US20160158317A1

US20160158317A1 - Antimicrobial Peptide Variants and Polynucleotides Encoding Same

Info

Publication number: US20160158317A1
Application number: US15/043,547
Authority: US
Inventors: Hans-Henrik Kristensen Hoegenhaug; Per Holse Mygind; Thomas Kruse; Dorotea Raventos Segura; Dorthe Hoj Sandvang; Soren Neve
Original assignee: ADENIUM BIOTECH AS
Current assignee: ADENIUM BIOTECH AS
Priority date: 2010-06-12
Filing date: 2016-02-13
Publication date: 2016-06-09
Also published as: ES2699734T3; EP2580235A1; TW201206353A; US20150031601A1; TWI654933B; BR112012031049A2; JP6120769B2; AR081866A1; RU2611173C2; US20110306750A1; CN103068840B; RU2012148781A; WO2011154525A1; US8835604B2; EP2580235B1; DK2580235T3; JP2013531491A; CN103068840A

Abstract

The present invention relates to variants of a parent antimicrobial peptide. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to variants of an antimicrobial peptide, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.
2. Description of the Related Art
Several classes of antimicrobial peptides (AMPs) have been described in literature, examples of which include defensins and alpha-helical peptides.
The present invention provides variants of an antimicrobial peptide isolated from Arenicola marina, and described in WO 2007/023163.
The variant antimicrobial peptides of the present invention exhibit improved antimicrobial activity as compared to the parent antimicrobial peptide. In particular, the variants exhibit improved antimicrobial activity in the presence of serum and blood proteins. Another advantage of the variant peptides of the invention is a reduced protein binding e.g. to serum and blood proteins, which results in an improved bioavailability as compared to the parent antimicrobial peptide.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising an alteration at one or more (several) of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity.
The present invention also relates to isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants.
The present invention also relates to a method of treating a microbial infection using the variants of the invention; and use of variants for manufacturing a medicament for the treatment of a microbial infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising an alteration at one or more (several) of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity.

DEFINITIONS

Antimicrobial activity: The term “antimicrobial activity” is defined herein as an activity which is capable of killing or inhibiting growth of microbial cells. In the context of the present invention the term “antimicrobial” is intended to mean that there is a bactericidal and/or a bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal effect, wherein the term “bactericidal” is to be understood as capable of killing bacterial cells. The term “bacteriostatic” is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells. The term “fungicidal” is to be understood as capable of killing fungal cells. The term “fungistatic” is to be understood as capable of inhibiting fungal growth, i.e. inhibiting growing fungal cells. The term “virucidal” is to be understood as capable of inactivating virus. The term “microbial cells” denotes bacterial or fungal cells (including yeasts).
In the context of the present invention the term “inhibiting growth of microbial cells” is intended to mean that the cells are in the non-growing state, i.e., that they are not able to propagate.
In a preferred embodiment, the term “antimicrobial activity” is defined as bactericidal and/or bacteriostatic activity. More preferably, “antimicrobial activity” is defined as bactericidal and/or bacteriostatic activity against Escherichia, preferably Escherichia coli.
For purposes of the present invention, antimicrobial activity may be determined according to the procedure described by Lehrer et al., 1991, Journal of Immunological Methods 137(2): 167-174. Alternatively, antimicrobial activity may be determined according to the NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards).
Peptides having antimicrobial activity may be capable of reducing the number of living cells of Escherichia coli (DSM 1576) to 1/100 after 8 hours (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and in particular after 30 minutes) incubation at 37° C. in a relevant microbial growth substrate at a concentration of 500 micrograms/ml; preferably at a concentration of 250 micrograms/ml; more preferably at a concentration of 100 micrograms/ml; even more preferably at a concentration of 50 micrograms/ml; most preferably at a concentration of 25 micrograms/ml; and in particular at a concentration of 10 micrograms/ml of the peptides having antimicrobial activity.
Peptides having antimicrobial activity may also be capable of inhibiting the outgrowth of Escherichia coli (DSM 1576) for 8 hours at 37° C. in a relevant microbial growth substrate, when added in a concentration of 500 micrograms/ml; preferably when added in a concentration of 250 micrograms/ml; more preferably when added in a concentration of 100 micrograms/ml; even more preferably when added in a concentration of 50 micrograms/ml; most preferably when added in a concentration of 10 micrograms/ml; and in particular when added in a concentration of 5 micrograms/ml.
The variant peptides of the present invention have improved antimicrobial activity compared to the antimicrobial peptide of SEQ ID NO: 2. In an embodiment, the variant peptides of the present invention have more than 100% of the antimicrobial activity of the peptide of SEQ ID NO: 2 in the presence of blood serum.
Variant: The term “variant” means a peptide having antimicrobial activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
Mutant: The term “mutant” means a polynucleotide encoding a variant.
Wild-type antimicrobial peptide: The term “wild-type” antimicrobial peptide means an antimicrobial peptide expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
Parent or Parent antimicrobial peptide: The term “parent” or “parent antimicrobial peptide” means an antimicrobial peptide to which an alteration is made to produce the enzyme variants of the present invention. The parent may be a naturally occurring (wild-type) peptide or a variant thereof.
Isolated variant: The term “isolated variant” means a variant that is modified by the hand of man. In one aspect, the variant is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE.
Substantially pure variant: The term “substantially pure variant” means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other peptide material with which it is natively or recombinantly associated. Preferably, the variant is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total peptide material present in the preparation. The variants of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant by well known recombinant methods or by classical purification methods.
Mature peptide: The term “mature peptide” means a peptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
Mature peptide coding sequence: The term “mature peptide coding sequence” means a polynucleotide that encodes a mature peptide having antimicrobial activity.
Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)
For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)
Fragment: The term “fragment” means a peptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature peptide; wherein the fragment has antimicrobial activity. In one aspect, a fragment contains at least 15 amino acid residues, e.g., at least 17 and at least 19 amino acid residues (e.g., amino acids 1 to 20 of SEQ ID NO: 2).
Subsequence: The term “subsequence” means a polynucleotide having one or more (several) nucleotides deleted from the 5′- and/or 3′-end of a mature peptide coding sequence; wherein the subsequence encodes a fragment having antimicrobial activity.
Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded peptide) or may encode peptides having altered amino acid sequences. An allelic variant of a peptide is a peptide encoded by an allelic variant of a gene.
Isolated polynucleotide: The term “isolated polynucleotide” means a polynucleotide that is modified by the hand of man. In one aspect, the isolated polynucleotide is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose electrophoresis. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
Substantially pure polynucleotide: The term “substantially pure polynucleotide” means a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered peptide production systems. Thus, a substantially pure polynucleotide contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′- and 3′-untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, e.g., at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form.
Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of its peptide product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.
cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
Control sequences: The term “control sequences” means all components necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
Expression: The term “expression” includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to additional nucleotides that provide for its expression.
Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Conventions for Designation of Variants

For purposes of the present invention, the mature peptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another antimicrobial peptide. The amino acid sequence of another antimicrobial peptide is aligned with the mature peptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature peptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.
Identification of the corresponding amino acid residue in another antimicrobial peptide can be confirmed by an alignment of multiple peptide sequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23: 2947-2948).
When the other enzyme has diverged from the mature peptide of SEQ ID NO: 2 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pain/vise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of peptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the peptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the peptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.
For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and implementations of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
In describing the antimicrobial peptide variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.
Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411 Phe” or “G205R+S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.
Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.
Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:


	Parent:	Variant:

	195	195 195a 195b
	G	G - K - A

Multiple alterations. Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively. Different substitutions. Where different substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of arginine with tyrosine or glutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Parent Antimicrobial Peptides

The parent antimicrobial peptide is (a) a peptide with at least 60% sequence identity with the mature peptide of SEQ ID NO: 2; (b) a peptide encoded by a polynucleotide that hybridizes under medium stringency conditions with (i) the mature peptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary strand of (i); or (c) a peptide encoded by a polynucleotide with at least 60% sequence identity with the mature peptide coding sequence of SEQ ID NO: 1.
In a first aspect, the parent has a sequence identity to the mature peptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have antimicrobial activity. In one aspect, the amino acid sequence of the parent differs by no more than ten amino acids, e.g., by five amino acids, by four amino acids, by three amino acids, by two amino acids, and by one amino acid from the mature peptide of SEQ ID NO: 2.
The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2.
In an embodiment, the parent is a fragment of the peptide of SEQ ID NO: 2 containing at least 15 amino acid residues, e.g., at least 17 and at least 19 amino acid residues.
In another embodiment, the parent is an allelic variant of the peptide of SEQ ID NO: 2.
In a second aspect, the parent peptide is encoded by a polynucleotide that hybridizes under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature peptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary strand of (i) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, e.g., at least 25, or at least 35 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleotide probe corresponding to the polynucleotide shown in SEQ ID NO: 1, its complementary strand, or a subsequence thereof, under low to very high stringency conditions. Molecules to which the probe hybridizes can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is SEQ ID NO: 1.
For long probes of at least 60 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C. (low stringency), 55° C. (medium stringency), 60° C. (medium-high stringency), 65° C. (high stringency), or 70° C. (very high stringency).
For short probes that are about 15 nucleotides to about 60 nucleotides in length, stringency conditions are defined as prehybridization and hybridization at about 5° C. to about 10° C. below the calculated T, using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_m.
In a third aspect, the parent is encoded by a polynucleotide with a sequence identity to the peptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which encodes a peptide having antimicrobial activity.
The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a cell in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.
The parent may be a bacterial antimicrobial peptide. For example, the parent may be a gram-positive bacterial peptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces antimicrobial peptide, or a gram-negative bacterial peptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma antimicrobial peptide.
In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis antimicrobial peptide.
In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus antimicrobial peptide.
In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans antimicrobial peptide.
The parent may be a fungal antimicrobial peptide. For example, the parent may be a yeast antimicrobial peptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia antimicrobial peptide. For example, the parent may be a filamentous fungal antimicrobial peptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria antimicrobial peptide.
In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis antimicrobial peptide.
In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonaturn, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride antimicrobial peptide.
In another aspect, the parent is an Arenicola marina antimicrobial peptide, e.g., the antimicrobial peptide of SEQ ID NO: 2.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding a parent may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with a probe(s), the polynucleotide may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
The parent may be a hybrid peptide in which a portion of one peptide is fused at the N-terminus or the C-terminus of a portion of another peptide.
The parent also may be a fused peptide or cleavable fusion peptide in which one peptide is fused at the N-terminus or the C-terminus of another peptide. A fused peptide is produced by fusing a polynucleotide encoding one peptide to a polynucleotide encoding another peptide. Techniques for producing fusion peptides are known in the art, and include ligating the coding sequences encoding the peptides so that they are in frame and that expression of the fused peptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
A fusion peptide can further comprise a cleavage site between the two peptides. Upon secretion of the fusion protein, the site is cleaved releasing the two peptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Preparation of Variants

The present invention also relates to methods for obtaining a variant having antimicrobial activity, comprising: (a) introducing into a parent antimicrobial peptide a substitution at one or more (several) corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity; and (b) recovering the variant.
The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
Site-directed mutagenesis is a technique in which one or more (several) mutations are created at one or more defined sites in a polynucleotide encoding the parent.
Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.
Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.
Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.
Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a peptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a peptide.
Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Variants

The present invention also provides variants of a parent antimicrobial peptide comprising a substitution at one or more (several) positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 (preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17), wherein the variant has antimicrobial activity. In an embodiment, the variant has improved antimicrobial activity compared to the peptide of SEQ ID NO: 2; preferably in the presence of blood or serum. In another embodiment, the variant exhibit less protein binding compared to the peptide of SEQ ID NO: 2. Preferably, the variant antimicrobial peptides exhibit at the most 99% serum protein binding. The variant antimicrobial peptides also exhibit improved bioavailability. Preferably the subcutaneous bioavailability is at least 30%, more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60%.
In an embodiment, the variant has sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to the amino acid sequence of the parent antimicrobial peptide.
In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature peptide of SEQ ID NO: 2.
In one aspect, the number of substitutions in the variants of the present invention is 1-11, e.g., 1-10 substitutions, 1-9 substitutions, 1-8 substitutions, 1-7 substitutions, 1-6 substitutions, 1-5 substitutions, 1-4 substitutions, 1-3 substitutions and 1-2 substitutions.
In one aspect, the variant comprises or consists of the amino acid sequence shown as SEQ ID NO: 3 to SEQ ID NO: 548.
The term “SEQ ID NO: 3 to SEQ ID NO: 548” is intended to mean any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 423, SEQ ID NO: 424, SEQ ID NO: 425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507, SEQ ID NO: 508, SEQ ID NO: 509, SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516, SEQ ID NO: 517, SEQ ID NO: 518, SEQ ID NO: 519, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, and/or SEQ ID NO: 548.
In one aspect, a variant comprises a substitution at one or more (several) positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at four positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at five positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at six positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17.
In another aspect, the variant comprises the substitution G1A,D,F,H,I,K,M,Q,R,S,T,V,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution F2A,G,H,I,L,M,P,S,V,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution C3L of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution W4A,E,F,G,I,L,M,N,Q,S,T,V,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y5E,F,G,H,K,N,R,S,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V6A,C,E,F,G,H,I,L,M,N,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution C7V of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V8A,F,G,H,I,L,N,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y9A,D,F,G,H,I,K,M,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution R10K,P,S,T of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution N11A,G,H,Q,R,S,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution G12A,D,E,F,H,K,N,R,S,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V13A,C,F,G,H,K,L,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V15A,C,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y17C,F,G,H,I,K,L,M,N,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution R19D,H,K,M,T,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution N21A,C,F,G,H,I,K,L,M,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 1. In another aspect, the amino acid at position 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Phe, His, Ile, Lys, Met, Gin, Arg, Ser, Thr, Val, Trp, or Tyr. In another aspect, the variant comprises the substitution G1A,D,F,H,I,K,M,Q,R,S,T,V,W,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 2. In another aspect, the amino acid at position 2 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Gly, His, Ile, Leu, Met, Pro, Ser, Val, Trp, or Tyr. In another aspect, the variant comprises the substitution F2A,G,H,I,L,M,P,S,V,W,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 3. In another aspect, the amino acid at position 3 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, the variant comprises the substitution C3L of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 4. In another aspect, the amino acid at position 4 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Glu, Phe, Gly, Ile, Leu, Met, Asn, Gin, Ser, Thr, Val, or Tyr. In another aspect, the variant comprises the substitution W4A,E,F,G,I,L,M,N,Q,S,T,V,Y of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 5. In another aspect, the amino acid at position 5 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu, Phe, Gly, His, Lys, Asn, Arg, Ser, or Trp. In another aspect, the variant comprises the substitution Y5E,F,G,H,K,N,R,S,W of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 6. In another aspect, the amino acid at position 6 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V6A,C,E,F,G,H,I,L,M,N,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 7. In another aspect, the amino acid at position 7 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, the variant comprises the substitution C7V of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 8. In another aspect, the amino acid at position 8 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V8A,F,G,H,I,L,N,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 9. In another aspect, the amino acid at position 9 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Phe, Gly, His, Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, or Trp. In another aspect, the variant comprises the substitution Y9A,D,F,G,H,I,K,M,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 10. In another aspect, the amino acid at position 10 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Lys, Pro, Ser, or Thr. In another aspect, the variant comprises the substitution R10K,P,S,T of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 11. In another aspect, the amino acid at position 11 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Gly, His, Gln, Arg, Ser, or Tyr. In another aspect, the variant comprises the substitution N11A,G,H,Q,R,S,Y of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 12. In another aspect, the amino acid at position 12 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Glu, Phe, His, Lys, Asn, Arg, Ser, or Tyr. In another aspect, the variant comprises the substitution G12A,D,E,F,H,K,N,R,S,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 13. In another aspect, the amino acid at position 13 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V13A,C,F,G,H,K,L,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at position 15. In another aspect, the amino acid at position 15 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V15A,C,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 17. In another aspect, the amino acid at position 17 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Trp. In another aspect, the variant comprises the substitution Y17C,F,G,H,I,K,L,M,N,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 19. In another aspect, the amino acid at position 19 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp, His, Lys, Met, Thr, or Tyr. In another aspect, the variant comprises the substitution R19D,H,K,M,T,Y of the mature peptide of SEQ ID NO: 2.
In one aspect, the variant comprises a substitution at position 21. In another aspect, the amino acid at position 21 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution N21A,C,F,G,H,I,K,L,M,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises a substitution at positions corresponding to positions 5 and 17, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5 and 9, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5 and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 17 and 9, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 17 and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 9 and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, and 9, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 9, and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 17, 9, and 15, such as those described above.
In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, 9, and 15, such as those described above.
In another aspect, the variant comprises one or more (several) substitutions selected from the group consisting of

G1A, G1D, G1F, G1H, G1I, G1K, G1M, G1Q, G1R, G1S, G1T, G1V, G1W, G1Y,

F2A, F2G, F2H, F2I, F2L, F2M, F2P, F2S, F2V, F2W, F2Y,

C3L,

W4A, W4E, W4F, W4G, W4I, W4L, W4M, W4N, W4Q, W4S, W4T, W4V, W4Y,

Y5E, Y5F, Y5G, Y5H, Y5K, Y5N, Y5R, Y5S, Y5W,

V6A, V6C, V6E, V6F, V6G, V6H, V6I, V6L, V6M, V6N, V6Q, V6R, V6S, V6T, V6W, V6Y,

C7V,

V8A, V8F, V8G, V8H, V8I, V8L, V8N, V8S, V8T, V8W, V8Y,

Y9A, Y9D, Y9F, Y9G, Y9H, Y9I, Y9K, Y9M, Y9Q, Y9R, Y9S, Y9T, Y9V, Y9W,

R10K, R10P, R10S, R10T,

N11A, N11G, N11H, N11Q, N11R, N11S, N11Y,

G12A, G12D, G12E, G12F, G12H, G12K, G12N, G12R, G12S, G12Y,

V13A, V13C, V13F, V13G, V13H, V13K, V13L, V13P, V13Q, V13R, V13S, V13T, V13W, V13Y,

V15A, V15C, V15F, V15G, V15H, V15I, V15K, V15L, V15M, V15N, V15P, V15Q, V15R, V15S, V15T, V15W, V15Y,

Y17C, Y17F, Y17G, Y17H, Y17I, Y17K, Y17L, Y17M, Y17N, Y17Q, Y17R, Y17S, Y17T, Y17V, Y17W,

R19D, R19H, R19K, R19M, R19T, R19Y,

N21A, N21C, N21F, N21G, N21H, N21I, N21K, N21L, N21M, N21P, N21Q, N21R, N21S, N21T, N21W, and N21Y;

preferably

W4A, Y5H, Y5N, Y5R, V6A, V6F, V8A, Y9K, Y9R, G12R, G12K, V13A, V15I, V15S, and Y17H.

In another aspect, the variant comprises the substitutions Y5N+Y17H of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y5N+Y9R of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y5N+Y9K of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y17H+Y9R of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y17H+Y9K of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y5N+Y17H+Y9R of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y5N+Y17H+Y9K of the mature peptide of SEQ ID NO: 2.
In another aspect, the variant comprises the substitutions Y5N+V6A+Y9K or V8A+Y9R+V13A or Y5N+Y9R+Y17H or Y9K+V15S or W4A+Y5R+Y9K or Y5N+G12R+Y17H or Y5N+V6F+Y17H or Y5N+V15I+Y17H or Y5H+V8A+Y9R or Y5N+G12K+Y17H of the mature peptide of SEQ ID NO: 2.
Essential amino acids in a parent can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for antimicrobial activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The identities of essential amino acids can also be inferred from analysis of identities with peptides that are related to the parent.

Polynucleotides

The present invention also relates to isolated polynucleotides that encode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
A polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter sequence, which is recognized by a host cell for expression of the polynucleotide. The promoter sequence contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular peptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are the promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter including a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include modified promoters including the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the variant-encoding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the variant. However, any signal peptide coding region that directs the expressed variant into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding region that encodes a propeptide positioned at the N-terminus of a variant. The resultant peptide is known as a proenzyme or propeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to an active peptide by catalytic or autocatalytic cleavage of the propeptide from the propeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide regions are present at the N-terminus of a variant, the propeptide region is positioned next to the N-terminus of the variant and the signal peptide region is positioned next to the N-terminus of the propeptide region.
It may also be desirable to add regulatory sequences that allow the regulation of the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the dal genes from Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra) to obtain substantially pure variants.

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.
The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
The bacterial host cell may be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonaturn, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.
The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the peptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.
The variant may be detected using methods known in the art that are specific for the variants. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant.
The variant may be recovered by methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.
In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing a variant is used as a source of the variant.

In Vitro Synthesis

The polypeptides of the invention may also 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., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms) e.g. D-alanine and D-isoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g. reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like.
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.
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.

Plants

The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising a polynucleotide of the present invention so as to express and produce the variant in recoverable quantities. The variant may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the variant may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats.
Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.
The transgenic plant or plant cell expressing a variant may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (several) expression constructs encoding a variant into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a variant operably linked with appropriate regulatory sequences required for expression of the polynucleotide in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying plant cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the variant is desired to be expressed. For instance, the expression of the gene encoding a variant may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiol. 86: 506.
For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.
A promoter enhancer element may also be used to achieve higher expression of a variant in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the polynucleotide encoding a variant. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformation methods for use in accordance with the present disclosure include those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by reference in their entirety).
Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.
In addition to direct transformation of a particular plant genotype with a construct prepared according to the present invention, transgenic plants may be made by crossing a plant having the construct to a second plant lacking the construct. For example, a construct encoding a variant can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present invention encompasses not only a plant directly regenerated from cells which have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a DNA construct prepared in accordance with the present invention, or a portion of a DNA construct prepared in accordance with the present invention. Crossing results in the introduction of a transgene into a plant line by cross pollinating a starting line with a donor plant line. Non-limiting examples of such steps are further articulated in U.S. Pat. No. 7,151,204.
Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid.
Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.
The present invention also relates to methods of producing a variant of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and (b) recovering the variant.

Methods and Uses

The present invention is also directed to methods for using the polypeptides having antimicrobial activity. The antimicrobial polypeptides are typically useful at any locus subject to contamination by microorganisms. Typically, loci are in aqueous systems such as cooling water systems, where microorganisms need to be killed or where their growth needs to be controlled. However, the present invention may also be used in all applications for which known antimicrobial compositions are useful, such as protection of wood, latex, adhesive, glue, paper, cardboard, textile, leather and feed.
Other uses include preservation of foods, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products or food ingredients.
In general it is contemplated that the antimicrobial polypeptides of the present invention are useful for cleaning, disinfecting or inhibiting microbial growth on any surface. Examples of surfaces, which may advantageously be contacted with the antimicrobial polypeptides of the invention, are surfaces of process equipment used e.g. dairies, chemical or pharmaceutical process plants. The antimicrobial polypeptides of the invention should be used in an amount, which is effective for cleaning, disinfecting or inhibiting microbial growth on the surface in question.
The antimicrobial polypeptides of the invention may additionally be used for cleaning surfaces and cooking utensils in food processing plants and in any area in which food is prepared or served such as hospitals, nursing homes and restaurants.
The invention also relates to the use of an antimicrobial polypeptide or composition of the invention as a medicament. Further, an antimicrobial polypeptide or composition of the invention may also be used for the manufacture of a medicament for controlling or combating microorganisms, such as fungal organisms or bacteria, preferably gram negative bacteria.
The composition and antimicrobial polypeptide of the invention may be used as an antimicrobial veterinarian or human therapeutic or prophylactic agent. Thus, the composition and antimicrobial polypeptide of the invention may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of microbial infections, such as bacterial or fungal infections, preferably gram positive bacterial infections. In particular the microbial infections may be associated with lung diseases including, but not limited to, tuberculosis, pneumonia and cystic fibrosis; skin infections and infections in the eye or the mouth; and sexually transmitted diseases including, but not limited to, gonorrhea and chlamydia.
The invention also relates to wound healing compositions or products such as bandages, medical devices such as, e.g., catheters.
The composition of the invention comprises an effective amount of the antimicrobial polypeptide of the invention.
The term “effective amount” when used herein is intended to mean an amount of the antimicrobial polypeptides of the invention, which is sufficient to inhibit growth of the microorganisms in question.
Formulations of the antimicrobial polypeptides of the invention are administered to a host suffering from or predisposed to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism, preferably it will be localized. Generally the dose of the antimicrobial polypeptides of the invention will be sufficient to decrease the microbial population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs of killing. The compounds of the present invention are administered at a dosage that reduces the microbial 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. The antimicrobial polypeptides of the invention are particularly useful for killing gram negative bacteria, including Pseudomonas aeruginosa, and Chlamydia trachomatis; and gram-positive bacteria, including streptococci such as Streptococcus pneumonia, S. uberis, S. hyointestinalis, S. pyogenes and S. agalactiae; and staphylococci such as Staphylococcus aureus, S. epidermidis, S. simulans, S. xylosus and S. carnosus.
Formulations of the antimicrobial polypeptides of the invention may be administered to a host suffering from or predisposed to a microbial lung infection, such as pneumonia; or to a microbial wound infection, such as a bacterial wound infection.
Formulations of the antimicrobial polypeptides of the invention may also be administered to a host suffering from or predisposed to a skin infection, such as acne, atopic dermatitis or seborrheic dermatitis; preferably the skin infection is a bacterial skin infection, e.g. caused by Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes, Pityrosporum ovale or Malassezia furfur.
The antimicrobial polypeptides of the invention are also useful for in vitro formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, the antimicrobial polypeptides of the invention may be added to animal and/or human food preparations; or they may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture.
The susceptibility of a particular microbe to killing with the antimicrobial polypeptides of the invention may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with the antimicrobial polypeptide at varying concentrations for a period of time sufficient to allow the protein to act, usually between about one hour and one day. The viable microbes are then counted, and the level of killing determined.
Microbes of interest include, but are not limited to, Gram-negative bacteria, for example: Citrobacter sp.; Enterobacter sp.; Escherichia sp., e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium; Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Y. enterocolitica; Franciscella sp.; Pasteurella sp.; Vibrio sp., e.g. V. cholerae, V. parahemolyticus; Campylobacter sp., e.g. C. jejuni; Haemophilus sp., e.g. H. influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other bacteria of interest include Legionella sp., e.g. L. pneumophila; Listeria sp., e.g. L. monocytogenes; Mycoplasma sp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M. leprae; Treponema sp., e.g. T. pallidum; Borrelia sp., e.g. B. burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., e.g. H. pylori, etc.
Non-bacterial pathogens of interest include fungal and protozoan pathogens, e.g. Plasmodia sp., e.g. P. falciparum, Trypanosoma sp., e.g. T. brucei; shistosomes; Entaemoeba sp., Cryptococcus sp., Candida sp., e.g. C. albicans; etc.
Various methods for administration may be employed. 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 antimicrobial polypeptide 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. For example, the carboxy terminus may be amidated.

Formulations

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, creams, foams, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The antimicrobial polypeptides of the invention 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.
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.
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.
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.
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.
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.
Unit dosage forms for oral 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.
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 the antimicrobial polypeptides of the invention 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.
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.
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.
Typical dosages for systemic administration range from 0.1 pg 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.
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.
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 intracellularly. 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.
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 seconds, 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 seconds 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
For use in the subject methods, the antimicrobial polypeptides of the invention 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 beta-lactamase inhibitors, cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; chloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.
Anti-mycotic agents are also useful, including polyenes, e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol and fluconazol. Antituberculotic drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in a formulation of the antimicrobial polypeptides of the invention, e.g. interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

NZ17074 is the antimicrobial peptide of SEQ ID NO: 70.

Example 1

Isolation of Variants of SEQ ID NO: 2 Having Improved Antimicrobial Activity

The cDNA encoding SEQ ID NO: 2 was fused to the proregion of plectasin (see Mygind et al., 2005, Nature 437: 975-980) and the Mating Factor alpha-leader from Saccharomyces cerevisiae and introduced into the inducible S. cerevisiae expression vector, pYES2, and transformed into S. cerevisiae. This system takes advantage of the GAL1 promoter which is repressed by glucose and activated by galactose.
Several strategies were used for variant generation of the polynucleotide of SEQ ID NO:1. The resulting libraries were cloned and expressed in S. cerevisiae. Transformed clones were screened on a plate assay containing growth media supplemented with 1.5% galactose and 0.5% glucose and either horse blood (2.5-5%) or serum (5%), overlayed with the target organism, E. coli ATCC 10536 (See Raventos et al., 2005, Comb Chem High Throughput Screen. 8:219-33).
The plate assay conditions fully inhibited the activity of the antimicrobial peptide of SEQ ID NO: 2 (the parent antimicrobial peptide). Variants exhibiting improved antimicrobial activity (giving rise to clearing zones) in the presence of 2.5% blood, 5% blood or 5% serum were picked and sequenced, and are shown in Table 1.

Plate Assay Screening Procedure

Approximately, 300 Saccharomyces cerevisiae colonies expressing arenicin variants were spread on screening plates containing either horse blood (2.5% or 5%) or 5% horse serum (see composition of the plates below). Plates were incubated 3 hours at 30° C. to allow them to dry. Next, 25 ml overlay temperate at 42° C. was added to the plates. After the media had solidified, the plates were incubated 3 days at 30° C.
On day 4, plates were overlayed with pre-warmed media at 42° C. containing either 2.5% or 5% horse blood or 5% horse serum and the target bacteria, E. coli ATCC 10536 (see below for details on media composition). After the overlay solidified, plates were incubated 16 hours at 37° C. Next day, plates were colored by adding 10 ml of 1.5 mM MTT to the plates and incubated at room temperature for 30 minutes. Clones giving rise to clearing zones were picked and sequenced.

Plate and Media Composition

Three different types of screening plates a), b) and c) were used in the screening:

a) Plates+2.5% Horse Blood

The bottom layer contains 50 ml of 1.5% agarose+¼ SC media+2.5% blood+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+¼ SC media+2.5% blood+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+2.5% horse blood and 1.25×10⁶colony forming units (cfu) of E. coli ATCC 10536.

b) Plates+5% Horse Blood

The bottom layer contains 50 ml of 1.5% agarose+¼ SC media+5% blood+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+¼ SC media+5% blood+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+5% horse blood and 1.25×10⁶colony forming units (cfu) of E. coli ATCC 10536.

c) Plates+5% Horse Serum

The bottom layer contains 50 ml of 1.5% agarose+½ SC media+5% serum+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+½ SC media+5% serum+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+5% horse serum and 1.25×10⁶colony forming units (cfu) of E. coli ATCC 10536.

Composition of SC Media (450 ml)

Yeast Nitrogen Base w/o amino acids: 3.75 g
Succinic acid: 5.65 g
Sodium hydroxide: 3.4 g
Casamino acid vitamin assay: 2.8 g

L-Tryptophan: 0.05 g

Water: 450 ml

pH was adjusted to 6 and the media was autoclaved and diluted ¼ when preparing the blood plates and ½ when preparing the serum plates.
MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma 13,503-8)

Determination of the Protein Binding

Protein binding assays were performed as follows. The purified peptides were mixed with 90% serum and centrifuged through a 30 kDa filter. The ultra-filtrate and the non filtrated serum samples were quantified by HPLC measurements and the protein binding was subsequently calculated.
The antimicrobial peptide of SEQ ID NO: 2 (the parent antimicrobial peptide) exhibited a protein binding of 99.5% in this assay. As shown in Table 1, all exemplified variants exhibit a lower protein binding than the antimicrobial peptide of SEQ ID NO: 2.

TABLE 1

Variants exhibiting improved antimicrobial activity and reduced protein binding.
The symbol “—” means “not analyzed”.

SEQ			Protein binding
ID NO:	Substitution	Amino acid sequence	(%)

3	F2A	GACWYVCVYRNGVRVCYRRCN	86

4	W4A	GFCAYVCVYRNGVRVCYRRCN	89

5	W4E	GFCEYVCVYRNGVRVCYRRCN

6	W4G	GFCGYVCVYRNGVRVCYRRCN

7	W4S	GFCSYVCVYRNGVRVCYRRCN	68

8	W4T	GFCTYVCVYRNGVRVCYRRCN

9	W4Y	GFCYYVCVYRNGVRVCYRRCN

10	Y5K	GFCWKVCVYRNGVRVCYRRCN	64

11	Y5N	GFCWNVCVYRNGVRVCYRRCN	73

12	Y5R	GFCWRVCVYRNGVRVCYRRCN

13	V6A	GFCWYACVYRNGVRVCYRRCN

14	V6E	GFCWYECVYRNGVRVCYRRCN

15	V6G	GFCWYGCVYRNGVRVCYRRCN	96

16	V6L	GFCWYLCVYRNGVRVCYRRCN

17	V6N	GFCWYNCVYRNGVRVCYRRCN

18	V6R	GFCWYRCVYRNGVRVCYRRCN

19	V6S	GFCWYSCVYRNGVRVCYRRCN	89

20	V6W	GFCWYWCVYRNGVRVCYRRCN	99

21	V8A	GFCWYVCAYRNGVRVCYRRCN	98.5

22	V8G	GFCWYVCGYRNGVRVCYRRCN

23	V8H	GFCWYVCHYRNGVRVCYRRCN	98

24	V8S	GFCWYVCGYRNGVRVCYRRCN	85

25	V8Y	GFCWYVCYYRNGVRVCYRRCN	98

26	Y9I	GFCWYVCVIRNGVRVCYRRCN

27	Y9K	GFCWYVCVKRNGVRVCYRRCN

28	Y9R	GFCWYVCVRRNGVRVCYRRCN	92

29	V13A	GFCWYVCVYRNGARVCYRRCN	99

30	V13G	GFCWYVCVYRNGGRVCYRRCN	99

31	V13K	GFCWYVCVYRNGKRVCYRRCN

32	V13L	GFCWYVCVYRNGLRVCYRRCN

33	V13P	GFCWYVCVYRNGPRVCYRRCN	99

34	V13Q	GFCWYVCVYRNGQRVCYRRCN

35	V13R	GFCWYVCVYRNGRRVCYRRCN

36	V13S	GFCWYVCVYRNGSRVCYRRCN	60

37	V15A	GFCWYVCVYRNGVRACYRRCN

38	V15G	GFCWYVCVYRNGVRGCYRRCN	93

39	V15H	GFCWYVCVYRNGVRHCYRRCN	98

40	V15K	GFCWYVCVYRNGVRKCYRRCN

41	V15N	GFCWYVCVYRNGVRNCYRRCN	97

42	V15Q	GFCWYVCVYRNGVRQCYRRCN	96

43	V15R	GFCWYVCVYRNGVRRCYRRCN

44	V15S	GFCWYVCVYRNGVRACYRRCN	85

45	V15T	GFCWYVCVYRNGVRACYRRCN	98

46	Y17H	GFCWYVCVYRNGVRVCHRRCN	88

47	Y17K	GFCWYVCVYRNGVRVCKRRCN	89

48	Y17N	GFCWYVCVYRNGVRVCNRRCN	68

49	Y17R	GFCWYVCVYRNGVRVCRRRCN

50	N21H	GFCWYVCVYRNGVRVCYRRCH

51	N21K	GFCWYVCVYRNGVRVCYRRCK

52	N21R	GFCWYVCVYRNGVRVCYRRCR

53	N21S	GFCWYVCVYRNGVRVCYRRCS	99

54	N21T	GFCWYVCVYRNGVRVCYRRCT	99

55	G1R + N21R	RFCWYVCVYRNGVRVCYRRCR

56	W4F + Y17R	GFCFYVCVYRNGVRVCRRRCN	94

57	W4A + Y5H	GFCAHVCVYRNGVRVCYRRCN

58	W4A + Y5R	GFCARVCVYRNGVRVCYRRCN

59	W4F + Y9R	GFCFYVCVRRNGVRVCYRRCN

60	W4G + Y5H	GFCGHVCVYRNGVRVCYRRCN	64

61	W4G + Y5R	GFCGRVCVYRNGVRVCYRRCN	72

62	W4S + V15A	GFCSYVCVYRNGVRACYRRCN

63	W4S + Y5R	GFCSRVCVYRNGVRVCYRRCN

64	W4Y + Y5R	GFCYRVCVYRNGVRVCYRRCN

65	Y5F + V15Q	GFCWFVCVYRNGVRQCYRRCN

66	Y5H + V15S	GFCWHVCVYRNGVRSCYRRCN

67	Y5H + Y17R	GFCWHVCVYRNGVRVCRRRCN

68	Y5K + Y17S	GFCWKVCVYRNGVRVCSRRCN	87

69	Y5N + V15S	GFCWNVCVYRNGVRSCYRRCN

70	Y5N + Y17H	GFCWNVCVYRNGVRVCHRRCN	79

71	Y5R + V15P	GFCWRVCVYRNGVRPCYRRCN

72	Y5R + V6A	GFCWRACVYRNGVRVCYRRCN	97

73	Y5R + V8G	GFCWRVCGYRNGVRVCYRRCN

74	Y5R + V8H	GFCWRVCHYRNGVRVCYRRCN	99

75	Y5R + V8S	GFCWRVCGYRNGVRVCYRRCN

76	Y5R + Y17H	GFCWRVCVYRNGVRVCHRRCN	92

77	Y5R + Y17N	GFCWRVCVYRNGVRVCNRRCN

78	Y5S + V15S	GFCWSVCVYRNGVRSCYRRCN

79	V6A + V13A	GFCWYACVYRNGARVCYRRCN

80	V6A + V13K	GFCWYACVYRNGKRVCYRRCN	96

81	V6A + V15A	GFCWYACVYRNGVRACYRRCN

82	V6A + Y17H	GFCWYACVYRNGVRVCHRRCN

83	V6M + V8G	GFCWYMCGYRNGVRVCYRRCN

84	V8A + V15A	GFCWYVCAYRNGVRACYRRCN

85	V8S + V13K	GFCWYACVYRNGKRVCYRRCN

86	Y9K + V13A	GFCWYVCVKRNGARVCYRRCN

87	Y9K + V15S	GFCWYVCVKRNGVRSCYRRCN	93

88	R10K + V15S	GFCWYVCVYKNGVRSCYRRCN

89	R10K + Y17H	GFCWYVCVYKNGVRVCHRRCN	90

90	R10P + V13G	GFCWYVCVYPNGGRVCYRRCN

91	N11R + V15Q	GFCWYVCVYRRGVRQCYRRCN

92	V13A + Y17C	GFCWYVCVYRNGARVCCRRCN

93	V13G + V15K	GFCWYVCVYRNGGRKCYRRCN

94	V15L + Y17H	GFCWYVCVYRNGVRLCHRRCN

95	G1A + Y5N + Y17H	AFCWNVCVYRNGVRVCHRRCN

96	G1D + Y5N + Y17H	DFCWNVCVYRNGVRVCHRRCN

97	G1F + Y5N + Y17H	FFCWNVCVYRNGVRVCHRRCN

98	G1H + Y5N + Y17H	HFCWNVCVYRNGVRVCHRRCN	77

99	G1I + Y5N + Y17H	IFCWNVCVYRNGVRVCHRRCN

100	G1K + Y5N + Y17H	KFCWNVCVYRNGVRVCHRRCN	78

101	G1M + Y5N + Y17H	MFCWNVCVYRNGVRVCHRRCN	81

102	G1Q + Y5N + Y17H	QFCWNVCVYRNGVRVCHRRCN

103	G1R + Y5N + Y17H	RFCWNVCVYRNGVRVCHRRCN

104	G1S + Y5N + Y17H	SFCWNVCVYRNGVRVCHRRCN

105	G1T + Y5N + Y17H	TFCWNVCVYRNGVRVCHRRCN

106	G1V + Y5N + Y17H	VFCWNVCVYRNGVRVCHRRCN

107	G1W + Y5N + Y17H	WFCWNVCVYRNGVRVCHRRCN

108	G1Y + Y5N + Y17H	YFCWNVCVYRNGVRVCHRRCN

109	F2G + Y5N + Y17H	GGCWNVCVYRNGVRVCHRRCN

110	F2H + Y5N + Y17H	GHCWNVCVYRNGVRVCHRRCN

111	F2I + Y5N + Y17H	GICWNVCVYRNGVRVCHRRCN

112	F2L + Y5N + Y17H	GLCWNVCVYRNGVRVCHRRCN

113	F2M + Y5N + Y17H	GMCWNVCVYRNGVRVCHRRCN

114	F2P + Y5N + Y17H	GPCWNVCVYRNGVRVCHRRCN

115	F2V + Y5N + Y17H	GVCWNVCVYRNGVRVCHRRCN

116	F2W + Y5N + Y17H	GWCWNVCVYRNGVRVCHRRCN

117	F2Y + Y5N + Y17H	GYCWNVCVYRNGVRVCHRRCN

118	C3L + W4Q + Y17H	GFLQYVCVYRNGVRVCHRRCN

119	W4A + V6A + Y9K	GFCAYACVKRNGVRVCYRRCN

120	W4A + Y5K + V15S	GFCAKVCVYRNGVRSCYRRCN	69

121	W4A + Y5R + V15A	GFCARVCVYRNGVRACYRRCN

122	W4A + Y5R + V15S	GFCARVCVYRNGVRACYRRCN	81

123	W4A + Y5R + V15T	GFCARVCVYRNGVRACYRRCN

124	W4A + Y5R + V8S	GFCARVCVYRNGVRACYRRCN

125	W4A + Y5R + Y9K	GFCARVCVKRNGVRVCYRRCN	90

126	W4A + Y5W + V15Q	GFCAWVCVYRNGVRQCYRRCN

127	W4A + Y9R + V15S	GFCAYVCVRRNGVRSCYRRCN

128	W4F + V8A + V15S	GFCFYVCAYRNGVRSCYRRCN

129	W4F + Y5H + V15S	GFCFHVCVYRNGVRSCYRRCN

130	W4F + Y5N + V15S	GFCFNVCVYRNGVRSCYRRCN

131	W4F + Y5N + Y17H	GFCFNVCVYRNGVRVCHRRCN

132	W4F + Y5R + V15K	GFCFRVCVYRNGVRKCYRRCN

133	W4F + Y5R + V15Q	GFCFRVCVYRNGVRQCYRRCN

134	W4F + Y5R + V15S	GFCFRVCVYRNGVRKCYRRCN

135	W4F + Y5R + Y17H	GFCFRVCVYRNGVRVCHRRCN

136	W4F + Y5R + Y17Q	GFCFRVCVYRNGVRVCQRRCN

137	W4F + Y9K + Y17H	GFCFYVCVKRNGVRVCHRRCN	86

138	W4G + Y5H + N21A	GFCGHVCVYRNGVRVCYRRCA

139	W4G + Y5H + N21K	GFCGHVCVYRNGVRVCYRRCK

140	W4G + Y5H + N21L	GFCGHVCVYRNGVRVCYRRCL

141	W4G + Y5H + N21M	GFCGHVCVYRNGVRVCYRRCM

142	W4G + Y5H + N21P	GFCGHVCVYRNGVRVCYRRCP

143	W4G + Y5H + N21R	GFCGHVCVYRNGVRVCYRRCR

144	W4G + Y5H + N21S	GFCGHVCVYRNGVRVCYRRCS	68

145	W4G + Y5H + N21Y	GFCGHVCVYRNGVRVCYRRCY

146	W4G + Y5H + V15A	GFCGHVCVYRNGVRACYRRCN

147	W4G + Y5H + V15F	GFCGHVCVYRNGVRFCYRRCN

148	W4G + Y5H + V15G	GFCGHVCVYRNGVRGCYRRCN

149	W4G + Y5H + V15H	GFCGHVCVYRNGVRHCYRRCN

150	W4G + Y5H + V15I	GFCGHVCVYRNGVRACYRRCN

151	W4G + Y5H + V15L	GFCGHVCVYRNGVRLCYRRCN

152	W4G + Y5H + V15M	GFCGHVCVYRNGVRMCYRRCN

153	W4G + Y5H + V15N	GFCGHVCVYRNGVRNCYRRCN

154	W4G + Y5H + V15Q	GFCGHVCVYRNGVRQCYRRCN

155	W4G + Y5H + V15R	GFCGHVCVYRNGVRRCYRRCN	83

156	W4G + Y5H + V15S	GFCGHVCVYRNGVRQCYRRCN

157	W4G + Y5H + V15T	GFCGHVCVYRNGVRQCYRRCN

158	W4G + Y5H + V15W	GFCGHVCVYRNGVRWCYRRCN

159	W4G + Y5H + V15Y	GFCGHVCVYRNGVRYCYRRCN

160	W4G + Y5H + Y17F	GFCGHVCVYRNGVRVCFRRCN

161	W4G + Y5H + Y17G	GFCGHVCVYRNGVRVCGRRCN

162	W4G + Y5H + Y17I	GFCGHVCVYRNGVRVCFRRCN

163	W4G + Y5H + Y17L	GFCGHVCVYRNGVRVCLRRCN

164	W4G + Y5H + Y17M	GFCGHVCVYRNGVRVCMRRCN

165	W4G + Y5H + Y17T	GFCGHVCVYRNGVRVCTRRCN

166	W4G + Y5H + Y17V	GFCGHVCVYRNGVRVCVRRCN

167	W4G + Y5H + Y17W	GFCGHVCVYRNGVRVCWRRCN

168	W4G + Y5K + V15H	GFCGKVCVYRNGVRHCYRRCN

169	W4G + Y5R + V15L	GFCGRVCVYRNGVRLCYRRCN

170	W4G + Y5R + V15R	GFCGRVCVYRNGVRRCYRRCN	87

171	W4G + Y5R + V15T	GFCGRVCVYRNGVRLCYRRCN	84

172	W4G + Y5R + Y9R	GFCGRVCVRRNGVRVCYRRCN	88

173	W4G + Y5S + V15A	GFCGSVCVYRNGVRACYRRCN

174	W4G + Y5S + V15K	GFCGSVCVYRNGVRKCYRRCN

175	W4G + Y5S + V15R	GFCGSVCVYRNGVRRCYRRCN

176	W4G + Y9R + V15S	GFCGYVCVRRNGVRSCYRRCN

177	W4I + Y5N + Y17H	GFCINVCVYRNGVRVCHRRCN

178	W4L + Y5H + V15Q	GFCLHVCVYRNGVRQCYRRCN

179	W4L + Y5K + V15K	GFCLKVCVYRNGVRKCYRRCN

180	W4L + Y5R + V15Q	GFCLRVCVYRNGVRQCYRRCN

181	W4L + Y5R + V15S	GFCLRVCVYRNGVRSCYRRCN

182	W4M + Y5E + Y17T	GFCMEVCVYRNGVRVCTRRCN

183	W4M + Y5H + V15S	GFCMHVCVYRNGVRSCYRRCN

184	W4M + Y5N + Y17H	GFCMNVCVYRNGVRVCHRRCN

185	W4M + Y5R + V15T	GFCMRVCVYRNGVRTCYRRCN

186	W4M + Y5S + V15Q	GFCMSVCVYRNGVRQCYRRCN

187	W4M + Y5S + V15R	GFCMSVCVYRNGVRRCYRRCN

188	W4N + Y5R + V15I	GFCNRVCVYRNGVRICYRRCN

189	W4S + Y5K + V15R	GFCSKVCVYRNGVRRCYRRCN

190	W4S + Y5R + V15I	GFCSRVCVYRNGVRICYRRCN

191	W4T + Y5N + V15A	GFCTNVCVYRNGVRACYRRCN

192	W4T + Y5R + V15R	GFCTRVCVYRNGVRRCYRRCN

193	W4T + Y5R + V15T	GFCTRVCVYRNGVRTCYRRCN

194	W4T + Y5S + V15H	GFCTSVCVYRNGVRHCYRRCN

195	W4T + Y9K + V15K	GFCTYVCVKRNGVRKCYRRCN	95

196	W4V + Y5H + V15P	GFCVHVCVYRNGVRPCYRRCN

197	W4V + Y5H + Y17H	GFCVHVCVYRNGVRVCHRRCN

198	W4V + Y5K + V15Q	GFCVKVCVYRNGVRQCYRRCN

199	W4V + Y5N + Y17H	GFCVNVCVYRNGVRVCHRRCN

200	W4V + Y5R + V15G	GFCVRVCVYRNGVRGCYRRCN

201	W4V + Y5R + V15P	GFCVRVCVYRNGVRPCYRRCN

202	W4V + Y5R + V15Q	GFCVRVCVYRNGVRQCYRRCN

203	W4V + Y5R + V15R	GFCVRVCVYRNGVRRCYRRCN

204	W4V + Y5R + V15T	GFCVRVCVYRNGVRTCYRRCN

205	W4Y + Y5H + V15Y	GFCYHVCVYRNGVRYCYRRCN

206	W4Y + Y5K + V15S	GFCYKVCVYRNGVRSCYRRCN	86

207	W4Y + Y5N + V15R	GFCYNVCVYRNGVRRCYRRCN	91

208	W4Y + Y5N + Y17H	GFCYNVCVYRNGVRVCHRRCN

209	W4Y + Y5R + V15T	GFCYRVCVYRNGVRTCYRRCN	89

210	W4Y + Y5R + Y17H	GFCYRVCVYRNGVRVCHRRCN	89

211	W4Y + Y5R + Y17S	GFCYRVCVYRNGVRVCSRRCN	96

212	W4Y + Y5W + V15S	GFCYWVCVYRNGVRSCYRRCN

213	Y5H + V13A + V15S	GFCWHVCVYRNGARSCYRRCN	95

214	Y5H + V13S + V15S	GFCWHVCVYRNGSRSCYRRCN

215	Y5H + V15S + Y17S	GFCWHVCVYRNGVRSCSRRCN

216	Y5H + V8A + V13K	GFCWHVCAYRNGKRVCYRRCN

217	Y5H + V8A + V15S	GFCWHVCAYRNGVRSCYRRCN

218	Y5H + V8A + Y9R	GFCWHVCARRNGVRVCYRRCN	99

219	Y5H + V8H + G12S	GFCWHVCHYRNSVRVCYRRCN	99

220	Y5H + Y9S + V15K	GFCWHVCVSRNGVRKCYRRCN

221	Y5K + Y9S + Y17S	GFCWKVCVSRNGVRVCSRRCN

222	Y5N + G12A + Y17H	GFCWNVCVYRNAVRVCHRRCN

223	Y5N + G12D + Y17H	GFCWNVCVYRNDVRVCHRRCN

224	Y5N + G12E + Y17H	GFCWNVCVYRNEVRVCHRRCN

225	Y5N + G12F + Y17H	GFCWNVCVYRNFVRVCHRRCN

226	Y5N + G12H + Y17H	GFCWNVCVYRNHVRVCHRRCN

227	Y5N + G12K + Y17H	GFCWNVCVYRNKVRVCHRRCN	80

228	Y5N + G12R + Y17H	GFCWNVCVYRNRVRVCHRRCN	86

229	Y5N + G12Y + Y17H	GFCWNVCVYRNYVRVCHRRCN	91

230	Y5N + N11A + Y17H	GFCWNVCVYRAGVRVCHRRCN	71

231	Y5N + N11G + Y17H	GFCWNVCVYRGGVRVCHRRCN	66

232	Y5N + N11H + Y17H	GFCWNVCVYRHGVRVCHRRCN	85

233	Y5N + N11Q + Y17H	GFCWNVCVYRQGVRVCHRRCN	78

234	Y5N + N11R + Y17H	GFCWNVCVYRRGVRVCHRRCN	86

235	Y5N + V13A + Y17H	GFCWNVCVYRNGARVCHRRCN	64

236	Y5N + V13F + Y17H	GFCWNVCVYRNGFRVCHRRCN

237	Y5N + V13H + Y17H	GFCWNVCVYRNGHRVCHRRCN	40

238	Y5N + V13Q + Y17H	GFCWNVCVYRNGQRVCHRRCN

239	Y5N + V13R + Y17H	GFCWNVCVYRNGRRVCHRRCN	65

240	Y5N + V13T + Y17H	GFCWNVCVYRNGTRVCHRRCN

241	Y5N + V13W + Y17H	GFCWNVCVYRNGWRVCHRRCN

242	Y5N + V13Y + Y17H	GFCWNVCVYRNGYRVCHRRCN	83

243	Y5N + V15A + Y17H	GFCWNVCVYRNGVRACHRRCN

244	Y5N + V15C + Y17H	GFCWNVCVYRNGVRCCHRRCN

245	Y5N + V15F + Y17H	GFCWNVCVYRNGVRFCHRRCN	85

246	Y5N + V15G + Y17H	GFCWNVCVYRNGVRGCHRRCN	79

247	Y5N + V15H + Y17H	GFCWNVCVYRNGVRHCHRRCN

248	Y5N + V15I + Y17H	GFCWNVCVYRNGVRICHRRCN	58

249	Y5N + V15L + Y17H	GFCWNVCVYRNGVRLCHRRCN	83

250	Y5N + V15M + Y17H	GFCWNVCVYRNGVRMCHRRCN

251	Y5N + V15N + Y17H	GFCWNVCVYRNGVRNCHRRCN	71

252	Y5N + V15R + Y17H	GFCWNVCVYRNGVRRCHRRCN	76

253	Y5N + V15W + Y17H	GFCWNVCVYRNGVRWCHRRCN	94

254	Y5N + V15Y + Y17H	GFCWNVCVYRNGVRYCHRRCN	77

255	Y5N + V6A + V15N	GFCWNACVYRNGVRNCYRRCN

256	Y5N + V6A + V15S	GFCWNACVYRNGVRNCYRRCN

257	Y5N + V6A + Y17H	GFCWNACVYRNGVRVCHRRCN

258	Y5N + V6A + Y9K	GFCWNACVKRNGVRVCYRRCN	89

259	Y5N + V6C + Y17H	GFCWNCCVYRNGVRVCHRRCN

260	Y5N + V6F + Y17H	GFCWNFCVYRNGVRVCHRRCN	84

261	Y5N + V6H + Y17H	GFCWNHCVYRNGVRVCHRRCN

262	Y5N + V6I + Y17H	GFCWNICVYRNGVRVCHRRCN	88

263	Y5N + V6L + Y17H	GFCWNLCVYRNGVRVCHRRCN	80

264	Y5N + V6M + Y17H	GFCWNMCVYRNGVRVCHRRCN

265	Y5N + V6Q + Y17H	GFCWNQCVYRNGVRVCHRRCN

266	Y5N + V6T + Y17H	GFCWNTCVYRNGVRVCHRRCN

267	Y5N + V6W + Y17H	GFCWNWCVYRNGVRVCHRRCN	85

268	Y5N + V6Y + Y17H	GFCWNYCVYRNGVRVCHRRCN

269	Y5N + V8A + V13A	GFCWNVCAYRNGARVCYRRCN

270	Y5N + V8A + V15S	GFCWNVCAYRNGARVCYRRCN

271	Y5N + V8A + Y17H	GFCWNVCAYRNGVRVCHRRCN

272	Y5N + V8F + Y17H	GFCWNVCAYRNGVRVCHRRCN	74

273	Y5N + V8G + Y17H	GFCWNVCGYRNGVRVCHRRCN

274	Y5N + V8I + Y17H	GFCWNVCAYRNGVRVCHRRCN

275	Y5N + V8L + Y17H	GFCWNVCLYRNGVRVCHRRCN	80

276	Y5N + V8W + Y17H	GFCWNVCWYRNGVRVCHRRCN

277	Y5N + V8Y + Y17H	GFCWNVCYYRNGVRVCHRRCN

278	Y5N + Y17H + N21A	GFCWNVCVYRNGVRVCHRRCA

279	Y5N + Y17H + N21C	GFCWNVCVYRNGVRVCHRRCC

280	Y5N + Y17H + N21F	GFCWNVCVYRNGVRVCHRRCF

281	Y5N + Y17H + N21G	GFCWNVCVYRNGVRVCHRRCG	66

282	Y5N + Y17H + N21H	GFCWNVCVYRNGVRVCHRRCH	82

283	Y5N + Y17H + N21I	GFCWNVCVYRNGVRVCHRRCI

284	Y5N + Y17H + N21K	GFCWNVCVYRNGVRVCHRRCK	72

285	Y5N + Y17H + N21L	GFCWNVCVYRNGVRVCHRRCL

286	Y5N + Y17H + N21M	GFCWNVCVYRNGVRVCHRRCM

287	Y5N + Y17H + N21P	GFCWNVCVYRNGVRVCHRRCP

288	Y5N + Y17H + N21Q	GFCWNVCVYRNGVRVCHRRCQ

289	Y5N + Y17H + N21R	GFCWNVCVYRNGVRVCHRRCR	83

290	Y5N + Y17H + N21S	GFCWNVCVYRNGVRVCHRRCS

291	Y5N + Y17H + N21W	GFCWNVCVYRNGVRVCHRRCW	99

292	Y5N + Y17H + N21Y	GFCWNVCVYRNGVRVCHRRCY

293	Y5N + Y17H + R19D	GFCWNVCVYRNGVRVCHRDCN

294	Y5N + Y17H + R19H	GFCWNVCVYRNGVRVCHRHCN

295	Y5N + Y17H + R19K	GFCWNVCVYRNGVRVCHRKCN

296	Y5N + Y17H + R19M	GFCWNVCVYRNGVRVCHRMCN

297	Y5N + Y17H + R19T	GFCWNVCVYRNGVRVCHRTCN

298	Y5N + Y17H + R19Y	GFCWNVCVYRNGVRVCHRYCN

299	Y5N + Y9A + Y17H	GFCWNVCVARNGVRVCHRRCN	74

300	Y5N + Y9D + Y17H	GFCWNVCVDRNGVRVCHRRCN

301	Y5N + Y9F + Y17H	GFCWNVCVFRNGVRVCHRRCN

302	Y5N + Y9G + Y17H	GFCWNVCVGRNGVRVCHRRCN

303	Y5N + Y9H + Y17H	GFCWNVCVHRNGVRVCHRRCN	43

304	Y5N + Y9I + Y17H	GFCWNVCVIRNGVRVCHRRCN

305	Y5N + Y9K + Y17H	GFCWNVCVKRNGVRVCHRRCN	76

306	Y5N + Y9M + Y17H	GFCWNVCVMRNGVRVCHRRCN

307	Y5N + Y9Q + Y17H	GFCWNVCVQRNGVRVCHRRCN

308	Y5N + Y9R + Y17H	GFCWNVCVRRNGVRVCHRRCN	94

309	Y5N + Y9S + Y17H	GFCWNVCVSRNGVRVCHRRCN

310	Y5N + Y9T + Y17H	GFCWNVCVTRNGVRVCHRRCN

311	Y5N + Y9V + Y17H	GFCWNVCVVRNGVRVCHRRCN

312	Y5N + Y9W + Y17H	GFCWNVCVWRNGVRVCHRRCN

313	Y5R + V13A + V15K	GFCWRVCVYRNGARKCYRRCN	98

314	Y5R + V13A + Y17S	GFCWRVCVYRNGARVCSRRCN

315	Y5R + V15K + Y17H	GFCWRVCVYRNGVRKCHRRCN	93

316	Y5R + V15S + Y17H	GFCWRVCVYRNGVRSCHRRCN	90

317	Y5R + V15S + Y17S	GFCWRVCVYRNGVRSCSRRCN

318	Y5R + V6A + G12S	GFCWRACVYRNSVRVCYRRCN

319	Y5R + V6A + V15A	GFCWRACVYRNGVRACYRRCN	40

320	Y5R + V6A + V15S	GFCWRACVYRNGVRACYRRCN	88

321	Y5R + V6A + Y17H	GFCWRACVYRNGVRVCHRRCN

322	Y5R + V6A + Y9K	GFCWRACVKRNGVRVCYRRCN

323	Y5R + V6C + V15S	GFCWRCCVYRNGVRSCYRRCN

324	Y5R + V6S + Y17H	GFCWRSCVYRNGVRVCHRRCN

325	Y5R + V8A + V15S	GFCWRCCVYRNGVRSCYRRCN

326	Y5R + V8G + Y17H	GFCWRVCGYRNGVRVCHRRCN

327	Y5R + V8H + G12S	GFCWRVCHYRNSVRVCYRRCN	98

328	Y5R + V8H + V13K	GFCWRVCHYRNGKRVCYRRCN

329	Y5R + V8S + V13A	GFCWRVCSYRNGSRVCYRRCN

330	Y5R + V8S + V13S	GFCWRVCSYRNGSRVCYRRCN

331	Y5R + V8S + V15K	GFCWRVCSYRNGVRKCYRRCN	95

332	Y5R + V8S + Y9R	GFCWRVCSRRNGVRVCYRRCN

333	Y5R + V8S + Y9S	GFCWRVCSSRNGVRVCYRRCN

334	Y5R + Y9R + V13A	GFCWRVCVRRNGARVCYRRCN

335	Y5R + Y9R + Y17H	GFCWRVCVRRNGVRVCHRRCN	95

336	Y5R + Y9S + Y17H	GFCWRVCVSRNGVRVCHRRCN

337	Y5R + Y9S + Y17S	GFCWRVCVSRNGVRVCSRRCN

338	V6A + G12S + V13K	GFCWYACVYRNSKRVCYRRCN

339	V6A + V13A + V15S	GFCWYACVYRNGKRACYRRCN

340	V6A + V13K + V15A	GFCWYACVYRNGKRACYRRCN	98

341	V6A + V13K + Y17H	GFCWYACVYRNGKRVCHRRCN

342	V6A + V15K + Y17H	GFCWYACVYRNGVRKCHRRCN

343	V6A + V8A + V15A	GFCWYACAYRNGVRACYRRCN	74

344	V6R + V8H + R10S	GFCWYRCHYSNGVRVCYRRCN

345	V6S + Y9R + V15S	GFCWYSCVRRNGVRSCYRRCN

346	V6T + Y9K + V13L	GFCWYTCVKRNGLRVCYRRCN	86

347	V8A + R10K + Y17H	GFCWYVCAYKNGVRVCHRRCN

348	V8A + V15A + Y17H	GFCWYVCAYRNGVRACHRRCN

349	V8A + V15S + Y17H	GFCWYVCAYRNGVRSCHRRCN

350	V8A + Y9R + V13A	GFCWYVCARRNGARVCYRRCN

351	V8A + Y9R + V15S	GFCWYSCVRRNGVRSCYRRCN

352	V8F + G12H + Y17H	GFCWYVCFYRNHVRVCHRRCN

353	Y9F + V15A + Y17H	GFCWYVCVFRNGVRACHRRCN

354	Y9K + V13A + Y17S	GFCWYVCVKRNGARVCSRRCN

355	Y9R + V15S + Y17S	GFCWYVCVRRNGVRSCSRRCN

356	R10K + V13K + V15S	GFCWYVCVYKNGKRSCYRRCN

357	V13K + V15A + Y17H	GFCWYVCVYRNGKRACHRRCN

358	G1R + Y5N + N11H + Y17H	RFCWNVCVYRHGVRVCHRRCN

359	G1R + Y5N + Y17H + R19H	RFCWNVCVYRNGVRVCHRHCN	80

360	W4A + V6A + Y9K + V13A	GFCAYACVKRNGARVCYRRCN

361	W4A + Y5H + V13A + V15K	GFCAHVCVYRNGARKCYRRCN

362	W4A + Y5H + Y9K + V13A	GFCAHVCVKRNGARVCYRRCN	76

363	W4A + Y5R + V8A + Y9K	GFCARVCAKRNGVRVCYRRCN	80

364	W4A + Y5R + Y9K + V15S	GFCARVCAKRNGVRVCYRRCN

365	W4A + Y5R + Y9R + V15K	GFCARVCVRRNGVRKCYRRCN

366	W4A + Y5R + Y9R + V15S	GFCARVCVRRNGVRKCYRRCN

367	W4F + V6A + Y9R + V15S	GFCFYACVRRNGVRSCYRRCN

368	W4F + V8A + Y9S + V15S	GFCFYVCASRNGVRSCYRRCN

369	W4F + Y5H + V8A + Y17H	GFCFHVCAYRNGVRVCHRRCN

370	W4F + Y5H + V8S + V15K	GFCFHVCSYRNGVRKCYRRCN

371	W4F + Y5N + V6A + V15S	GFCFNACVYRNGVRSCYRRCN

372	W4F + Y5N + V8A + Y9R	GFCFNVCARRNGVRVCYRRCN

373	W4F + Y5R + V15K + Y17H	GFCFRVCVYRNGVRKCHRRCN

374	W4F + Y5R + V15S + Y17S	GFCFRVCVYRNGVRSCSRRCN

375	W4F + Y5R + V6A + Y9K	GFCFRACVKRNGVRVCYRRCN

376	W4F + Y5R + V6S + V13A	GFCFRSCVYRNGARVCYRRCN

377	W4F + Y5R + V6S + Y9R	GFCFRSCVRRNGVRVCYRRCN	65

378	W4F + Y5R + V8A + V15K	GFCFRVCAYRNGVRKCYRRCN

379	W4F + Y5R + V8A + Y17H	GFCFRVCAYRNGVRVCHRRCN

380	W4F + Y5R + V8A + Y9S	GFCFRVCASRNGVRVCYRRCN

381	W4F + Y5R + Y9K + V15K	GFCFRVCVKRNGVRKCYRRCN	98

382	W4F + Y9R + V15S + Y17H	GFCFYVCVRRNGVRSCHRRCN

383	W4G + Y5H + V15M + N21H	GFCGHVCVYRNGVRMCYRRCH

384	W4G + Y5K + Y9R + V13L	GFCGKVCVRRNGLRVCYRRCN

385	W4G + Y5R + V13K + V15A	GFCGRVCVYRNGKRACYRRCN

386	W4G + Y5R + V13K + V15A	GFCGRVCVYRNGKRACYRRCN	81

387	W4G + Y5R + V13K + V15S	GFCGRVCVYRNGKRACYRRCN

388	W4G + Y5R + Y9R + V15K	GFCGRVCVRRNGVRKCYRRCN

389	W4T + Y5R + Y9R + V13L	GFCTRVCVRRNGLRVCYRRCN

390	Y5H + R10K + V13K + V15S	GFCWHVCVYKNGKRSCYRRCN	87

391	Y5H + V6A + Y9R + V15S	GFCWHVCVRRNGARKCYRRCN

392	Y5H + V8A + V13K + V15A	GFCWHVCAYRNGKRACYRRCN

393	Y5H + V8A + Y9K + V13L	GFCWHVCAKRNGLRVCYRRCN

394	Y5H + V8A + Y9R + V13A	GFCWHVCARRNGARVCYRRCN	80

395	Y5H + V8A + Y9R + Y17H	GFCWHVCARRNGVRVCHRRCN

396	Y5H + V8S + Y9K + V13L	GFCWHVCSKRNGLRVCYRRCN	92

397	Y5H + Y9R + V13A + V15K	GFCWHVCVRRNGARKCYRRCN

398	Y5H + Y9R + V13A + V15S	GFCWHVCVRRNGARKCYRRCN

399	Y5H + Y9R + V13A + Y17S	GFCWHVCVRRNGARVCSRRCN

400	Y5H + Y9S + V13L + V15K	GFCWHVCVSRNGLRKCYRRCN

401	Y5K + V6A + R10K + Y17H	GFCWKACVYKNGVRVCHRRCN

402	Y5K + V6A + V13A + V15S	GFCWKACAYRNGARVCYRRCN

403	Y5K + V6A + V8A + V13A	GFCWKACAYRNGARVCYRRCN

404	Y5K + Y9K + V13A + V15K	GFCWKVCVKRNGARKCYRRCN

405	Y5K + Y9R + V13A + Y17S	GFCWKVCVRRNGARVCSRRCN

406	Y5N + V6A + V8A + V13K	GFCWNACAYRNGKRVCYRRCN

407	Y5N + V6A + V8A + Y9R	GFCWNACARRNGVRVCYRRCN	88

408	Y5N + V6A + Y9R + V15S	GFCWNACARRNGVRVCYRRCN

409	Y5N + V6F + N11Y+ Y17H	GFCWNFCVYRYGVRVCHRRCN

410	Y5N + V8A + N11Q + Y17H	GFCWNVCAYRQGVRVCHRRCN

411	Y5N + V8A + V13K + V15A	GFCWNVCAYRNGKRACYRRCN	86

412	Y5N + Y9R + V13A + V15S	GFCWNVCVRRNGARSCYRRCN

413	Y5R + G12S + V13K + Y17H	GFCWRVCVYRNSKRVCHRRCN

414	Y5R + R10K + V13K + Y17H	GFCWRVCVYKNGKRVCHRRCN

415	Y5R + V6A + V13K + V15S	GFCWRACAYRNGKRVCYRRCN

416	Y5R + V6A + V15A + Y17H	GFCWRACVYRNGVRACHRRCN

417	Y5R + V6A + V8A + V13K	GFCWRACAYRNGKRVCYRRCN	95

418	Y5R + V6A + V8A + V15A	GFCWRACAYRNGVRACYRRCN	92

419	Y5R + V6A + V8A + Y17H	GFCWRACAYRNGVRVCHRRCN

420	Y5R + V6A + V8A + Y9R	GFCWRACARRNGVRVCYRRCN

421	Y5R + V6A + V8H + V15S	GFCWRACHYRNGVRSCYRRCN

422	Y5R + V6A + Y9R + V15K	GFCWRACVRRNGVRKCYRRCN

423	Y5R + V6S + V13A + V15S	GFCWRSCVYRNGARSCYRRCN

424	Y5R + V8A + G12S + V15S	GFCWRVCAYRNSVRSCYRRCN

425	Y5R + V8A + R10S + V13K	GFCWRVCAYSNGKRVCYRRCN	69

426	Y5R + V8A + V13A + Y17H	GFCWRVCAYRNGARVCHRRCN

427	Y5R + V8A + V13K + V15A	GFCWRVCAYRNGKRACYRRCN

428	Y5R + V8A + V13K + Y17H	GFCWRVCAYRNGKRVCHRRCN

429	Y5R + V8A + V15A + Y17H	GFCWRVCAYRNGVRACHRRCN

430	Y5R + V8A + V15S + Y17H	GFCWRVCAYRNGVRSCHRRCN

431	Y5R + V8A + Y9K + V15K	GFCWRVCAKRNGVRKCYRRCN

432	Y5R + V8A + Y9K + V15S	GFCWRVCAKRNGVRKCYRRCN

433	Y5R + V8A + Y9K + Y17H	GFCWRVCAKRNGVRVCHRRCN

434	Y5R + V8A + Y9R + V15S	GFCWRACARRNGVRVCYRRCN

435	Y5R + V8A + Y9S + V13L	GFCWRVCASRNGLRVCYRRCN	85

436	Y5R + V8G + G12S + V13K	GFCWRVCGYRNSKRVCYRRCN

437	Y5R + V8H + G12S + V13K	GFCWRVCHYRNSKRVCYRRCN

438	Y5R + V8H + R10K + V13K	GFCWRVCHYKNGKRVCYRRCN

439	Y5R + V8H + R10S + V13K	GFCWRVCHYSNGKRVCYRRCN	92

440	Y5R + V8S + Y9K + V15K	GFCWRVCSKRNGVRKCYRRCN

441	Y5R + Y9K + V13A + V15K	GFCWRVCVKRNGARKCYRRCN

442	Y5R + Y9K + V15S + Y17S	GFCWRVCVKRNGVRSCSRRCN

443	Y5R + Y9R + V13A + V15K	GFCWRVCVRRNGARKCYRRCN

444	Y5R + Y9R + V13A + V15S	GFCWRVCVRRNGARKCYRRCN

445	Y5R + Y9R + V13L + V15K	GFCWRVCVRRNGLRKCYRRCN

446	Y5R + Y9R + V13L + Y17H	GFCWRVCVRRNGLRVCHRRCN

447	Y5R + Y9R + V15S + Y17S	GFCWRVCVRRNGVRSCSRRCN

448	Y5R + Y9R + Y17S + R19H	GFCWRVCVRRNGVRVCSRHCN

449	Y5R + Y9S + V13A + Y17S	GFCWRVCVSRNGARVCSRRCN

450	V6A + V8A + Y9K + V13L	GFCWYACAKRNGLRVCYRRCN	98

451	V6A + Y9S + V13L + V15S	GFCWYACVSRNGLRSCYRRCN

452	V8A + R10S + V15S + Y17H	GFCWYVCAYSNGVRSCHRRCN

453	V8A + Y9R + V13L + V15S	GFCWYVCARRNGLRSCYRRCN	92

454	Y9K + V13A + V15K + Y17H	GFCWYVCVKRNGARKCHRRCN	90

455	W4A + Y5R + Y9K + V13A + V15K	GFCARVCVKRNGARKCYRRCN

456	W4A + Y5R + Y9K + V13A + Y17H	GFCARVCVKRNGARVCHRRCN

457	W4A + Y5R + Y9S + V13L + V15K	GFCARVCVSRNGLRKCYRRCN

458	W4A + Y9S + V13A + V15S + Y17H	GFCAYVCVSRNGARSCHRRCN

459	W4F + Y5H + V8A + Y9R + V15K	GFCFHVCARRNGVRKCYRRCN

460	W4F + Y5H + V8A + Y9R + Y17H	GFCFHVCARRNGVRVCHRRCN

461	W4F + Y5H + Y9K + V15K + Y17S	GFCFHVCVKRNGVRKCSRRCN

462	W4F + Y5K + V8S + V13L+ V15K	GFCFKVCSYRNGLRKCYRRCN

463	W4F + Y5K + V8S + Y9K + V15K	GFCFKVCSKRNGVRKCYRRCN

464	W4F + Y5N + V8A + Y9R + Y17S	GFCFNVCARRNGVRVCSRRCN

465	W4F + Y5R + V13A + V15K + Y17S	GFCFRVCVYRNGARKCSRRCN

466	W4F + Y5R + V6A + V8A + Y17H	GFCFRACAYRNGVRVCHRRCN

467	W4F + Y5R + V6A + V8T + Y9S	GFCFRACVSRNGVRSCYRRCN

468	W4F + Y5R + V6A + Y9K + V13A	GFCFRACVKRNGARVCYRRCN

469	W4F + Y5R + V6A + Y9R + Y17H	GFCFRACVRRNGVRVCHRRCN

470	W4F + Y5R + V6A + Y9S + V15S	GFCFRACVSRNGVRSCYRRCN

471	W4F + Y5R + V6S + V8A + Y9R	GFCFRSCARRNGVRVCYRRCN

472	W4F + Y5R + V6S + V8A + Y9S	GFCFRSCASRNGVRVCYRRCN

473	W4F + Y5R + V8A + V13A + V15S	GFCFRVCSYRNGARSCYRRCN

474	W4F + Y5R + V8A + V13A + Y17H	GFCFRVCAYRNGARVCHRRCN

475	W4F + Y5R + V8A + V13A + Y17S	GFCFRVCAYRNGARVCSRRCN

476	W4F + Y5R + V8A + V15K + Y17H	GFCFRVCAYRNGVRKCHRRCN

477	W4F + Y5R + V8A + Y9K + V13A	GFCFRVCAKRNGARVCYRRCN

478	W4F + Y5R + V8A + Y9K + V15K	GFCFRVCAKRNGVRKCYRRCN

479	W4F + Y5R + V8A + Y9K + Y17H	GFCFRVCAKRNGVRVCHRRCN

480	W4F + Y5R + V8A + Y9S + V15K	GFCFRVCASRNGVRKCYRRCN

481	W4F + Y5R + V8S + V13A + V15S	GFCFRVCSYRNGARSCYRRCN

482	W4F + Y5R + V8S + V13L + V15K	GFCFRVCSYRNGLRKCYRRCN

483	W4F + Y5R + V8S + Y9K + V13A	GFCFRVCSKRNGARVCYRRCN

484	W4F + Y5R + V8S + Y9K + Y17H	GFCFRVCSKRNGVRVCHRRCN

485	W4F + Y5R + V8S + Y9R + V13L	GFCFRVCSRRNGLRVCYRRCN

486	W4F + Y5R + V8S + Y9S + V13A	GFCFRVCSSRNGARVCYRRCN

487	W4F + Y5R + V8S + Y9S + V15K	GFCFRVCSSRNGVRKCYRRCN

488	W4F + Y5R + Y9K + V13A + V15S	GFCFRVCVKRNGARSCYRRCN

489	W4F + Y5R + Y9R + V15K + Y17S	GFCFRVCVRRNGVRKCSRRCN

490	W4F + Y9S + V13A + V15K + Y17H	GFCFYVCVSRNGARKCHRRCN

491	W4G + Y5K + Y9K + V13L + V15K	GFCGKVCVKRNGLRKCYRRCN

492	W4G + Y5R + G12S + V13K + V15S	GFCGRVCVYRNSKRSCYRRCN

493	W4G + Y5R + R10K + V13K + V15A	GFCGRVCVYKNGKRACYRRCN	77

494	W4S + V6S + V8A + V13S + V15S	GFCSYSCAYRNGSRSCYRRCN

495	W4S + Y5N + V6S + Y9K + Y17S	GFCSNSCVKRNGVRVCSRRCN

496	W4T + Y5N + V8A + Y9K + V13A	GFCTNVCAKRNGARVCYRRCN

497	Y5H + V6A + Y9R + V13A + V15S	GFCWHACVRRNGARSCYRRCN

498	Y5H + V6A + Y9R + V13L + V15K	GFCWHACVRRNGLRKCYRRCN

499	Y5H + V6A + Y9R + V13L + Y17H	GFCWHACVRRNGLRVCHRRCN

500	Y5H + V8A + R10K + V15A + Y17H	GFCWHVCAYKNGVRACHRRCN

501	Y5H + V8A + Y9R + V13L + Y17S	GFCWHVCARRNGLRVCSRRCN

502	Y5K + V6R + V13L + V15K + Y17H	GFCWKRCVYRNGLRKCHRRCN

503	Y5K + V6S + Y9R + V13L + V15S	GFCWKSCVRRNGLRSCYRRCN

504	Y5K + V8A + Y9R + V13A + V15S	GFCWKVCARRNGARSCYRRCN	95

505	Y5K + V8S + Y9S + V13L+ V15S	GFCWKVCSSRNGLRSCYRRCN

506	Y5K + Y9S + V13A + V15K + Y17S	GFCWKVCVSRNGARKCSRRCN

507	Y5N + V8A + Y9R + V13L + Y17H	GFCWNVCARRNGLRVCHRRCN

508	Y5N + Y9R + V13L + V15K + Y17H	GFCWNVCVRRNGLRKCHRRCN

509	Y5R + R10S + V13K + V15S + Y17H	GFCWRVCVYSNGKRSCHRRCN	87

510	Y5R + V6A + R10K + G12S + V13K	GFCWRACVYKNSKRVCYRRCN	_

511	Y5R + V6A + R10K + V15A + Y17H	GFCWRACVYKNGVRACHRRCN

512	Y5R + V6A + V8A + V13K + V15A	GFCWRACAYRNGKRACYRRCN

513	Y5R + V6A + V8A + V13K + V15S	GFCWRACAYRNGKRSCYRRCN

514	Y5R + V6A + V8A + Y9S + Y17H	GFCWRACASRNGVRVCHRRCN

515	Y5R + V6A + V8S + Y9R + V13L	GFCWRACSRRNGLRVCYRRCN

516	Y5R + V6A + Y9K + V13A + Y17S	GFCWRACVKRNGARVCSRRCN

517	Y5R + V6A + Y9K + V13L + V15S	GFCWRACVKRNGLRSCYRRCN

518	Y5R + V6A + Y9R + V13A + V15S	GFCWRACVRRNGARSCYRRCN

519	Y5R + V6A + Y9R + V13L + V15S	GFCWRACVRRNGLRSCYRRCN

520	Y5R + v6A + Y9S + V13A + V15S	GFCWRACVSRNGARSCYRRCN

521	Y5R + V6A + Y9S + V13L + Y17S	GFCWRACVSRNGLRVCSRRCN

522	Y5R + V6R + Y9R + V13A + V15S	GFCWRRCVRRNGARSCYRRCN

523	Y5R + V8A + R10K + V13K + V15S	GFCWRVCAYKNGKRSCYRRCN	90

524	Y5R + V8A + Y9R + V13L + Y17H	GFCWRVCARRNGLRVCHRRCN

525	Y5R + V8A + Y9S + V13A + Y17S	GFCWRVCASRNGARVCSRRCN

526	Y5R + V8G + R10K + V15A + Y17H	GFCWRVCGYKNGVRACHRRCN

527	Y5R + V8G + V13K + V15A + Y17H	GFCWRVCGYRNGKRACHRRCN

528	Y5R + V8S + R10S + G12S + V13K	GFCWRVCSYSNSKRVCYRRCN

529	Y5R + V8S + Y9R + V13L + V15S	GFCWRVCSRRNGLRSCYRRCN

530	Y5R + Y9R + V13L + V15S + Y17S	GFCWRVCVRRNGLRSCSRRCN

531	Y5R + Y9S + V13A + V15K + Y17S	GFCWRVCVSRNGARKCSRRCN

532	Y5R + Y9S + V13L + V15S + Y17S	GFCWRVCVSRNGLRSCSRRCN

533	F2L + Y5R + V8A + R10S + V13K + V15A	GLCWRVCAYSNGKRACYRRCN

534	W4F + Y5N + Y9S + V13A + V15K + Y17H	GFCFNVCVSRNGARKCHRRCN

535	W4F + Y5R + V6A + V8A + Y9R + V15S	GFCFRACARRNGVRSCYRRCN

536	W4F + Y5R + V6A + V8S + Y9K + V13L	GFCFRACSKRNGLRVCYRRCN

537	W4F + Y5R + V6A + Y9K + V13L + V15K	GFCFRACVKRNGLRKCYRRCN

538	W4F + Y5R + V6A + Y9K + V13L + Y17H	GFCFRACVKRNGLRVCHRRCN

539	W4F + Y5R + V8A + Y9K + V13A + V15S	GFCFRVCAKRNGARSCYRRCN

540	W4F + Y5R + V8A + Y9S + V15K + Y17H	GFCFRVCASRNGVRKCHRRCN

541	W4F + Y5R + V8S + Y9K + V13A + V15K	GFCFRVCSKRNGARKCYRRCN

542	W4G + Y5H + V6R + V8S + Y9R + V15K	GFCGHRCSRRNGVRKCYRRCN

543	W4S + Y5H + V6R + V8S + G12S + V15A	GFCSHRCSYRNSVRACYRRCN

544	W4S + Y5R + V8S + V13S + V15A + Y17H	GFCSRVCSYRNGSRACHRRCN

545	Y5R + V6A + V8A + Y9S + V13L + Y17S	GFCWRACASRNGLRVCSRRCN

546	W4S + Y5N + V6R + V8H + R10S + V13S +	GFCSNRCHYSNGSRACHRRCN
	V15A + Y17H

547	Y5G + V6A + C7V + V8N + R10T + N11S +	GFCWGAVNYTSNCRACKRRCN
	G12N + V13C + V15A + Y17K

548	F2S + Y5G + V6A + C7V + V8N + R10T +	GSCWGAVNYTSNCRACKRRCN
	N11S + G12N + V13C + V15A + Y17K

Example 2

Efficacy of NZ17074 Against Escherichia coli AID#172 in the Neutropenic Murine Peritonitis/Sepsis Model and Estimation of ED50

Introduction

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic peritonitis model. Treatment with 40 mg/kg meropenem was included as a positive control group. The colony counts in blood and peritoneal fluid were determined at 5 hours after treatment.
The murine peritonitis/sepsis model is a well-recognized model for studies of antimicrobial activity as described by N. Frimodt-Møller and J. D. Knudsen in Handbook of Animal Models of Infection (1999), ed. by O. Zak & M. A. Sande, Academic Press, San Diego, US.

Materials and Methods

- 30 outbred, NMRI female mice, 25-30 grams (Harlan Scandinavia)
- E. coli AID#172 from Statens Serum Institute, Copenhagen, Denmark. Clinical isolate from a human wound from 2003. Multiresistant (Ampicillin, Ceftazidime, Aztreonam, Gentamicin, Ciprofloxacin)
- NZ17074 in Ringer Acetate, pH 6: 1.2 mg/ml, 6.0 ml. The solution was stored at 4° C. until use. Analyses of the dose formulations used were performed after completion of the in-life phase of the study and gave the following results:


	Intended concentration	Measured concentration

1.2	mg/ml	1.11	mg/ml
0.6	mg/ml	0.51	mg/ml
0.3	mg/ml	0.24	mg/ml
0.15	mg/ml	0.14	mg/ml
0.075	mg/ml	0.043	mg/ml
0.03	mg/ml	0.010	mg/ml
0.016	mg/ml	0.002	mg/ml

- Vehicle (Ringer Acetate pH 6). The solution was stored at 4° C. until use
- MERONEM®(AstraZeneca, 500 mg infusion substance, meropenem). Lot no. 09466C Date of expire: August 2013
- Water, sterile
- 0.9% saline, sterile
- Cyclophosphamide, APODAN® (A-Pharma, 1 g) Batch nr. 928491
- Date of expire: May 2012
- 5% Horse Blood Agar plates
- Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m-6 a.m.
The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 3 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage. Mice were weighed the day before dosing.

Preparation of NZ17074 Solutions

The solution of 1.2 mg/ml was further diluted in PBS vehicle as follows:
0.6 mg/ml˜7.5 mg/kg: 1.5 ml of 1.2 mg/ml NZ17074+1.5 ml vehicle
0.3 mg/ml˜5.0 mg/kg: 1.5 ml of 0.6 mg/ml NZ17074+1.5 ml vehicle
0.15 mg/ml˜2.5 mg/kg: 1.5 ml of 0.3 mg/ml NZ17074+1.5 ml vehicle
0.075 mg/ml˜1.25 mg/kg: 1.5 ml of 0.15 mg/ml NZ17074+1.5 ml vehicle
0.03 mg/ml˜0.63 mg/kg: 1.5 ml of 0.075 mg/ml NZ17074+2.25 ml vehicle
0.016 mg/ml˜0.16 mg/kg: 1.5 ml of 0.03 mg/ml NZ17074+1.5 ml vehicle

Preparation Meropenem Solution

Treatment with meropenem 40 mg/kg was included as a positive control group. A total of 500 mg meropenem (one ampoule) was dissolved in 10 ml water˜50 mg/ml This stock solution was further diluted to 4 mg/ml (0.4 ml 50 mg/ml+4.6 ml saline).

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule (APODAN® (A-Pharma, 1 g) 1 g) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml 20 mg/ml+13.5 ml saline) for use on day −4 or to 5 mg/kg (8.25 ml 20 mg/ml+21.75 ml saline)) for use on day −1.
Treatment of Mice with Cyclophosphamide
The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁶CFU/ml. One hour before start of treatment (time −1 hr) mice were inoculated intraperitoneally with 0.5 ml of the E. coli suspension in the lateral lower quadrant of the abdomen. Approximately ½-1 hour after treatment, mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml corresponding to 30 mg/kg) as a pain relief.

Treatment of Mice

The mice were treated i.v. in the lateral tail vein over approximately 30 seconds with 10 ml/kg with a single dose of NZ17074, meropenem or vehicle at time 0 hour (see Table 1). The dosing was based on a mean weight of 30 g. Mice that weighed 28-32 g received 0.30 ml solution. Mice that weighed 27-28 g received 0.25 ml solution and mice that weighed 32.1-36 g received 0.35 ml solution.

TABLE 1

Treatment and sampling schedule in the murine peritonitis model.

Inoculation

Intravenous treatment

Sampling and mouse no.

i.p. at - 1 hr	at 0 hr	0 hour	5 hours

0.5 ml of	Vehicle, Ringer acetate		1-2-3
E. coli	NZ17074 0.16 mg/kg		4-5-6
AID#172	NZ17074 0.30 mg/kg		7-8-9
1 × 10⁶CFU/ml	NZ17074 0.75 mg/kg		10-11-12
	NZ17074 1.5 mg/kg		13-14-15
	NZ17074 3.0 mg/kg		16-17-18
	NZ17074 6.0 mg/kg		19-20-21
	NZ17074 12 mg/kg		22-23-24
	meropenem 40 mg/kg		25-26-27
	No treatment	28-29-30

T indicates the time in relation to treatment.
Numbers in the sampling columns are mouse identification numbers.

Clinical Scoring of Mice

The mice were observed during the study and scored 0-5 based on their behaviour and clinical signs.

- Score 0: Healthy.
- Score 1: Minor clinical signs of infection and inflammation e.g. observations of minor signs of distress or changed activity.
- Score 2: Clear signs of infection like, social withdrawal, lack of curiosity, changed body position, piloerection, or changes in pattern of movement.
- Score 3: Severe signs of infection like stiff movements, lack of curiosity, changed body position, piloerection, pain, or changes in pattern of movement.
- Score 4: Severe pain and the mouse was sacrificed immediately to minimize the suffering of the animal.
- Score 5: The mouse was dead.

Sampling

Colony counts were determined from blood and peritoneal fluid at 0 and 5 hours. The mice were anaesthetized with CO₂+O₂and blood was collected from axillary cutdown in 1.5 ml EDTA coated eppendorf tubes. The mice were sacrificed immediately after blood sampling and a total of 2 ml sterile saline was injected i.p. and the abdomen gently massaged before it was opened and fluid sampled with a pipette. Each sample was then 10 fold diluted in saline and 20-microliter spots were applied on blue agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts were performed at the start of treatment and 5 hours after treatment. The CFU counts and the clinical score of the mice are shown in Table 3. The CFU numbers are log₁₀transformed before performing calculations.
The CFU/ml in the inoculum was determined to 6.29 log₁₀. At start of treatment the mean log₁₀CFU/ml in peritoneal fluid was 5.76 and in blood 5.13 and the CFU levels remained at a similar level in the vehicle group (5.72 and 4.65 log₁₀CFU/ml in the peritoneum and blood respectively) at 5 hours after treatment. Slightly lower CFU levels were observed in blood and peritoneal fluid after treatment with NZ17074 0.16-3.0 mg/kg. Treatment with 6 and 12 mg/kg NZ17074 resulted in CFU levels significantly lower (p<0.001) than after vehicle treatment both in peritoneal fluid and in blood (Table 3). Also the meropenem treatment, 40 mg/kg, resulted in significant reduction compared to the vehicle treated mice both in blood (p<0.05) and peritoneal fluid (p<0.01).
The dose-response curves (data not shown) were calculated in GraphPad Prism using Sigmoidal dose-response (variable slope). From these ED50 values were determined to 3.09±2.07 mg/kg in peritoneal fluid and 3.17±0.53 mg/kg in blood.
The maximum effect of NZ17074, E_max, was defined as the log CFU difference between no response and maximum response. No response was characterised as colony counts at the same level as determined for vehicle treated mice. The E_maxwas calculated as the difference between the “Top plateau” and “Bottom plateau” in GraphPad Prism using Sigmoidal dose-response to be 4.72 log₁₀CFU for the peritoneal fluid and 3.15 log₁₀CFU for the blood.
In addition the 1, 2 and 3 log killing, defined as the dose required to obtain 1, 2 or 3 log reduction in bacterial loads compared to start of treatment, were estimated using GraphPad Prism. The 1, 2 and 3 log killing of NZ17074 was 1.11 mg/kg, 2.95 mg/kg and 4.73 mg/kg respectively in peritoneal fluid and 0.25 mg/kg, 2.75 mg/kg and 3.78 mg/kg respectively in blood.
No or only mild clinical score was observed in all of the treatment groups (Table 3).

Discussion and Conclusion

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic peritonitis/sepsis model.
The ED50 values for NZ17074 were determined to 3.09±2.07 mg/kg in the peritoneal fluid and 3.17±0.53 mg/kg in the blood. The 1 log killing was estimated to be 1.11 mg/kg in the peritoneal fluid and 0.25 mg/kg in the blood. The 2 log killing was estimated to be 2.95 mg/kg in the peritoneal fluid and 2.76 mg/kg in the blood. The 3 log killing was estimated to be 4.73 mg/kg in the peritoneal fluid and 3.78 mg/kg in the blood.

TABLE 2

Efficacy values for NZ17074 against E. coli AID#172
calculated in Graph Pad Prism.

	NZ17074	Peritoneal fluid	Blood

TOP	0.325	CFU/ml	−0.985	CFU/ml
BOTTOM	−4.486	CFU/ml	−4.138	CFU/ml
Emax	4.811	CFU/ml	3.153	CFU/ml
ED50	3.086	mg/kg	3.168	mg/kg

R²

0.7524

0.6889

1 log killing	1.11	mg/kg	0.25	mg/kg
2 log killing	2.95	mg/kg	2.76	mg/kg
3 log killing	4.73	mg/kg	3.78	mg/kg

TABLE 3

Colony counts of E. coli AID#172 in blood and peritoneal fluid from neutropenic mice
treated with a single dose of NZ17074, meropenem or vehicle.

log₁₀CFU

Treatment

Mouse

Clinical score

mean

T = 0 hour	no.	Time	T = 0 hour	T = 5 hours	PF	PF	Blood	Blood

Vehicle	1	T = 5	1	1	5.74	5.72	5.05	4.65
	2	T = 5	1	0	5.54		4.78
	3	T = 5	1	1	5.88		4.11
NZ17074	4	T = 5	1	1	5.16	5.31	4.27	4.54
0.16 mg/kg	5	T = 5	1	1	4.78		4.19
	6	T = 5	1	0	5.98		5.16
NZ17074	7	T = 5	1	0	2.76	4.26	1.40	2.88
0.30 mg/kg	8	T = 5	1	1	5.74		4.63
	9	T = 5	1	0	4.27		2.60
NZ17074	10	T = 5	1	0	5.74	5.16	5.07	4.46
0.75 mg/kg	11	T = 5	1	1	4.95		4.30
	12	T = 5	1	1	4.78		4.00
NZ17074	13	T = 5	1	0	3.33	4.41	3.51	3.99
1.5 mg/kg	14	T = 5	1	1	4.72		3.92
	15	T = 5	1	0	5.18		4.54
NZ17074	16	T = 5	1	1	4.74	3.91	3.86	2.81
3.0 mg/kg	17	T = 5	1	1	4.74		3.57
	18	T = 5	1	0	2.24		1.00
NZ17074	19	T = 5	1	1	2.18	2.12***	1.00	1.00***
6.0 mg/kg	20	T = 5	1	1	2.18		1.00
	21	T = 5	1	1	2.00		1.00
NZ17074	22	T = 5	1	1	1.00	1.36***	1.00	1.00***
12 mg/kg	23	T = 5	1	0	1.69		1.00
	24	T = 5	1	0	1.40		1.00
Meropenem	25	T = 5	1	1	3.92	2.64**	2.48	2.38*
40 mg/kg	26	T = 5	1	1	1.70		1.70
	27	T = 5	1	0	2.30		2.95
None	28	T = 0	1		5.84	5.76	5.08	5.13
	29	T = 0	1		5.78		4.98
	30	T = 0	1		5.65		5.34

Stars indicate significantly different from vehicle group (Anova; multiple comparison).
*corresponds to p < 0.05;
**corresponds to p < 0.01;
***corresponds to p < 0.001.
Detection limit 1.4 log₁₀CFU/ml.
Samples with no detectable bacteria is presented as 1.0 log₁₀CFU/ml.

Example 3

Peritonitis/Sepsis Model

Effect Over Time of 7.5 mg/kg NZ17074 Against Escherichia coli AID#172 in Neutropenic NMRI Mice

Introduction

The purpose of this study was to investigate the in vivo efficacy of NZ17074 following intravenous (i.v.) administration of a single dose of 7.5 mg/kg. The effect was tested against Escherichia coli AID#172 in the peritonitis model in neutropenic NMRI mice to avoid the use of mucin as normally applied in the murine peritonitis model. The mice were rendered neutropenic by cyclophosphamide injections. Treatment with 40 mg/kg meropenem was included as a positive control group and treatment with vehicle was included as a negative control group. The colony counts in peritoneal fluid and blood were determined at 2 and 5 hours after treatment.

Materials and Methods

- 30 outbred, NMRI female mice, 28-32 grams (Harlan Scandinavia)
- Escherichia coli AID#172 from Statens Serum Institute, Copenhagen, Denmark. Clinical isolate from a human wound from 2003. Multiresistant (Ampicillin, Ceftazidime, Aztreonam, Gentamicin, Ciprofloxacin)
- NZ17074 in Ringer Acetate pH 6, 1.2 ml 0.75 mg/ml. Analyses of the dose formulation performed after the study showed a concentration of approx. 0.78 mg/ml.
- Vehicle (Ringer Acetate pH 6) 3 ml.
- MERONEM®(AstraZeneca, 500 mg meropenem infusion substance). Lot no. 09466C Date of expiration: 08-2013
- APODAN®(A-Pharma, 1 g cyclophosphamide) Batch nr. 928491
- Date of expiration: May 2012
- Water, sterile
- 0.9% saline, sterile
- 5% Horse Blood Agar plates
- Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m.-6 a.m. The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 3 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage.

NZ17074 Solution

A solution of 0.75 mg/ml of each test compound was stored at +4° C. until one hour before injection, thereafter at room temperature.

Preparation of Meropenem Solution

A total of 500 mg meropenem (one ampoule) was dissolved in 10 ml water˜50 mg/ml the day of use. This stock solution was further diluted to 4 mg/ml (0.4 ml 50 mg/ml+4.6 ml saline).

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule APODAN®(A-Pharma)) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml of 20 mg/ml+13.5 ml saline) for use on day −4 or to 5.5 mg/ml (8.25 ml of 20 mg/ml+21.75 ml saline) for use on day −1.
Treatment of Mice with Cyclophosphamide
The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁶CFU/ml.
One hour before start of treatment (time −1 hour) mice were inoculated intraperitoneally with 0.5 ml of the E. coli suspension in the lateral lower quadrant of the abdomen.
2.5 hours after treatment, when clinical signs of infection were significant, mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml, corresponding to 30 mg/kg) as a pain relief.

Scoring of Mice

The mice were clinically scored for signs of infection at the time of each sampling.

- Score 0: Healthy.
- Score 1: Minor clinical signs of infection and inflammation e.g. observations of minor signs of distress or changed activity.
- Score 2: Clear signs of infection like, social withdrawal, lack of curiosity, changed body position, piloerection, or changes in pattern of movement.
- Score 3: Severe signs of infection like stiff movements, lack of curiosity, forced ventilation, changed body position, piloerection, pain, or changes in pattern o movement.
- Score 4: Severe pain and the mouse was sacrificed immediately to minimize the suffering of the animal.
- Score 5: The mouse was dead.

Treatment of Mice

The mice were treated i.v. in the lateral tail vein over approximately 30 seconds with a single dose of NZ17074, meropenem or vehicle at time 0 hour (see Table 1). The dosing was based on a mean weight of 30 g. Mice that weighed 28-32 g received 0.30 ml solution. Mice that weighed 27-28 g received 0.25 ml solution and mice that weighed 32.1-36 g received 0.35 ml solution. Mouse 17 accidently received 0.35 ml although it weighed 29.5 g. This does not seem to have influenced the results as the CFU levels in this mouse was very similar to the other two mice in the group.

TABLE 4

Treatment and sampling schedule in the murine peritonitis model.

		Sampling
Inoculation	Treatment	T = 0	Sampling	Sampling
T = −1 hour	T = 0 hour	hour	T = 2 hours	T = 5 hours

0.5 ml of	NZ17074			4, 5, 6
E. coli	Meropenem			7, 8, 9
AID#172	Vehicle			10, 11, 12
10⁶CFU/ml	(Ringer
	Acetate)
	NZ17074		16, 17, 18
	Meropenem		19, 20, 21
	Vehicle		22, 23, 24
	(Ringer
	Acetate)
	None		25, 26, 27
	None	28, 29, 30

T indicates the time in relation to treatment.
Numbers in the sampling columns are mouse identification numbers.

Sampling

Colony counts were determined from blood and peritoneal fluid at 0, 2 and 5 hours after treatment according to Table 1.
The mice were anesthetized with O₂+CO₂and blood was collected by axilliary cut down. The mice were sacrificed by cervical dislocation and a total of 2 ml sterile saline was injected i.p. and the abdomen gently massaged before it was opened and fluid sampled with a pipette. Each sample was 10 fold diluted in saline and 20-microliter spots were applied on blood agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts and the clinical scores of the mice are shown in Table 2. The CFU numbers are log₁₀transformed before performing calculations to obtain a normal distribution.
The CFU/ml in the inoculum was determined to 6.30 log₁₀. At start of treatment the mean log₁₀CFU/ml in the peritoneal fluid was 3.57 and in the blood 3.54 and the CFU level increased to 5.43 and 4.58 in the peritoneal fluid and the blood respectively after 2 hours in vehicle treated animals and to 5.72 and 4.74 in the peritoneal fluid and the blood respectively after 5 hours in vehicle treated mice, which was as expected.
At 2 hours after treatment with NZ17074 significantly lower CFU levels were observed both in the blood and the peritoneal fluid compared to the vehicle treatment (p<0.001).
A further reduction of the CFU levels was observed at 5 hours after treatment with NZ17074 both in the blood and in the peritoneal fluid (p<0.001 compared to vehicle control). The CFU levels were more the 3 log₁₀CFU/ml lower than after vehicle treatment.
Also meropenem treatment resulted in significantly (p<0.01) reduced CFU levels compared to vehicle treatment in the peritoneal fluid at both 2 and 5 hours after treatment but in the blood only at 5 hours after treatment. The lack of significance in the blood at 2 hours after treatment may reflect the large variability in the vehicle group rather than poor effect of meropenem.
The difference in CFU levels after NZ17074 or meropenem treatment compared to vehicle treatment was:


NZ17074,	2 hours: peritoneum −1.63 log cfu/ml	blood −2.50 log
7.5 mg/kg		cfu/ml
	5 hours: peritoneum −3.76 log cfu/ml	blood −3.74 log
		cfu/ml
Meropenem	2 hours: peritoneum −1.51 log cfu/ml	blood −0.82 log
40 mg/kg		cfu/ml
	5 hours: peritoneum −1.51 log cfu/ml	blood −1.64 log
		cfu/ml

All mice had only mild or no symptoms of infection (Table 2).

Discussion and Conclusion

The purpose of this study was to investigate the efficacy of NZ17074 following intravenous (i.v.) administration of a single dose of 7.5 mg/kg in the neutropenic peritonitis model in NMRI mice. A significant (p<0.001) reduction of more the 3 log₁₀CFU/ml compared to vehicle treatment was observed for NZ17074 in blood and peritoneal fluid at 5 hours after treatment. Also at 2 hours after treatment with NZ17074 a significant reduction (p<0.001) both in the blood and peritoneal fluid was observed. Meropenem showed a significant reduction compared to the vehicle group (p<0.01) both in the blood and in the peritoneal fluid at 5 hours but at 2 hours after treatment only in the peritoneal fluid.

TABLE 5

Colony counts of E. coli AID#172 in mice treated with a single dose of NZ17074,
vehicle or meropenem

Score

log₁₀CFU

	id	Time of	T = 0	T = 2	T = 5		mean		Mean
Treatment	no.	sampling	hour	hours	hours	PF	PF	Blood	Blood

NZ17074	4	T = 5	0		0	2.18	1.96***	1.00	1.00***
7.5 mg/kg	5	T = 5	0		1	2.30		1.00
	6	T = 5	0		1	1.40		1.00
Meropenem	7	T = 5	0		0	4.38	4.21**	3.20	3.10**
40 mg/kg	8	T = 5	0		0	4.00		2.85
	9	T = 5	0		0	4.26		3.26
Vehicle	10	T = 5	0		0	5.39	5.72	4.63	4.74
	11	T = 5	0		0	5.99		5.24
	12	T = 5	0		0	5.78		4.36
NZ17074	16	T = 2	0	1		4.24	3.79**	2.04	2.08**
7.5 mg/kg	17	T = 2	0	1		3.60		2.20
	18	T = 2	0	1		3.54		2.00
Meropenem	19	T = 2	0	1		4.12	3.92**	3.60	3.76
40 mg/kg	20	T = 2	0	1		3.40		3.57
	21	T = 2	0	1		4.24		4.11
Vehicle	22	T = 2	0	0		4.89	5.43	3.21	4.58
	23	T = 2	0	1		5.65		5.39
	24	T = 2	0	0		5.74		5.15
None	25	T = 2	0	0		4.45	5.08	4.39	4.33
	26	T = 2	0	0		5.42		4.57
	27	T = 2	0	0		5.38		4.02
None	28	T = 0	0			1.88	3.57	1.00	3.54
	29	T = 0	0			3.71		4.27
	30	T = 0	0			5.13		5.35

PF: peritoneal fluid. Used inoculum: 1.97 × 10⁶CFU/ml.
^# Mouse received 0.35 ml instead of 0.30 ml of test compound
*p < 0.05,
**p < 0.01,
***p < 0.001 compared to vehicle group.

Example 4

Neutropenic Thigh Infection Model

Efficacy of NZ17074 Against Escherichia coli AID#172 and Estimation of ED50

Introduction

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic thigh model. Treatment with 40 mg/kg meropenem was included as a positive control group. The colony counts in thighs were determined at 5 hours after treatment.
The thigh infection model is a well-recognized model for studies of antimicrobial effect and tissue penetration as described by S. Gudmundsson & H. Erlensdóttir: Handbook of Animal Models of Infection (1999), ed. by O. Zak & M. A. Sande, Academic Press, San Diego, US and in several publications. Reviewed by D. Andes & C. Craig: Animal model pharmacokinetics and pharmacodynamics: a critical review. International Journal of Antimicrobial Agents 19(4): 261-268.

Materials and Methods

- 40 outbred, NMRI female mice, 25-30 grams (Harlan Scandinavia)
- E. coli AID#172 from Statens Serum Institute, Copenhagen, Denmark: Clinical isolate from a human wound from 2003. Multiresistant (Ampicillin, Ceftazidime, Aztreonam, Gentamicin, Ciprofloxacin)
- NZ17074 in Ringer Acetate, pH 6: 1.2 mg/ml, 6.0 ml. The solution was stored at 4° C. until use. Analyses of the dose formulations used were performed after completion of the in-life phase of the study and gave the following results:


	Intended concentration	Measured concentration

- Vehicle (Ringer Acetate pH 6). The solution was stored at 4° C. until use
- MERONEM®(AstraZeneca, 500 mg infusion substance, meropenem). Lot no. 09466C Date of expire: August 2013
- Water, sterile
- 0.9% saline, sterile
- SENDOXAN®(Cyclophosphamide, Baxter, 1 g) Batch nr. 0A671C Date of expire: January 2013
- 5% Horse Blood Agar plates
- Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m-6 a.m.
The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 4 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage. Mice were weighed the day before dosing.

Preparation of NZ17074 Solutions

Preparation Meropenem Solution

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule SENDOXAN® (Cyclosphosphamide, Baxter, 1 g) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml 20 mg/ml+13.5 ml saline) for use on day −4 or to 5 mg/kg (8.25 ml 20 mg/ml+21.75 ml saline)) for use on day −1.
Treatment of Mice with Cyclophosphamide
The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁷CFU/ml. One hour before start of treatment (time −1 hour) mice were inoculated intramuscularly with 0.05 ml of the E. coli suspension in the left hind leg. Approximately ½ hour before inoculation mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml corresponding to 30 mg/kg) as a pain relief.

Treatment of Mice

TABLE 6

Treatment and sampling schedule in the murine thigh model.

Inoculation

Intravenous treatment

Sampling and mouse no.

i.m. at - 1 hr	at 0 hr	0 hr	5 hours

0.05 ml of	Vehicle, Ringer acetate		1-2-3-4
E. coli	NZ17074 0.16 mg/kg		5-6-7-8
AID#172	NZ17074 0.30 mg/kg		9-10-11-12
2 × 10⁷CFU/ml	NZ17074 0.75 mg/kg		13-14-15-16
	NZ17074 1.5 mg/kg		17-18-19-20
	NZ17074 3.0 mg/kg		21-22-23-24
	NZ17074 6.0 mg/kg		25-26-27-28
	NZ17074 12 mg/kg		29-30-31-32
	meropenem 40 mg/kg		33-34-35-36
	No treatment	37-38-39-40

T indicates the time in relation to treatment.
Numbers in the sampling columns are mouse identification numbers.

Clinical Scoring of Mice

Sampling

Colony counts were determined from thighs at 0 and 5 hours. The mice were anaesthetized with CO₂+O₂and sacrificed. Immediately after, skin was removed and the left hind leg was collected and frozen at −70° C. After thawing, the thighs were homogenized using a Dispomix® Drive. Each sample was then 10 fold diluted in saline and 20-microliter spots were applied on blue agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts were performed at the start of treatment and 5 hours after treatment. The CFU counts are shown in Table 3. The CFU numbers are log₁₀transformed before performing calculations.
The CFU/ml in the inoculum was determined to 7.35 log₁₀corresponding to 6.05 log₁₀CFU/mouse. The high variability observed may be caused by suboptimal inoculation of some mice and resulting in too low CFU values. The lowest value in each group was therefore excluded from graphs and calculations (se Table 3). At start of treatment the mean log₁₀CFU/ml was 4.93 and increased to 6.49 log₁₀CFU/ml in the vehicle group at 5 hrs after treatment. Slightly lower CFU levels were observed after treatment with NZ17074 0.16-3.0 mg/kg. Significantly lower CFU levels were observed after treatment with 6 mg/kg (p<0.05) and 12 mg/kg (p<0.01) NZ17074 compared to vehicle treatment (Table 3). Meropenem treatment, 40 mg/kg, resulted in slight but not significant reduction compared to the vehicle treated mice.
The dose-response curves (not shown) were calculated in GraphPad Prism using Sigmoidal dose-response (variable slope). From this the ED50 value was determined to 5.9 mg/kg. However, a bottom plateau was not obtained and this value may therefore be underestimated.
The maximum effect of NZ17074, E_max, was defined as the log CFU difference between no response and maximum response. No response was characterised as colony counts at the same level as determined for vehicle treated mice. The E_maxwas calculated as the difference between the “Top plateau” and “Bottom plateau” in GraphPad Prism using Sigmoidal dose-response to be 2.4 Δ log₁₀CFU/ml. In addition the 1 log killing, defined as the dose required to obtain 1 log reduction in bacterial loads compared to start of treatment, was estimated using GraphPad Prism to 6.1 mg/kg. A 2 and 3 log killing was not obtained.
No clinical signs of infection were observed at any time point in any of the mice.

TABLE 7

Efficacy values for NZ17074 against E. coli AID#172
calculated in Graph Pad Prism

	TOP	1.1 Dlog₁₀CFU/ml
	BOTTOM	−1.3 Dlog₁₀CFU/ml
	Emax	2.4 Dlog₁₀CFU/ml
	ED50	5.9 mg/kg
	R²	0.46
	1 log killing	6.1 mg/kg

TABLE 8

Colony counts of E. coli AID#172 in thighs from neutropenic
mice treated with a single dose of NZ17074, meropenem or vehicle.

			log₁₀
Treatment	mouse	Time of	CFU
T = 0 hour	no.	sampling	thigh	mean

Vehicle	1	T = 5	5.16¤	6.49
	2	T = 5	6.47
	3	T = 5	6.13
	4	T = 5	6.86
NZ17074	5	T = 5	3.18¤	5.16
0.16 mg/kg	6	T = 5	6.03
	7	T = 5	3.30
	8	T = 5	6.15
NZ17074	9	T = 5	2.00¤	5.09
0.30 mg/kg	10	T = 5	5.40
	11	T = 5	3.10
	12	T = 5	6.78
NZ17074	13	T = 5	2.9¤	6.33
0.75 mg/kg	14	T = 5	5.72
	15	T = 5	7.27
	16	T = 5	6.00
NZ17074	17	T = 5	2.56¤	5.62
1.5 mg/kg	18	T = 5	6.23
	19	T = 5	4.93
	20	T = 5	5.70
NZ17074	21	T = 5	5.30	6.07
3.0 mg/kg	22	T = 5	6.03
	23	T = 5	4.85¤
	24	T = 5	6.89
NZ17074	25	T = 5	2.75	4.10*
6.0 mg/kg	26	T = 5	4.54
	27	T = 5	1.48¤
	28	T = 5	5.01
NZ17074	29	T = 5	2.48¤	3.32**
12 mg/kg	30	T = 5	3.27
	31	T = 5	3.19
	32	T = 5	3.51
Meropenem	33	T = 5	3.08¤	4.25
40 mg/kg	34	T = 5	3.81¤
	35	T = 5	4.99
	36	T = 5	3.94
None	37	T = 0	4.98	4.93
	38	T = 0	3.81¤
	39	T = 0	4.79
	40	T = 0	5.01

¤This value was excluded from calculations as it was considered an outlier.
Stars indicate significantly different from vehicle group (Annova; multiple comparison).
*corresponds to p < 0.05;
**corresponds to p < 0.01.
Detection limit 1.4 log₁₀CFU/ml.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

What is claimed is:

1. An isolated variant of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising a substitution at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the amino acid sequence of SEQ ID NO: 2; wherein the variant has antimicrobial activity, and the number of substitutions is 1-11.

2. The variant of claim 1, wherein the number of substitutions are 1-10, 1-9 substitutions, 1-8 substitutions, 1-7 substitutions, 1-6 substitutions, 1-5 substitutions, 1-4 substitutions, 1-3 substitutions, or 1-2 substitutions.

3. The variant of any claim 1, which further comprises one or more substitutions selected from the group consisting of

G1A, G1D, G1F, G1H, G1I, G1K, G1M, G1Q, G1R, G1S, G1T, G1V, G1W, G1Y,

F2A, F2G, F2H, F2I, F2L, F2M, F2P, F2S, F2V, F2W, F2Y,

C3L,

W4A, W4E, W4F, W4G, W4I, W4L, W4M, W4N, W4Q, W4S, W4T, W4V, W4Y,

Y5E, Y5F, Y5G, Y5H, Y5K, Y5N, Y5R, Y5S, Y5W,

V6A, V6C, V6E, V6F, V6G, V6H, V6I, V6L, V6M, V6N, V6Q, V6R, V6S, V6T, V6W, V6Y, C7V,

V8A, V8F, V8G, V8H, V8I, V8L, V8N, V8S, V8T, V8W, V8Y,

Y9A, Y9D, Y9F, Y9G, Y9H, Y9I, Y9K, Y9M, Y9Q, Y9R, Y9S, Y9T, Y9V, Y9W,

R10K, R10P, R10S, R10T,

N11A, N11G, N11H, N11Q, N11R, N11S, N11Y,

G12A, G12D, G12E, G12F, G12H, G12K, G12N, G12R, G12S, G12Y,

R19D, R19H, R19K, R19M, R19T, R19Y,

N21A, N21C, N21F, N21G, N21H, N21I, N21K, N21L, N21M, N21P, N21Q, N21R, N21S, N21T, N21W, and N21Y.

4. The variant of claim 1, which comprises a substitution at two of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21.

5. The variant of claim 1, which comprises a substitution at three of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21.

6. The variant of claim 1, which comprises a substitution at four of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21.

7. The variant of claim 1, which comprises a substitution at one or more (several) of positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21.

8. An isolated polynucleotide encoding the variant of claim 1.

9. A nucleic acid construct comprising the polynucleotide of claim 8.

10. An expression vector comprising the polynucleotide of claim 8.

11. A host cell comprising the polynucleotide of claim 8.

12. A method of producing a variant of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising:

a) cultivating the host cell of claim 11 under conditions suitable for the expression of the variant; and

b) recovering the variant.

13. A method of treating a microbial infection in a subject in need thereof comprising administering an amount of the variant of claim 1 effective to treat said microbial infection.

16. A composition comprising the variant of claim 1 and an antibiotic.