequence similarity, e.g., at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as gapped BLAST, described in Altschul et al. Nucl. Acids Res. (1997) 25:3389-3402.

[36] The subject nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active polypeptide and/or are useful in the methods disclosed herein. The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide of the invention.

[37] A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It can further include the 3' and 5' untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' and 3' end of the transcribed region. The genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3' and 5', or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue, stage-specific, or disease- state specific expression.

[38] The nucleic acid compositions of the subject invention can encode all or a part of the subject polypeptides. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. Isolated nucleic acids and nucleic acid fragments of the invention comprise at least about 18, about 50, about 100, to about 200 contiguous nt selected from the nucleic acid sequence. For the most part, fragments will be of at least 18 nt, usually at least 25 nt, and up to at least about 50 contiguous nt in length or more.

[39] Probes specific to the nucleic acid of the invention can be generated using the nucleic acid sequence encoding the polypeptides. The probes are preferably at least about 18 nt, 25 nt or more of the corresponding contiguous sequence. The probes can be synthesized chemically or can be generated from longer nucleic acids using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Preferably, probes are designed based upon an identifying sequence of one of the provided sequences. More preferably, probes are designed based on a contiguous sequence of one of the subject nucleic acids that remain unmasked following application of a masking program for masking low complexity (e.g., BLASTX) to the sequence, i.e., one would select an unmasked region, as indicated by the nucleic acids outside the poly-n stretches of the masked sequence produced by the masking program.

[40] The nucleic acids of the invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

[41 ] Hapivirin or diprovirin encoding nucleic acids can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

[42] Expression vectors may be used to introduce a hapivirin or diprovirin coding sequence into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

[43] The gene or hapivirin or diprovirin peptide may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992) Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermal^ by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the protein or DNA, then bombarded into skin cells.

METHODS OF USE

[44] Formulations of hapivirin or diprovirins are administered to a host suffering from an ongoing bacterial or viral infection or who faces exposure to a bacterial or viral infection. Administration may be topical, localized or systemic, depending on the specific patient needs. Generally the dosage will be sufficient to decrease the microbial or viral population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs. The compounds of the present invention are administered at a dosage that reduces the pathogen population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use. Hapivirin or diprovirins are particularly useful for preventing infection by certain viruses.

[45] Hapivirin or diprovirins are also useful for in vitro formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, hapivirin or diprovirins may be added to animal and/or human food preparations, or to blood products intended for transfusion to reduce the risk of consequent bacterial or viral infection. Hapivirin or diprovirins may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture.

[46] The susceptibility of a particular microbe or virus to killing or inhibition by hapivirin or diprovirins may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with hapivirin or diprovirins at varying concentrations for a period of time sufficient to allow the protein to act, usually ranging from about one hour to one day. The viable microbes are then counted, and the level of killing determined. Two stage radial diffusion assay is a convenient alternative to determining the MIC or minimum inhibitory concentration of an antimicrobial agent.

[47] Various methods for administration may be employed. For the prevention of virus infection, administration to mucosal surfaces is of particular interest, e.g. vaginal, rectal, etc. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific hapivirin or diprovirin or demi-defensin to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration.

Formulations

[48] The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, vaginal, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration. The hapivirin or diprovirins may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.

[49] The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.) In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting.

[50] For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[51 ] The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[52] The compounds can be utilized in aerosol formulation to be administered via inhalation.

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[53] The compounds can be used as lotions, for example to prevent infection of burns, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[54] Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[55] Unit dosage forms for oral, vaginal or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[56] Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing hapivirin or diprovirins is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body.

[57] The term "unit dosage form", as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host.

[58] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[59] Typical dosages for systemic administration range from 0.1 μg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

[60] Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

[61 ] The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellular^. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will be normally be neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[62] For preparing the liposomes, the procedure described by Kato et al. (1991 ) J. Biol.

Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing peptides are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1 -10 weight percent. After intense agitation for short periods of time, from about 5-60 sec, the tube is placed in a warm water bath, from about 25-40° C and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1 -10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1 -2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules.

Formulations with Other Active Agents

[63] For use in the subject methods, hapivirin or diprovirins may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with β-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.

[64] Cytokines may also be included in a hapivirin or diprovirin formulation, e.g. interferon γ, tumor necrosis factor a, interleukin 12, etc.

[65] Antiviral agents, e.g. acyclovir, gancyclovir, etc., and other circular mini-defensins (theta defensins) may also be included in hapivirin or diprovirin formulations.

EXPERIMENTAL

[66] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Hapivirins and Diprovirins: Novel Θ-Defensin Analogs With Potent Activity Against Influenza

A Virus

[67] θ-defensins are cyclic octadecapeptides whose broad antiviral-spectrum includes VIRUS-1 , HSV-1 , SARS, and avian and nonavian strains of influenza A virus (IAV). Although θ-defensin peptides have been found only in some non-human primates, we previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This report describes two families of peptides, hapivirins (HpVs) and diprovirins (DpVs), whose design was inspired by θ-defensins. Since HpVs contain 13 residues, and DpVs have 13 or 14, both are smaller than θ-defensins, which have eighteen. Seventy-two HpV or DpV peptides are described in this report, including several whose anti-IAV activity equals or exceeds that of normal a- or θ-defensins. These new peptides were active against H3N2 and H1 N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum half-life in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection and when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogues provides an approach for creating novel antiviral agents for IAV infections.

[68] In contrast to θ-defensins, HNP1 and 2, which bind strongly to surfactant protein D (SP- D) and interfere with its antiviral activity, retrocyclins and SP-D have additive antiviral activity when combined. Based upon the favorable antiviral profile of retrocyclins, we developed two series of novel θ-defensin analogs and tested their interactions with IAV.

[69] Even though relatively small (18 Xaa), θ-defensins are difficult to synthesize. They belong to cysteine-rich peptides containing a circular backbone and characteristic ladder of three disulfide bridges forming β-hairpin structure. Such compounds require two postsynthetic steps, as follows: oxidation (disulfide bonds' formation) and circularization via amide bond, resulting in a low final yield of the desired product. Therefore, our current studies focused on the modification of θ-defensins with the general goal of simplifying their structure and preserving and/or possibly enhancing their antiviral properties. Hapivirins (HpVs) have the trisulfide ladder of θ-defensins, but did not have a fully circular backbone (see Fig. 1 ). The HpVs had various hydrophobic substitutions at a single amino acid site (position Xi). In case of diprovirins (DpVs), their b-hairpin structure was imposed by structural element, -(D)Pro-(L)Pro- moiety, making positions previously occupied by four cysteines (positions X∑and Xs) available for probing. In turn, substitutions in positions X₅, and Xe investigated the role of hydrophobicity, the presence of a "clipping" disulfide bridge, and the feasibility of N-terminal substitutions, respectively. MATERIALS AND METHODS

[70] Preparation of IA V, bacteria, and fungi. IAV was grown in the chorioallantoic fluid of 10- d-old chicken eggs and purified on a discontinuous sucrose gradient, as previously described. The virus was dialyzed against PBS to remove sucrose, aliquoted, and stored at 280°C until needed. A/Phillipines/82 (H3N2) (Phil82) was provided by E. Anders (Department of Microbiology, University of Melbourne, Melbourne, Australia). The A/PR/8/34/H1 N1 (PR-8) strain was a gift of J. Abramson (Bowman Gray School of Medicine, Winston-Salem, NC). The hemagglutinin titer of each virus preparation was determined by titration of virus samples in PBS with thoroughly washed human type O, Rh(-) RBC. Post-thawing the viral stocks contained ~5 X 10⁸ PFU/ml. Staphylococcus aureus (Wood 46 strain; ATCC 10832) and Candida albicans (Berkhout anamorph; ATCC MYA-3573) were obtained from American Type Culture Collection. S. aureus was grown overnight by inoculating the lyophilized bacteria in LB broth and used at a dilution that yielded -100 colonies on agar culture dishes after 24 h. To test for inhibition of bacterial growth, the diluted bacteria were incubated for 30 min at 37°C with peptides in PBS and then inoculated on agar plates using sterile glass beads to spread bacteria evenly over the surface. Results were expressed as percentage of control colonies in peptide-treated samples. Inhibition of growth of C. albicans was tested in a similar manner. Fluorescent bioparticles of S. aureus and zymosan were obtained from Molecular Probes for use in studies of particle aggregation using fluorescent microscopy.

[71 ] Hapivirin/Diprovirin preparation. All Hapivirin (HpV) and Diprovirin (DpV) peptides were synthesized by the solid phase method using CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), applying 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry and standard, commercially available amino acid derivatives and reagents (EMD Biosciences, San Diego, CA and Chem-lmpex International, Inc., Wood Dale, IL). Rink Amide MBHA resin (EMD Biosciences, San Diego, CA) was used as a solid support. Peptides were cleaved from resin using modified reagent K (TFA 94% (v/v); phenol, 2% (w/v); water, 2% (v/v); TIS, 1 % (v/v); EDT, 1 % (v/v); 2 hours) and precipitated by addition of ice-cold diethyl ether. Reduced peptides were purified by preparative reverse- phase high performance liquid chromatography (RP-HPLC) to >90% homogeneity and their purity evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) as well as analytical RP-HPLC. For analytical details, please see Supporting Information.

[72] Disulfide Bond Formation - Peptides were dissolved at a final concentration 0.25 mg/ml in 50% solution of DMSO in H₂0 and stirred overnight at room temperature. Subsequently peptides were lyophilyzed and re-purified on a preparative C18 SymmetryShield™ RP- HPLC column to >95% homogeneity and their purity evaluated by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) as well as analytical RP-HPLC. [73] Analytical HPLC - Analytical RP-HPLC was performed on a Varian ProStar 210 HPLC system equipped with ProStar 325 Dual Wavelength UV-Vis detector with the wavelengths set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). Mobile phases consisted of solvent A, 0.1 % TFA in water, and solvent B, 0.1 % TFA in acetonitrile. Analyses of peptides were performed with an analytical reversed phase C18 SymmetryShield™ column, 4.6 x 250 mm, 5 μηι (Waters Corp., Milford, MA) or analytical RP C18 Vydac 218TP54 column, 4.6x 250 mm, 5 μηι (Grace, Deerfield, IL) applying a linear gradient of solvent B from 0 to 100% over 100 min (flow rate: 1 ml/min).

[74] SP-D preparation. Recombinant human SP-D was produced in stably transfected CHO- K1 cells as previously described. For these studies the dodecameric fraction of recombinant human SP-D was used unless otherwise specified. The collectin preparations used in this report were tested for endotoxin using a quantitative endotoxin assay (Limulus Amebocyte Lysate; BioWhittaker). The final concentrations of endotoxin in protein samples containing the highest concentrations of collectins were -20-100 pg/ml (or 6-12 endotoxin U/ml using an internal assay standard).

[75] Fluorescent focus assay of IA V infectivity. Viral neutralization was measured using a fluorescent focus reduction assay. In brief, A549 (respiratory epithelial cell line) or Madin Darby Canine Kidney (MDCK) cell monolayers were prepared in 96-well plates and grown to confluency. These layers were then infected with diluted IAV preparations at a multiplicity of infection of -1 :100. The infection was conducted for 45 min at 37°C in PBS and tested for presence of IAV infected cells after 7 h using a monoclonal antibody directed against the influenza A viral nucleoprotein, provided by Dr. N. Cox (Centers for Disease Control, Atlanta, GA) as previously described. IAV was preincubated for 30 min at 37 °C with defensins, SP-D or control buffer, followed by addition of these viral samples to the A549 or MDCK cells. The viral incubation with defensins was conducted in PBS with added calcium and magnesium (Dulbecco's PBS; Life Technologies) with no serum added during the incubation.

[76] Measurement of IA V aggregation. Aggregation of IAV particles was assessed following addition of various concentrations of antimicrobial peptides by monitoring increases in light absorbance by viral suspensions at 350nm. This was done using a Perkin Elmer Lambda 35 UV/Vis spectrophotometer. Confirmation of IAV aggregation was obtained by electron microscopy, as described below. Aggregation of fluorescent bacteria and zymosan was assessed by fluorescence microscopy.

[77] Electron microscopy. Antimicrobial peptides were incubated with Phil82 IAV at 37°C for 30 min, and a 4-μΙ sample was placed on each copper grid. After the unbound virus was blotted off, the grid was fixed with 4 μΙ of 2.5% glutaraldehyde for 5 min. Samples were stained with 1 % sodium phosphotungstate (pH 7.3) (Sigma-Aldrich) for 10 s, and excess stain was blotted off. The grids were then air dried and stored in a grid box until examined with a Phillips 300 electron microscope.

[78] Human neutrophil preparation Neutrophils from healthy volunteers were isolated to >95% purity by dextran sedimentation, followed by Ficoll-Paque gradient separation for the removal of mononuclear cells, and then hypotonic lysis to eliminate any contaminating erythrocytes, as previously described. Cell viability was determined to be >98% by trypan blue staining. The isolated neutrophils were resuspended at the appropriate concentrations in control buffer (PBS) and used within 2 h. Neutrophil collection was done with informed consent, as approved by the Institutional Review Board of Boston University School of Medicine.

[79] Measurement of IAV uptake by Neutrophils and RAW cells. FITC-labeled IAV (Phil82 strain) was prepared and uptake of virus by neutrophils or RAW 264.7 cells was measured as previously described. Briefly, IAV was incubated with neutrophils or RAW cells for 30 min at 37°C in the presence of control buffer or RCs. Extracellular fluorescence was quenched by addition of trypan blue (0.2 mg/ml) to the samples. After they were washed, the neutrophils were fixed with 1 % paraformaldehyde, and neutrophil/RAW cell-associated fluorescence was measured using flow cytometry. The mean intracellular fluorescence (2000 cells/sample) was measured. Neutrophil uptake by adherent neutrophils was performed as previously described. In brief, neutrophils were allowed to adhere to glass slides for 1 h at 37°C followed by washing and addition of Alexa-Fluor-labeled Phil82 IAV. After 45 min of incubation with the virus at 37 ^<Ό, the neutrophils were again washed, and trypan blue was added to quench extracellular fluorescence. The cells were then examined under fluorescent microscopy at a magnification of x40.

[80] Measurement of human monocytes tumor necrosis alpha (TNFct) production in response to IA V - Human peripheral blood monocytes were isolated by magnetic bead separation as described above and infected with an MOI of -50 of Phil82 IAV for 45 min. at 37 ^<Ό. The virus was either used alone or after 45 min. incubation with various concentrations of LL-37. After this the cells were pelleted, washed with PBS and then cultured for 24 hrs. at 37^<Ό in RPMI with 10% autologous serum in a C0₂ incubator. After 24 hrs, supernatant was collected and assayed for TNFa using a sandwich ELISA method (catalogue number MTNFAI; Endogen), following the manufacturer's instructions.

[81 ] Statistics. Statistical comparisons were made using Student's paired, two-tailed f test or ANOVA with Tukey's post hoc test.

RESULTS

[82] Viral Neutralization by Hapivirins. Figure 1 shows the amino acid sequences of the Hapivirins and Diprovirins that were synthesized for these studies. The Hapivirins all have a relatively rigid hairpin structure due to the presence of three disulfide bond ladder with alternating hydrophobic and basic amino acids adjacent to the cysteines. Variants were made simply by changing one amino acid in the hairpin loop (Xi , the red residue in Figure 1 ). Several of the unusual amino acids used to substitute for X_! are shown in Figure 2. Figure 3 shows results of screening assays for the antiviral activity of the Hapivirin peptides 1 to 16. Neutralization was assessed using a fluorescent focus assay and the seasonal H3N2 strain, Phil82. Obvious differences in activity were found among the Hapivirin variants and several caused >50% neutralization of viral infectivity at a concentration of ^g/ml, including HpV1 , HpV8, and HpVs 1 1 -15. In our prior experiments the most effective defensins at inhibiting this viral strain on a weight basis were RC2 and RC101 each of which had an approximate 50% neutralizing dose of 1 .25 μg ml; hence, several of the HpVs had activity equal to or greater than the most potent natural defensins.

[83] Several Hapivirins with increased hydrophobicity in the connecting loop (e.g., HpVs1 1 , 12, and 14) of the peptide had increased activity. HpV1 1 had particularly strong activity so a further modification of this peptide was made in which all lie residues in the structure were replaced with cyclohexylglycine (Chg) to further increase hydrophobicity of the analog called HpV19. As shown in figure 4A and B, this further change caused a slight reduction in activity compared to HpV1 1 , but both had strong neutralizing activity when tested in either MDCK cells as in Figure 3 or A549 cells, an alveolar epithelial cell line. Since several HpVs with hydrophobic amino acids at position X_! (HpV1 1 , 12, 14, 15, and 19) had strong activity, we prepared and tested analogs containing cyclohexylalanine (HpV17) or napthylalanine (HpV18) at this position. Structures of these amino acids are shown in Figure 2. As shown in Figure 4, both analogues had strong neutralizing activity, causing 50% neutralization at -0.4 μg/ml. The strong activity of several HpVs was also manifested in tests with the H1 N1 PR-8 strain, which lacks glycosylation on the head region of its hemagglutinin (Figure 4D).

[84] Viral neutralizing activity of Diprovirins. The Diprovirins (DpVs) are a series of synthetic antimicrobial peptides each containing 13 residues with cysteines at positions 2 and 13, arginines at positions 3 and 10 and a (D)Pro-(L)Pro moiety at positions 7 and 8. The β- hairpin-inducing diproline moiety is commemorated in the Diprovirin name. The structures of these analogues are shown schematically in Figure 1 . DpVs 1 -21 are variants in which amino acids 4, 6, 9, and 1 1 (positions occupied by cysteines in HpVs) are substituted simultaneously with various amino acids: glycine (DpV1 ), alanine (DpV2), D-alanine (DpV3), and so on. Of special note are DpVs 13 and 16 in which these four amino acids or isoleucine or leucine, respectively. A large set of variants of DpV16 (DpVs 1601 through 1622) retain these leucines but vary amino acids 1 , 5, and 12. DpVsl 623-1629 are identical to DpV16 except for modification or substitutions of cysteines 2 and 13. Finally DpV1630-32 are identical to DpV16 except for having methylsulfonyl, PEG₅ or Aoa-PEG₅ attachments. As shown in Table 2, several of these compounds, including DpVs 13, 16, 1607, 1609, 1615, 1616, 1623, and 1630-1632, had strong neutralizing activity in initial screening assays. In follow-up dose response experiments (Figure 5) we found that Diprovirins 13, 16, 1623 and 1632 had particularly strong activity. These results are of interest since DpVs13 and 16 have the hydrophobic isoleucine and leucine substitutions in positions 4, 6, 9, and 1 1 (although note that tert-Leu substitutions as in DpVs 14 and 15 did not confer as much activity). DpV1623 is identical to DpV16 apart from having no disulfide bond, since cysteines in positions 2 and 13 possess free sulfhydryl groups (SH). DpVs 1630-32 are identical to DpV16 apart from having PEG₅ (21 -amino-4,7, 10,13,16,19- hexaoxaheneicosanoic acid) or methylsulfonyl attachments, indicating that such attachments may increase activity. This was particularly evident in the case of DpV1632, in which the PEG₅ substituent was additionally modified with aminooxyacetic acid (Aoa).

[85] Effect of delayed addition of potent Hapivirins or Diprovirins on viral neutralizing activity.

Prior reports have indicated that some antimicrobial peptides inhibit IAV principally through interacting with epithelial cells rather than with the virus. To test whether this is true for the Hapivirins or Diprovirins, we compared the effects of varying the timing of addition of HpV1 1 , HpV17, and DpV1632 with respect addition of virus to either MDCK cells or A549 cells. We also used RC2 for comparison. As shown in Figure 6, allowing the virus to infect the cells for 15 or 45 min. prior to adding the peptides caused a marked diminution of their viral neutralizing activity.

[86] Interactions of Hapivirins and Diprovirins with SP-D. HNPs 1 -3 and retrocyclins bind to pulmonary surfactant protein D. In the case of the HNPs (but not of the retrocyclins) this leads to competitive effects such that the IAV neutralizing activity of combinations of HNPs and SP-D are less than occurred with SP-D alone. As shown in Table 2, DpVs 1607 and 1632 had additive activity when combined with SP-D. Similar results were obtained with HpVs 6 and 1 1 .

[87] Viral aggregation by Hapivirins and Diprovirins. As we have reported for HNPs and retrocyclins, some of the active Hapivirins and Diprovirins were also able to induce viral aggregation as assessed using the Phil82 viral strain (Figure 7). Certain Hapivirins were particularly effective in this regard, including HpV1 1 and HpV17, both of which had strong neutralizing activity as well. Note that these were dramatically more potent in this assay than RC2 (which was the most active retrocyclin in this assay in former studies). Other Hapivirins with lesser but still significant viral aggregating activity in this assay included HpV15 (Figure 7) and HpV18. Electron micrographs confirmed the presence of viral aggregates as shown in Figure 7. Several Diprovirins were also tested for viral aggregating activity. DpV16 and DpV1632 had aggregating activity (see electron micrographs in Figure 7), but DpV2 did not cause viral aggregation (data not shown). Of interest, Hapivirins and Diprovirins also caused aggregation of Staphylococcus aureus and Zymosan particles (Figure 8) indicating that this aggregating property applies to other microorganisms besides IAV.

[88] Hapivirins and Diprovirins increase neutrophil and monocyte uptake of IAV. Preincubation of IAV with several of the most highly neutralizing Hapivirins and Diprovirins also resulted in increased neutrophil uptake of the virus (Figure 9A and B), including HpVs 17 and 18, and DpVs13, 1631 and 1632. In further studies, HpV14 and DpV1622 also increased neutrophil uptake of IAV significantly compared to control buffer; whereas peptides that had weaker antiviral activity (e.g., HpV6 and DpVs 2 and 1617) did not (Table 5). Hence, the ability of HpVs and DpVs to promote neutrophil uptake of IAV correlated overall with their antiviral activity. Note that both HpV17 and HpV18 increased viral uptake by RAW cells as well (Figure 9C).

[89] Hapivirins and Diprovirins reduce human monocyte generation of TNFa in response to IAV. Some of the peptides were also tested for their ability to inhibit lAV-induced TNFa production by human monocytes. DpVs16 and 1630 and HpVs 1 1 and 19 inhibited the TNF response to varying degrees (Figure 10).

[90] Synthetic θ-defensin analogs are provided that have equal or greater activity against IAV than their natural counterparts. The analogs comprised two distinct subgroups, Hapivirins and Diprovirins. Some members of each group had neutralizing activity comparable to that of wild type defensins. The Hapivirins resembled primate retrocyclins in having an intramolecular ladder of three evenly spaced disulfide bonds. Strikingly, changing a single amino acid in the hairpin loop of HpVs resulted in marked changes in antiviral activity. Several HpVs had 50% neutralizing activity at concentrations of -500 ng/ml. Several DpVs could have similar activity since they reduced viral infectivity to <20% of control at 3 μg ml and DpV1632 had a 50% neutralizing dose <0.4 μg ml (Table 2). In our prior studies, the most active θ-defensins RC2 and RC101 showed 50% neutralizing activity of -1 .25 μg/ml.

[91 ] Structure-function analysis of the HpV and DpV variants tested provides insights into the structural features that are most important for antiviral activity. While cationic charge has been considered the most important determinant of defensin-mediated antimicrobial activity, our findings suggest that increasing hydrophobicity can have a marked impact on antiviral activity. The feature of HpVs conferring the best antiviral activity was increased "non- aromatic type" hydrophobicity in the loop of the molecule (position 7) delivered by L- cyclohexylalanine (Cha) or (S)-octahydroindole-2-carboxylic acid (Oic) - which can be considered a constrained analog of Cha. Because HpV1 1 and HpV17 were among the most active HpVs, their side chains may represent optimal hydrophobicity as well as spatial arrangements for maximal antiviral effect, especially considering that fairly similar substituents did not confer similar effects (see Figure 2).

[92] DpVs 1 -21 were designed to simplify the β-hairpin structure of HpVs, which is imparted and rigidified by a "tri-disulfide ladder". The β-hairpin structure of DpVs was imparted by incorporating the -(D)Pro-(L)Pro- moiety. In this group of analogues, the most effective substitutions were L-isoleucine (DpV13) and L-leucine (DpV16), with isoleucine being superior to leucine. Substitution with L-valine also led to increased activity compared to alanine or polar amino acids although this increase was modest compared to the L- isoleucine or L-leucine substitutions. Of interest, the D-isomers of leucine, valine or alanine all had lower activity than the L-isomers.

[93] Given the strong activity of the L-leucine variant (DpV16) this peptide became the starting point for a series of additional peptides having substitutions at positions 1 , 5, and 12. In this case hydrophobic substitutions were not clearly advantageous, although Tie, Cpg, Chg, Tyr analogues showed considerable activity. Increased activity was seen with more polar substitutions: i.e., threonine, serine and arginine. Since the parental compound (DpV16) has limited solubility in the aqueous solutions, further increase in hydrophobicity may be detrimental, while polar residues are beneficial.

[94] Retaining the intact sequence of DpV16 but reducing cysteines 2 and 13 or adding PEG₅, methylsulfonyl attachments or most importantly Aoa-PEG₅ to the molecule gave the greatest increase in activity. The fact that addition of PEG₅ or methylsulfonyl group to the peptide significantly increased activity is useful, since these attachments may also prolong the half life of the peptide in physiological conditions.

[95] The presence of the aminooxyacetic acid is particularly beneficial. Its effects may be partially explained by the presence of modified hydroxylamine group. Hydroxylamine was shown to inactivate influenza virus by cleaving fatty acids from viral haemagglutinin, lowering fusogenic and hemolytic activity. Although the concentration of hydroxylamine in the aforementioned experiments was much higher (1 M), perhaps DpV1632 augments its intrinsic antiviral activity by delivering the Aoa moiety to sites that are conducive to allow fatty acid cleavage. Replacing the cysteines with various polar or charged amino acids generally resulted in loss of activity.

[96] HNPs were reported to exert effects on epithelial cells that inhibit replication of IAV. In the present experiments, IAV was generally pre-incubated with defensins before infection of epithelial cells. To ascertain if the activity of HpV and DpV resulted from events before or after viral internalization, we compared pre-incubating IAV with the peptide, to introducing the peptides after the virus had interacted with cells for 15 or 45 minutes. Surprisingly, delaying peptide addition for as little as 15 minutes markedly diminished neutralizing activity. This suggests that the HpVs and DpVs exerted their antiviral effects by interacting with the virus itself. The ability of collectins or antimicrobial peptides to induce viral aggregation is an important correlate of antiviral activity. Viral aggregation can reduce particle numbers and promote clearance of virus from the airway through mucociliary action or uptake by phagocytes. We studied IAV aggregation mainly with peptides that showed strong activity in the neutralization assays. In general, highly neutralizing HpVs and DpVs also had strong viral aggregating activity, so these properties may be closely related. Note that the aggregating activity of the most potent peptides tested (e.g., HpV1 1 or HpV17) greatly exceeded that of retrocyclin RC2, which had the strongest aggregating activity among the Θ- defensins tested in our prior studies. The ability of the peptides to induce viral aggregation suggests that they may oligomerize as has been reported for some defensins and retrocyclins. We have found that oligomerization is important for the antiviral activity of collectins.

[97] HNPs and retrocyclins have been reported to have pro- and anti-inflammatory effects in the lung and to promote viral or bacterial uptake by phagocytes. The opsonizing activity of defensins may relate to the ability of these defensins to induce viral or bacterial aggregation. HpVs and DpVs showed opsonizing activity for IAV comparable to that shown by HNPs and RCs. Significantly, peptides that displayed strong aggregating activity, such as HpV17, also had the strongest opsonizing activity. It is notable that the HpVs and DpVs were also capable of inhibiting IAV induced TNFa responses in monocytes. Production of TNFa and other strong pro-inflammatory cytokines may be deleterious during IAV infection so this effect could be beneficial during severe IAV infection in vivo.

[98] Overall, our findings show that developing synthetic θ-defensin analogs as therapeutics for IAV is promising. Recent reports showed that retrocyclins have significant protective activity against serious respiratory pathogens including SARS and avian influenza (H5N1 ) in mice. Since retrocyclins are also highly effective against other viruses, including VIRUS and HSV, HpVs and DpVs may be similarly active against these other viruses. If so, the small size and simple structures of DpVs and HpVs makes them excellent candidates for use as topical microbicides as well as potential systemic therapeutics. Of note, HpVs and DpVs appeared to have additive neutralizing activity when combined with SP-D, quite unlike the competitive effects seen when HNP1 and SP-D were combined. These findings suggest that HpVs or DpVs would not interfere with functional activities of SP-D if used to enhance lung immunity in vivo.

Table 1 Neutralizing activity of Diprovirins

Peptide % Infect. Foci Peptide % Infect. Foci

a All peptides were tested at 40 μg/ml

Table 2 Neutralizing activity of SP-D alone or in combination with DpV1607, DpV1632, or HpV1 1 DpV1607 DpV1632

Control

1.5 Mg/ml 0.4 g/ml

Control 100% 58±6 28±2

SP-D

74±10 34±14* 15±5*

5 ng/ml

SP-D

50±6 32±13 14±7*

10 ng/ml

Mean±SEM of 4 experiments using A549 eel s in the fluorescent ocus assay

*p<0.05 compared with SP-D alone

Table 3. Ability of selected peptides to increase viral uptake by neutrophils

Example 2

Antiviral Activity

[99] To test whether thesepeptides possess an antiviral potential they were screened using standard, simple and robust assays allowing for fast testing of a large number of compounds in the relatively short period of time. To screen HpV and DpV peptides against HIV-1 we utilized luciferase reporter assay and for testing against HSV-2 we employed plaque forming assay. The data for HpV peptides is shown in Table 4.

[100] The diprovirins were then tested, and it was found that DpV13 and DpV16 showed prominent antiviral properties against both HIV-1 and HSV-2 viruses (Table 5). Further antiviral testing revealed that most active compound DpV16 possesses antiviral activity comparable to parental θ-defensins (RC1 and RC101 ). Hydrophobicity in positions X₂ and X₃ is important for antiviral activity since only analogues carrying lie & Leu in X₂ and Ach, Tie, Cpg, Chg &Tyr in X₃ were active. Only one additional permissive substitution utilizing Arg residues was found (DpV1622). Oxidation via disulfide bond appears not to be crucial for activity, however the presence of cysteines in positions X₄ is. In addition, N-terminal modifications in DpV-molecule are generally permissive and may be used to introduce fairly large moieties, such as PEG₅ spacer. To characterize further antiviral activity of selected analogues, we performed additional dose response experiments. Our studies revealed that in the case of anti-H IV-1 activity new compounds are slightly less active than control peptide RC101 (IC₅₀=3.4±0.2 μΜ) showing following IC₅₀ values: DpV16, IC₅₀=8.8±1 .9 μΜ, DpV1616, IC₅₀=7.8±1 .0 μΜ; DpV1 622, IC₅₀=8.6±1 .3 μΜ. Surprisingly in case of HSV-2 our new analogues showed enhanced activity compared to control (RC1 , IC₅₀=1 1 .5±2.0 μΜ) with the most active compound DpV1 6 displaying IC₅₀=0.14±0.02 μΜ.

Table 4. Antiviral activity of Hapivirins against H IV-1 and HSV-2.

% InhibitioniSEM

Peptide HIV-1 HSV-2

at 10 g/mL at 50 g/ml

HpV1 25.4±2.3 100.0±0.0

HpV2 3.5±1.5 11.3+7.1

HpV3 20.4±3.5 8.5±7.9

HpV4 43.5±2.9 8.1 ±5.2

HpV5 57.3±2.8 94.1 ±4.2

HpV6 60.1 +1.8 96.6±1.2

HpV7 56.0±1.3 98.1 +1.1

HpV8 N.T. 99.0±1.3

HpV9 14.7±4.6 99.2±1.3

HpV10 16.0±3.2 100.0±0.0

HpV11 N.T. 10.7±0.0

HpV12 38.6±1.8 100.0±0.0

HpV13 14.2±1.9 100.0±0.0

HpV14 N.T. 100.0±0.0

HpV15 37.2±2.3 85.4±3.5

HpV16 26.2±1.7 21.7+11.3

RC101 95.6±0.5 100.0±0.0

Table 5. Antiviral activity of Diprovirins against H IV-1 and HSV-2.

% InhibitioniSEM % InhibitioniSEM

Peptide HIV-1 HSV-2 Peptide HIV-1 HSV-2

at 10 g/mL at 50 g/ml at 10 g/ml at 50 g/ml

DpV1 -6.7±8.8 8.3+1.1 DpV1601 0.0±0.4 -21.0±2.9

DpV2 2.5±3.1 9.2±2.5 DpV1602 -12.6±2.6 -15.2±4.8

DpV3 -2.7±2.3 3.0±0.3 DpV1603 -27.5±5.9 17.1 +8.6

DpV4 2.6±3.3 6.2±1.8 DpV1604 3.6±5.1 9.5±1.0

DpV5 -8.2±3.8 5.7±2.8 DpV1605 -29.0±2.5 1.0±3.8

DpV6 -4.2±7.0 9.7±0.5 DpV1606 79.4±2.2 16.8±3.6 DpV7 -1.2±9.9 2.1 ±1.1 DpV1607 36.4+0.7 78.1 +1.0

DpV8 -9.9±6.1 1.0±1.1 DpV1608 10.0+0.6 38.1 +1.0

DpV9 -15.3±4.1 82.2±4.6 DpV1609 84.1 +1.0 99.5+0.5

DpV10 2.7±2.1 7.4±0.3 DpV1610 26.4+3.8 34.3+6.7

DpV11 -6.6±9.3 3.1 ±1.1 DpV1611 15.0+4.1 27.6+1.9

DpV12 9.8±3.5 5.511.6 DpV1612 -21.1 +1.5 24.2+1.9

DpV13 88.8±0.0 95.4±2.6 DpV1613 -10.5+5.0 4.8+1.9

DpV14 -16.3±3.9 6.1 +4.1 DpV1614 87.0+3.1 73.5+1.0

DpV15 -8.8±5.6 4.311.3 DpV1615 96.3+0.3 99.0+0.0

DpV16 93.410.2 91.3+4.6 DpV1616 97.4+0.2 99.5+0.5

DpV17 -31.7±2.4 7.212.1 DpV1617 -20.1 +7.1 10.9+1.6

DpV18 -22.1 ±8.9 4.210.5 DpV1618 -11.4+3.3 16.4+2.3

DpV19 13.6±7.9 4.412.3 DpV1619 -20.6+5.6 50.8+0.8

DpV20 -8.4±2.6 96.7+0.8 DpV1620 53.6+24.2 18.8+0.0

DpV21 -2.3±5.4 100.0+0.0 DpV1621 -19.0+2.3 30.5+7.1

DpV1622 87.9+0.2 97.4+0.5

DpV1623 85.3+1.5 60.2+4.1

DpV1624 -17.4+3.2 10.9+1.6

DpV1625 23.9+0.2 64.1 +0.0

DpV1626 1.5+0.3 13.3+0.8

DpV1627 -0.5+2.1 12.5+0.0

DpV1628 -9.1 +0.4 21.9+1.6

DpV1629 -15.6+2.3 14.8+5.5

DpV1630 88.8+1.1 98.7+1.3

DpV1631 83.3+0.8 99.6+0.4

DpV1632 73.7+0.3 98.2+0.9

Methods

Binding Studies. Binding studies were performed by surface plasmon resonance (SPR) on a BIAcore 3000 system (BiaCore AB, Piscataway, NJ). BIAcore CM5 sensor chip was utilized in all experiments and binding studies were performed in HBS-EP running buffer (pH=7.4) containing 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, and 0.005% polysorbate 20. Proteins/peptides were immobilized on a CM5 sensor chip using the amine coupling method. The chip was activated by mixing 400 mM A/-ethyl-/V-(3-dimethylaminopropyl)- carbodiimide hydrochloride and 100 mM /V-hydroxysuccinimide. Residual reactive groups on the chip surface were blocked using 1 .0 M ethanolamine/HCI (pH=8.5). The flow cell-1 chip, which served as a control, lacked immobilized protein but was treated with A/-ethyl-/V- (3-dimethylaminopropyl)-carbodiimide hydrochloride, /V-hydroxysuccinimide, and ethanolamine/HCI. Binding signals were corrected for nonspecific binding by subtracting the flow cell-1 signal. To regenerate chip surfaces, bound ligands were removed with 10 mM HCI. Data were analyzed with BIAevaluation 4.1 software (Biacore, Piscataway, NJ).

[i 02]Binding studies were performed by surface plasmon resonance (SPR) on a BIAcore 3000 system (BiaCore AB, Piscataway, NJ). BIAcore CM5 sensor chip was utilized in all experiments and binding studies were performed in HBS-EP running buffer (pH=7.4) containing 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, and 0.005% polysorbate 20. Proteins/peptides were immobilized on a CM5 sensor chip using the amine coupling method. The chip was activated by mixing 400 mM /V-ethyl-/V-(3-dimethylaminopropyl)- carbodiimide hydrochloride and 100 mM /V-hydroxysuccinimide. Residual reactive groups on the chip surface were blocked using 1 .0 M ethanolamine/HCI (pH=8.5). The flow cell-1 chip, which served as a control, lacked immobilized protein but was treated with /V-ethyl-/V- (3-dimethylaminopropyl)-carbodiimide hydrochloride, /V-hydroxysuccinimide, and ethanolamine/HCI. Binding signals were corrected for nonspecific binding by subtracting the flow cell-1 signal. To regenerate chip surfaces, bound ligands were removed with 10 mM HCI. Data were analyzed with BIAevaluation 4.1 software (Biacore, Piscataway, NJ).

[103] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[104] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1 . An isolated hapivirin or diprovirin peptide.

2. The peptide of Claim 1 , wherein the peptide comprises a sequence set forth in Figure 1 .

3. The peptide of Claim 1 consisting essentially of a sequence set forth in Figure 1 .

4. The peptide of Claim 1 , wherein the peptide is selected from HpV17, HpV18, and HpV19.

5. The peptide of Claim 1 , wherein the peptide is HpV1 1 .

6. A formulation comprising a peptide of any one of Claims 1 -5, and a pharmaceutically acceptable excipient.

7. The formulation of Claim 6, comprising two or more hapivirin or diprovirin peptides.

8. A method for reducing virus infection of a cell, the method comprising:

contacting the virus or and/or the cell with an effective dose of a formulation according to Claim 6 or Claim 7, wherein infection is reduced.

9. The method of Claim 8, wherein the virus is an influenza virus.

10. The method of Claim 8, wherein the cell is present in an animal.

1 1 . The method of Claim 8, wherein the virus is present in an animal.

12. A method for administering hapivirin or diprovirin as a prophylactic agent to prevent a viral infection in a patient at risk of developing such infection.