Antimicrobial cationic peptides

A novel class of cationic peptides having antimicrobial activity is provided. Examples of such peptides include NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH (SEQ ID NO:1) and NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (SEQ ID NO:2).

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates generally to antimicrobial peptides and specifically 
to a new class of antimicrobial cationic peptides referred to as 
bactolysins. 
2. Description of Related Art 
In 1981, the self-promoted uptake hypothesis was first proposed to explain 
the mechanism of action of polycationic antibiotics in Pseudomonas 
aeruginosa. According to this hypothesis, polycations interact with sites 
on the outer membranes of Gram-negative bacteria at which divalent cations 
cross-bridge adjacent lipopolysaccharide molecules. Due to their higher 
affinity for these sites, polycations displace the divalent cations and, 
since the polycations are bulkier than the divalent cations, cause 
structural perturbations in the outer membrane. These perturbations result 
in increased outer membrane permeability to compounds such as the 
.beta.-lactam antibiotic nitrocefin, the eukaryotic non-specific defense 
protein lysozyme and to hydrophobic substances. By analogy, molecules 
accessing this pathway are proposed to promote their own uptake. 
It has been clearly demonstrated that the outer membranes of Gram-negative 
bacteria are semipermeable molecular "sieves" which restrict access of 
antibiotics and host defense molecules to their targets within the 
bacterial cell. Thus, cations and polycations which access the 
self-promoted uptake system are, by virtue of their ability to interact 
with and break down the outer membrane permeability barrier, capable of 
increasing the susceptibility of Gram-negative pathogenic bacteria to 
antibiotics and host defense molecules. Hancock and Wong demonstrated that 
a broad range of such compounds could overcome the permeability barrier 
and coined the name "permeabilizers" to describe them (Hancock and Wong, 
Antimicrob. Agents Chemother., 26:48, 1984). While self-promoted uptake 
and permeabilizers were first described for P. aeruginosa, they have now 
been described for a variety of Gram-negative bacteria. 
Over the past decade, non-specific defense molecules have been described in 
many animals, including insects and humans. One subset of these molecules 
have in common the following features: (a) they are small peptides, 
usually 15-35 amino acids in length, (b) they contain 4 or more positively 
charged amino acid residues, either lysines or arginines, and (c) they are 
found in high abundance in the organisms from which they derive. Several 
of these molecules have been isolated, amino acid sequenced and described 
in the patent literature (e.g., cecropins: WO8900199, WO 8805826, 
WO8604356, WO 8805826; defensins: EP 193351, EP 85250, EP 162161, U.S. 
Pat. No. 4,659,692, WO 8911291). However, only limited amounts of these 
peptides can be isolated from the host species. For example, Sawyer, et 
al., (Infect. Immun. 56:693, 1988) isolated 100-200 mg of rabbit 
neutrophil defensins 1 and 2 from 10.sup.9 primed peritoneal neutrophils 
or lipopolysaccharide-elicited alveolar macrophages (i.e., the numbers 
present in a whole animal). 
The gene for human defensin has been cloned and sequenced, but no 
successful expression has been demonstrated, as yet. Furthermore, 
production of these peptides using peptide synthesis technology produces 
peptides in limited amounts and is expensive when scaled up or when many 
variant peptides must be produced. Also, structural analysis is difficult 
without specific incorporation of .sup.15 N and .sup.13 C tagged amino 
acids which is prohibitively expensive using amino acid synthesis 
technology. 
There is a need to develop polypeptides having a broad range of potent 
antimicrobial activity against a plurality of microorganisms, including 
gram negative bacteria, gram positive bacteria, fungi, protozoa, viruses 
and the like. 
SUMMARY OF THE INVENTION 
The present invention provides a novel class of cationic peptides, referred 
to as bactolysins, which have antimicrobial activity. Two representative 
peptides are provided and include 
MBI 29, NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLIS-COOH (SEQ ID NO:1) and 
MBI 26, NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (SEQ ID NO:2), analogs, 
derivatives and conservative variations thereof. 
The invention also provides a method of inhibiting the growth of bacteria 
comprising contacting the bacteria with an inhibiting effective amount of 
a peptide having an amino acid sequence of MBI 29 (SEQ ID NO:1) or MBI 26 
(SEQ ID NO:2) alone, or in combination with an antibiotic. 
In another embodiment, the invention provides a method of inhibiting an 
endotoxemia or sepsis associated disorder in a subject having or at risk 
of having such a disorder, comprising administering to the subject a 
therapeutically effective amount of a peptide of the invention.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a novel classs of cationic peptides, called 
bactolysins, which have antimicrobial activity and have the ability to 
significantly reduce the level of lipopolysaccharide (LPS)-induced tumor 
necrosis factor (TNF). These peptides are useful for inhibiting microbial 
infection or growth, as well reducing the effects of endotoxemia and are 
often synergistic with conventional antibiotics and/or lysozyme. In 
addition, such peptides are useful as antifungal agents, antitumor agents, 
or antiviral agents. 
The term "antimicrobial" as used herein means that the peptides of the 
present invention inhibit, prevent, or destroy the growth or proliferation 
of microbes such as bacteria, fungi, viruses or the like. The term 
"antiviral" as used herein means that the peptides of the present 
invention inhibit, prevent or destrroy the growth or proliferation of 
viruses or of virally-infected cells. The term "anti-tumor" as used herein 
means that the peptides of the present invention may be used to inhibit 
the growth of or destroy tumors. The term "antifungal" as used herein 
means that the peptides of the present inventon may be used to inhibt the 
growth of or destroy fungi. 
In a first embodiment, the invention provides an isolated antimicrobial 
peptide having an amino acid sequence: 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLIS-COOH (SEQ ID NO:7), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH (SEQ ID NO:8), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLIS-COOH (SEQ ID NO:9), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH (SEQ ID NO:10), 
or 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH (SEQ ID NO:11) 
wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a 
hydrophilic amino acid residue. 
Examples of such peptides of the invention include but are not limited to: 
NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLIS-COOH (MBI 29) (SEQ ID NO:1), 
NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (MBI 26) (SEQ ID NO:2), 
NH.sub.2 -KWKSFIKNLTKGGSKILTTGLIS-COOH (MBI 201) (SEQ ID NO:3), 
NH.sub.2 -KWKKFIKNLTKGGSKILTTGLIS-COOH (MBI 202) (SEQ ID NO:4), 
NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH (MBI 203) (SEQ ID NO:5), and 
NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH (MBI 204) (SEQ ID NO:6), 
analogs, derivatives and conservative variations thereof, wherein the 
peptides have antimicrobial activity. The peptides of the invention 
include SEQ ID NO:1-6, as well as the broader groups of peptides having 
hydrophilic and hydrophobic substitutions, and conservative variations 
thereof. 
The sequence of SEQ ID NO:1 contains helix-forming hydrophilic amino acids 
at residues 4, 8, 10, 11, and 14. Amino acids 6 and 9 are hydrophobic 
residues (see FIG. 1). The helical nature of the first 10 amino acids is 
depicted in FIGS. 1 and 2. The cationic charge of the peptide of SEQ ID 
NO:1 was achieved by placing a lysine at positions 8 and 14, thereby 
increasing the total positive charge to 6. The carboxy terminus of SEQ ID 
NO:1 was converted to the methyl ester and the antibacterial activity of 
this derivative was the same as that of the unmodified peptide. The 
peptide of SEQ ID NO:1 also has antifungal activity. Amino acid residues 
3, 4, 7, 8, 10, 11, 14, and 15 are preferably hydrophilic residues, while 
amino acid residues 2, 5, 6, 9, 12, and 13 are preferably hydrophobic 
residues. 
SEQ ID NO:1 was modified to increase the alpha helical nature by changing 
amino acids in the C-terminal tail, thereby resulting in SEQ ID NO:2. 
Amino acids 11 (neutral hydrophilic) and 13 (hydrophobic) of SEQ ID NO:1 
were changed to another neutral hydrophilic and hydrophobic amino acid, 
respectively, in order to increase alpha helicity in this region. The 
C-terminal tail (residue 20-26) was modified to include a positive charge 
and increase alpha helicity. Therefore residues 20, 21, 23, 24 and 25 were 
changed (See FIGS. 1 and 2). SEQ ID NO:3 was obtained by changing amino 
acids 8, 11, 12, 13, 14, and 16 of SEQ ID NO:1. The first 15 amino acid 
residues contain lysine at every fourth residue of the alpha helix. Thus, 
the lysines at positions 8 and 14 in SEQ ID NO:1 were replaced by 
asparagine and serine, respectively, and the serine at position 11 was 
changed to lysine. Further changes were made at positions 12, 13 and 16 to 
conserve the helical nature. The C-terminal tail was unchanged. SEQ ID 
NO:4 is identical to SEQ ID NO:3, except for the amino acid at position 4, 
which incorporates an additional positive charge. SEQ ID NO:5 and SEQ ID 
NO:6 represent peptides having the same amino acid sequence as SEQ ID NO:3 
for the first 9 amino acids. The amino acid at position 10 is glutamic 
acid. Residues 10, 11, 12, and 13 in both SEQ ID NO:5 and SEQ ID NO:6 are 
different than SEQ ID NO:1, but remain either hydrophilic or hydrophobic 
as described above. 
Residue 14 in SEQ ID NO:5 is the same as in SEQ ID NO:1. All residues 
thereafter, 15-26, are completely changed to construct a hydrophobic 
helical tail, and bears no resemblance to the tail of SEQ ID NO:1. SEQ ID 
NO:6 contains the same residues as SEQ ID NO:1 at positions 14 and 15. 
Residues 16-26 have been changed to form a helical tail. 
The term "isolated" as used herein refers to a peptide substantially free 
of proteins, lipids, nucleic acids, for example, with which it is 
naturally associated. Those of skill in the art can make similar 
substitutions to achieve peptides with greater antibacterial activity and 
a broader host range. For example, the invention includes the bactolysin 
peptides depicted in SEQ ID NO:1-6, as well as analogues or derivatives 
thereof, as long as the bioactivity of the peptide remains. Minor 
modifications of the primary amino acid sequence of the peptides of the 
invention may result in peptides which have substantially equivalent 
activity as compared to the specific peptides described herein. Such 
modifications may be deliberate, as by site-directed mutagenesis, or may 
be spontaneous. All of the peptides produced by these modifications are 
included herein as long as the biological activity of the original peptide 
still exists. 
Further, deletion of one or more amino acids can also result in a 
modification of the structure of the resultant molecule without 
significantly altering its biological activity. This can lead to the 
development of a smaller active molecule which would also have utility. 
For example, amino or carboxy terminal amino acids which may not be 
required for biological activity of the particular peptide can be removed. 
Peptides of the invention include any analog, homolog, mutant, isomer or 
derivative of the peptides disclosed in the present invention, so long as 
the bioactivity as described herein is remains. All peptides were 
synthesized using L amino acids, however, all D forms of the peptides 
(e.g., see Table 1B, CEMA) can be synthetically produced. In addition, 
C-terminal derivatives can be produced, such as C-terminal methyl esters, 
in order to increase the antimicrobial activity of a peptide of the 
invention. 
The peptide of the invention include peptides which are conservative 
variations of those peptides specifically exemplified herein. The term 
"conservative variation" as used herein denotes the replacement of an 
amino acid residue by another, biologically similar residue. Examples of 
conservative variations include the substitution of one hydrophobic 
residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, 
phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for 
another, or the substitution of one polar residue for another, such as the 
substitution of arginine for lysine, glutamic for aspartic acids, or 
glutamine for asparagine, and the like. Neutral hydrophilic amino acids 
which can be substitued for one another include asparagine, glutamine, 
serine and threonine. The term "conservative variation" also includes the 
use of a substituted amino acid in place of an unsubstituted parent amino 
acid provided that antibodies raised to the substituted polypeptide also 
immunoreact with the unsubstituted polypeptide. Such conservative 
substitutions are within the definition of the classes of the peptides of 
the invention with respect to R.sub.1 and R.sub.2. 
The biological activity of the peptides can be determined by standard 
methods known to those of skill in the art, such as "minimal inhibitory 
concentration (MIC)" assay described in the present examples, whereby the 
lowest concentration at which no change in OD is observed for a given 
period of time is recorded as MIC. Alternatively, "fractional inhibitory 
concentration (FIC)" is also useful for determination of synergy between 
the peptides of the invention, or the peptides in combination with known 
antibiotics. FICs are performed by checkerboard titrations of peptides in 
one dimension of a microtiter plate, and of antibiotics in the other 
dimension, for example. The FIC is calculated by looking at the impact of 
one antibiotic on the MIC of the other and vice versa. An FIC of one 
indicates that the influence of the compounds is additive and an FIC of 
less than one indicates synergy. 
Peptides of the invention can be synthesized by such commonly used methods 
as t-BOC or FMOC protection of alpha-amino groups. Both methods involve 
stepwise syntheses whereby a single amino acid is added at each step 
starting from the C terminus of the peptide (See, Coligan, et al., Current 
Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of 
the invention can also be synthesized by the well known solid phase 
peptide synthesis methods described Merrifield, J. Am. Chem. Soc., 
85:2149, 1962), and Stewart and Young, Solid Phase Peptides Synthesis, 
(Freeman, San Francisco, 1969, pp.27-62), using a 
copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. 
On completion of chemical synthesis, the peptides can be deprotected and 
cleaved from the polymer by treatment with liquid HF-10% anisole for about 
1/4-1 hours at 0.degree. C. After evaporation of the reagents, the 
peptides are extracted from the polymer with 1% acetic acid solution which 
is then lyophilized to yield the crude material. This can normally be 
purified by such techniques as gel filtration on Sephadex G-15 using 5% 
acetic acid as a solvent. Lyophilization of appropriate fractions of the 
column will yield the homogeneous peptide or peptide derivatives, which 
can then be characterized by such standard techniques as amino acid 
analysis, thin layer chromatography, high performance liquid 
chromatography, ultraviolet absorption spectroscopy, molar rotation, 
solubility, and quantitated by the solid phase Edman degradation. 
The invention includes polynucleotides encoding: 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLIS-COOH (SEQ ID NO:7), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH (SEQ ID NO:8), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLIS-COOH (SEQ ID NO:9), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH (SEQ ID NO:10), 
and 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH (SEQ ID NO:11) 
wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a 
hydrophilic amino acid residue. More specifically, the invention also 
includes an isolated polynucleotide which encodes the MBI 29 peptide of 
SEQ ID NO:1, an isolated polynucleotide which encodes the MBI 26 peptide 
of SEQ ID NO:2, and isolated polynucleotides which encode SEQ ID NO:3-6. 
In addition, the invention includes polynucleotides which encode analogs, 
mutants and variants of the peptides of the invention. The term "isolated" 
as used herein refers to a polynucleotide substantially free of proteins, 
lipids, nucleic acids, for example, with which it is naturally associated. 
As used herein, "polynucleotide" refers to a polymer of 
deoxyribonucleotides or ribonucleotides, in the form of a separate 
fragment or as a component of a larger construct. DNA encoding a peptide 
of the invention can be assembled from cDNA fragments or from 
oligonucleotides which provide a synthetic gene which is capable of being 
expressed in a recombinant transcriptional unit. Polynucleotide sequences 
of the invention include DNA, RNA and cDNA sequences. A polynucleotide 
sequence can be deduced from the genetic code, however, the degeneracy of 
the code must be taken into account. Polynucleotides of the invention 
include sequences which are degenerate as a result of the genetic code. 
Such polynucleotides are useful for the recombinant production of large 
quantities of a peptide of interest, such as the peptide of SEQ ID NO:1, 
2, 3, 4, 5, and 6. 
In the present invention, the polynucleotides encoding the cationic 
peptides of the invention may be inserted into a recombinant "expression 
vector". The term "expression vector" refers to a plasmid, virus or other 
vehicle known in the art that has been manipulated by insertion or 
incorporation of cationic genetic sequences. Such expression vectors of 
the invention are preferably plasmids which contain a promoter sequence 
which facilitates the efficient transcription of the inserted genetic 
sequence in the host. The expression vector typically contains an origin 
of replication, a promoter, as well as specific genes which allow 
phenotypic selection of the transformed cells. For example, the expression 
of the peptides of the invention can be placed under control of E. coli 
chromosomal DNA comprising a lactose or lac operon which mediates lactose 
utilization by elaborating the enzyme beta-galactosidase. The lac control 
system can be induced by IPTG. A plasmid can be constructed to contain the 
lac Iq repressor gene, permitting repression of the lac promoter until 
IPTG is added. Other promoter systems known in the art include beta 
lactamase, lambda promoters, the protein A promoter, and the tryptophan 
promoter systems. While these are the most commonly used, other microbial 
promoters, both inducible and constitutive, can be utilized as well. The 
vector contains a replicon site and control sequences which are derived 
from species compatible with the host cell. In addition, the vector may 
carry specific gene(s) which are capable of providing phenotypic selection 
in transformed cells. For example, the beta-lactamase gene confers 
ampicillin resistance to those transformed cells containing the vector 
with the beta-lactamase gene. 
Transformation of a host cell with the polynucleotide may be carried out by 
conventional techniques well known to those skilled in the art. For 
example, where the host is prokaryotic, such as E. coli, competent cells 
which are capable of DNA uptake can be prepared from cells harvested after 
exponential growth and subsequently treated by the CaCl.sub.2 method using 
procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl could 
be used. 
In addition to conventional chemical methods of transformation, the plasmid 
vectors of the invention may be introduced into a host cell by physical 
means, such as by electroporation or microinjection. Electroporation 
allows transfer of the vector by high voltage electric impulse, which 
creates pores in the plasma membrane of the host and is performed 
according to methods well known in the art. Additionally, cloned DNA can 
be introduced into host cells by protoplast fusion, using methods well 
known in the art. 
DNA sequences encoding the cationic peptides can be expressed in vivo by 
DNA transfer into a suitable host cell. "Host cells" of the invention are 
those in which a vector can be propagated and its DNA expressed. The term 
also includes any progeny of the subject host cell. It is understood that 
not all-progeny are identical to the parental cell, since there may be 
mutations that occur during replication. However, such progeny are 
included when the terms above are used. Preferred host cells of the 
invention include E. coli S. aureus and P. aeruginosa, although other 
Gram-negative and Gram-positive organisms known in the art can be utilized 
as long as the expression vectors contain an origin of replication to 
permit expression in the host. 
The cationic peptide polynucleotide sequence used according to the method 
of the invention can be isolated from an organism or synthesized in the 
laboratory. Specific DNA sequences encoding the cationic peptide of 
interest can be obtained by: 1) isolation of a double-stranded DNA 
sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence 
to provide the necessary codons for the cationic peptide of interest; and 
3) in vitro synthesis of a double-stranded DNA sequence by reverse 
transcription of mRNA isolated from a donor cell. In the latter case, a 
double-stranded DNA complement of mRNA is eventually formed which is 
generally referred to as cDNA. 
The synthesis of DNA sequences is frequently the method of choice when the 
entire sequence of amino acid residues of-the desired peptide product is 
known. In the present invention, the synthesis of a DNA sequence has the 
advantage of allowing the incorporation of codons which are more likely to 
be recognized by a bacterial host, thereby permitting high level 
expression without difficulties in translation. In addition, virtually any 
peptide can be synthesized, including those encoding natural cationic 
peptides, variants of the same, or synthetic peptides. 
When the entire sequence of the desired peptide is not known, the direct 
synthesis of DNA sequences is not possible and the method of choice is the 
formation of cDNA sequences. Among the standard procedures for isolating 
cDNA sequences of interest is the formation of plasmid or phage containing 
cDNA libraries which are derived from reverse transcription of mRNA which 
is abundant in donor cells that have a high level of genetic expression. 
When used in combination with polymerase chain reaction technology, even 
rare expression products can be cloned. In those cases where significant 
portions of the amino acid sequence of the cationic peptide are known, the 
production of labeled single or double-stranded DNA or RNA probe sequences 
duplicating a sequence putatively present in the target cDNA may be 
employed in DNA/DNA hybridization procedures which are carried out on 
cloned copies of the cDNA which have been denatured into a single stranded 
form (Jay, et al., Nuc. Acid Res., 11:2325, 1983). 
The invention also provides a method of inhibiting the growth of bacteria 
comprising contacting the bacteria with an inhibiting effective amount of 
a peptide of the invention, including: 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLIS-COOH (SEQ ID NO:7), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH (SEQ ID NO:8), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLIS-COOH (SEQ ID NO:9), 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH (SEQ ID NO:10), 
or 
NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2 
R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH (SEQ ID NO:11) 
wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a 
hydrophilic amino acid residue. More specifically, the invention provides 
a method of inhibiting the growth of bacteria comprising contacting the 
bacteria with an inhibiting effective amount of a peptide of the 
invention, such as MBI 29, 
NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLIS-COOH (SEQ ID NO:1), 
NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (SEQ ID NO:2), 
NH.sub.2 -KWKSFIKNLTKGGSKILTTGLIS-COOH (SEQ ID NO:3), 
NH.sub.2 -KWKKFIKNLTKGGSKILTTGLIS-COOH (SEQ ID NO:4), 
NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH (SEQ ID NO:5), and 
NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH (SEQ ID NO:6), and 
analogs, derivatives, or conservative variations thereof. 
The term "contacting" refers to exposing the bacteria to the peptide so 
that the peptide can effectively inhibit, kill, or lyse bacteria, bind 
endotoxin (LPS), or permeabilize gram-negative bacterial outer membranes. 
Contacting may be in vitro, for example by adding the peptide to a 
bacterial culture to test for susceptibility of the bacteria to the 
peptide. Contacting may be in vivo, for example administering the peptide 
to a subject with a bacterial disorder, such as septic shock. "Inhibiting" 
or "inhibiting effective amount" refers to the amount of peptide which is 
required to cause a bacteriostatic or bactericidal effect. Examples of 
bacteria which may be inhibited include E. coli, P. aeruginosa, E. 
cloacae, S. typhimurium, and S. aureus. The method of inhibiting the 
growth of bacteria may further include the addition of antibiotics for 
combination or synergistic therapy. The appropriate antibiotic 
administered will typically depend on the susceptibility of the bacteria 
such as whether the bacteria is gram negative or gram positive, and will 
be easily discernable by one of skill in the art. The peptides and/or 
analogues or derivatives thereof may be administered to any host, 
including a human or non-human animal, in an amount effective to inhibit 
not only growth of a bacterium, but also a virus or fungus. These peptides 
are useful as antimicrobial agents, antiviral agents, and antifungal 
agents. 
The peptide of the invention can be administered parenterally by injection 
or by gradual infusion over time. The peptide can be administered 
intravenously, intraperitoneally, intramuscularly, subcutaneously, 
intracavity, or transdermally. Preferred methods for delivery of the 
peptide include orally, by encapsulation in microspheres or proteinoids, 
by aerosol delivery to the lungs, or transdermally by iontophoresis or 
transdermal electroporation. Other methods of administration will be known 
to those skilled in the art. 
Preparations for parenteral administration of a peptide of the invention 
include sterile aqueous or non-aqueous solutions, suspensions, and 
emulsions. Examples of non-aqueous solvents are propylene glycol, 
polyethylene glycol, vegetable oils such as olive oil, and injectable 
organic esters such as ethyl oleate. Aqueous carriers include water, 
alcoholic/aqueous solutions, emulsions or suspensions, including saline 
and buffered media. Parenteral vehicles include sodium chloride solution, 
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or 
fixed oils. Intravenous vehicles include fluid and nutrient replenishers, 
electrolyte replenishers (such as those based on Ringer's dextrose), and 
the like. Preservatives and other additives may also be present such as, 
for example, antimicrobials, antioxidants, chelating agents, and inert 
gases and the like. 
The invention provides a method of treating or ameliorating an endotoxemia 
or septic shock (sepsis) associated disorder, or one or more of the 
symptoms of sepsis comprising administering to a subject displaying 
symptoms of sepsis or at risk for developing sepsis, a therapeutically 
effective amount of a cationic peptide of the invention, for example, SEQ 
ID NO:1, or analogs, derivatives, or conservative variations thereof. The 
term "ameliorate" refers to a decrease or lessening of the symptoms of the 
disorder being treated. Such symptoms which may be ameliorated include 
those associated with a transient increase in the blood level of TNF, such 
as fever, hypotension, neutropenia, leukopenia, thrombocytopenia, 
disseminated intravascular coagulation, adult respiratory distress 
syndrome, shock and multiple organ failure. Patients who require such 
treatment include those at risk for or those suffering from toxemia, such 
as endotoxemia resulting from a gram-negative bacterial infection, venom 
poisoning, or hepatic failure, for example. In addition, patients having a 
gram-positive bacterial, viral or fungal infection may display symptoms of 
sepsis and may benefit from such a therapeutic method as described herein. 
Those patients who are more particularly able to benefit from the method 
of the invention are those suffering from infection by E. coli, 
Haemophilus influenza B, Neisseria meningitides, staphylococci, or 
pneumococci. Patients at risk for sepsis include those suffering from 
gunshot wounds, renal or hepatic failure, trauma, burns, immunocompromised 
(HIV), hematopoietic neoplasias, multiple myeloma, Castleman's disease or 
cardiac myxoma. 
The term "therapeutically effective amount" as used herein for treatment of 
endotoxemia refers to the amount of cationic peptide used is of sufficient 
quantity to decrease the subject's response to LPS and decrease the 
symptoms of sepsis. The term "therapeutically effective" therefore 
includes that the amount of cationic peptide sufficient to prevent, and 
preferably reduce by at least 50%, and more preferably sufficient to 
reduce by 90%, a clinically significant increase in the plasma level of 
TNF. The dosage ranges for the administration of cationic peptide are 
those large enough to produce the desired effect. Generally, the dosage 
will vary with the age, condition, sex, and extent of the infection with 
bacteria or other agent as described above, in the patient and can be 
determined by one skilled in the art. The dosage can be adjusted by the 
individual physician in the event of any contraindications. In any event, 
the effectiveness of treatment can be determined by monitoring the level 
of LPS and TNF in a patient. A decrease in serum LPS and TNF levels should 
correlate with recovery of the patient. 
In addition, patients at risk for or exhibiting the symptoms of sepsis can 
be treated by the method as described above, further comprising 
administering, substantially simultaneously with the therapeutic 
administration of cationic peptide, an inhibitor of TNF, an antibiotic, or 
both. For example, intervention in the role of TNF in sepsis, either 
directly or indirectly, such as by use of an anti-TNF antibody and/or a 
TNF antagonist, can prevent or ameliorate the symptoms of sepsis. 
Particularly preferred is the use of an anti-TNF antibody as an active 
ingredient, such as a monoclonal antibody with TNF specificity as 
described by Tracey, et al. (Nature, 330:662, 1987). 
A patient who exhibits the symptoms of sepsis may be treated with an 
antibiotic in addition to the treatment with cationic peptide. Typical 
antibiotics include an aminoglycoside, such as gentamicin or a beta-lactam 
such as penicillin, or cephalosporin. Therefore, a preferred therapeutic 
method of the invention includes administering a therapeutically effective 
amount of cationic peptide substantially simultaneously with 
administration of a bactericidal amount of an antibiotic. Preferably, 
administration of cationic peptide occurs within about 48 hours and 
preferably within about 2-8 hours, and most preferably, substantially 
concurrently with administration of the antibiotic. 
The term "bactericidal amount" as used herein refers to an amount 
sufficient to achieve a bacteria-killing blood concentration in the 
patient receiving the treatment. The bactericidal amount of antibiotic 
generally recognized as safe for administration to a human is well known 
in the art, and as is known in the art, varies with the specific 
antibiotic and the type of bacterial infection being treated. 
Because of the antibiotic, antimicrobial, and antiviral properties of the 
peptides, they may also be used as preservatives or sterillants of 
materials susceptible to microbial or viral contamination. The peptides of 
the invention can be utilized as broad spectrum antimicrobial agents 
directed toward various specific applications. Such applications include 
use of the peptides as preservatives in processed foods (organisms 
including Salmonella, Yersinia, Shigella), either alone or in combination 
with antibacterial food additives such as lysozymes; as a topical agent 
(Pseudomonas, Streptococcus) and to kill odor producing microbes 
(Micrococci). The relative effectiveness of the cationic peptides of the 
invention for the applications described can be readily determined by one 
of skill in the art by determining the sensitivity of any organism to one 
of the peptides. 
The following examples are intended to illustrate but not limit the 
invention. While they are typical of those that might be used, other 
procedures known to those skilled in the art may alternatively be used. 
EXAMPLE 1 
MIC Values for Cationic Peptides 
The minimum inhibitory concentrations of (MIC) of CEME, CEMA, the first 8 
amino acid residues of CEME (NH.sub.2 -KWKLFKKIGIGAVLKVLTTGLISCOOH; SEQ 
ID NO:1 with changes at residues 4, 6, 8-11, 14) and CEMA (NH.sub.2 
-KWKLFKKIGIGAVLKVLTTGLKLTK-COOH; SEQ NO:1 with changes at residues 4, 
6, 8-11, 14, and 25-28), (Piers, K. and Hancock, R., Molec. Microbiology, 
12(6), 1994) 20 carboxy terminal amino acids of melittin (MA), 8 amino 
terminal amino acids from cecropin, and the peptides shown in SEQ ID NO:1, 
MBI 29 and SEQ ID NO:2, MBI 26, were determined for a number of different 
bacteria (Table 1a and 1b). Briefly, cells were grown overnight at 
37.degree. C. in LB-S (Luria broth without any salt supplement) and 
diluted one in 10,000 in the same medium to give concentrations of about 
10.sup.4 to 10.sup.5 CFU/ml. The broth dilutions were set up in a 96 well 
microtiter plate by putting 200 .mu.l of LB-S containing the initial 
concentration of antibiotic or compound in column 1 and 100 .mu.l of the 
same medium in columns 2-12. The compounds were diluted by taking 100 
.mu.l of broth from column 1 and mixing it with column 2, resulting in a 
one in two dilution. This was continued to column 10. Finally, 10 .mu.l of 
bacteria were pipetted into columns 1-11, and the plates incubated 
overnight at 37.degree. C. The next day the plates were scored for growth 
in the wells, and the MIC determined. 
TABLE 1a 
______________________________________ 
MINIMUM INHIBITORY CONCENTRATION 
(MIC) VALUES FOR CATIONIC PEPTIDES 
STRAIN CEME CEMA MBI 29 
MBI 26 
CE-8 MA-20 
______________________________________ 
S. aureus 32 .gtoreq.64 
20 .gtoreq.64 
.gtoreq.64 
.gtoreq.64 
(K147) 
S. aureus 24 .gtoreq.64 
18 .gtoreq.64 
.gtoreq.64 
.gtoreq.64 
(SAP0017) 
S. epidermis 
16 .gtoreq.64 
10 .gtoreq.64 
.gtoreq.64 
.gtoreq.64 
E. coli 5 10 3 3 .gtoreq.64 
.gtoreq.64 
(UB1005) 
P. aeruginosa 
8 26 5 11 .gtoreq.64 
.gtoreq.64 
(H187) 
Candida albicans 
.gtoreq.64 
.gtoreq.64 
40 .gtoreq.64 
.gtoreq.64 
.gtoreq.64 
(CAN105) 
______________________________________ 
In a separate set of experiments, the following MIC values were obtained: 
TABLE 1b 
______________________________________ 
MINIMUM INHIBITORY CONCENTRATION 
(MIC) VALUES FOR CATIONIC PEPTIDES 
MBI MBI MBI MBI 
STRAIN CEMA CEMA.sup.me 
29 29.sup.me 
26D 26L 
______________________________________ 
S. aureus 
.gtoreq.64 
.gtoreq.64 
32 16 .gtoreq.64 
.gtoreq.64 
(K147) 
S. aureus 
.gtoreq.64 
.gtoreq.64 
32 16 .gtoreq.64 
.gtoreq.64 
(SAP0017) 
S. epidermis 
43 43 16 8 8-16 32 
P. aeruginosa 
32 53 16 8 16 32 
(H187) 
E. coli 13 32 4 4 4-8 4 
(UB1005) 
E. faecalis 
.gtoreq.64 
.gtoreq.64 
32 .gtoreq.64 
.gtoreq.64 
.gtoreq.64 
Candida .gtoreq.64 
.gtoreq.64 
32 32 .gtoreq.64 
.gtoreq.64 
albicans 
(CAN105) 
______________________________________ 
The results show that both MBI 29 (SEQ ID NO:1) and MBI 26 (SEQ ID NO:2) 
are effective antimicrobial agents for a variety of gram positive and gram 
negative bacteria. In addition, all peptides were effective against 
Candida albicans. Modification of the peptides, such as methyl ester 
modification, or L to D amino acids provided a broader class of 
antimicrobial agents. 
EXAMPLE 2 
Inhibition of LPS-Mediated TNF Induction in Macrophages by Cationic 
Peptides 
The effect of cationic peptides, CEME (KWKLFKKIGIGAVLKVLTTGLIS) (SEQ ID 
NO:14) and CEMA (KWKLFKKIGIGAVLKVLTTGLKLTK) (SEQ ID NO:15) and the 
peptides of the invention, MBI 29, NH.sub.2 
-KWKSFIKKLTTAVKKVLTTGLIS-COOH (SEQ ID NO:1) and MBI 26, NH.sub.2 
-KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (SEQ ID NO:2), on LPS-induced TNF in 
macrophages was examined. RAW 264.7 macrophage cells were grown by seeding 
10.sup.6 cells into a 162 cm.sup.2 cell culture flask and incubated at 
37.degree. C., 5% CO.sub.2 for 1 week. RAW cell media (Dulbecco's 
Modified Eagle Medium with Hepes buffer 450 ml; 2.4 mM L-glutamine 3 ml 
(400 mM); Pen/Strep 3 ml (10.sup.4 U/ml of Pen, 1 mg/ml strep); and 10% 
heat inactivated fetal bovine serum (FBS) 50 ml)! was then completely 
removed from flasks. 10 mls of cell dissociation solution (Sigma) was 
added to each flask and incubated at 37.degree. C. for 10 minutes. Cells 
were removed from flasks, diluted in RAW cell media and centrifuged for 6 
minutes. The cell pellet was resuspended in 5 ml of media/flask used. 100 
.mu.l cell suspension was removed and added to 400 .mu.l of trypan blue 
and cells were counted using a hemocytometer. The cell suspension was 
diluted to 1.times.10.sup.6 cells/ml and 1 ml of suspension was added per 
well of a 24 well plate. The 24 well plates were incubated at 37.degree. 
C., 5% CO.sub.2 overnight for use in the assay. 
After an overnight incubation, the media was aspirated from all the wells. 
100 .mu.l of Lipopolysaccharide (LPS) was added at 100 ng/100 .mu.l. CEME, 
CEMA or MBI 29 was added at the desired concentration/100 .mu.l to 
specified wells. RAW cell media was added to all the wells so they all had 
a final volume of 1 ml. The plates were then incubated for six hours at 
37.degree. C., 5% CO.sub.2. The supernatant was then removed from the 
wells and stored overnight at 4.degree. C. For those wells in which whole 
bacteria were added directly to the wells, the supernatant was centrifuged 
in 0.2 .mu.m filter eppendorf tubes for 5 minutes. 
The supernatants were then used in cell cytotoxic L929 assay. The samples 
were transferred to 96 well plates. 50 .mu.l of TNF media was added to all 
the wells in all the plates except to those wells in the first row. 10 
.mu.l of murine TNF standard (20 ng/ml) and 90 .mu.l of TNF media was 
added in duplicate to the plate and diluted 1:2 down the plate to the 
second to last row. Test samples (75 .mu.l), comprising of the 
supernatants from the RAW cell assays, were added to separate rows in 
duplicate and diluted 1:3 to the second to last rows. 
TNF-sensitive L929 mouse fibroblast cells were grown by seeding 10.sup.6 
cells into a 162 cm.sup.2 cell culture flask and left to grow for 1 week. 
L929 cells were removed from the flask with 10 mls of trypsin-EDTA/flask 
and incubated 3-5 minutes. Cell suspension was diluted and centrifuged for 
6 minutes. The pellet was resuspended in 5 mls of fresh L929 media/flask 
and counted (same as RAW cells). Cell suspension was diluted to 10.sup.6 
cells/ml. 100 .mu.l was used to inoculate each well of the 96 well plates 
with the supernatants. (L929 Growth Media was the same as RAW cell media 
except instead of FBS, 50 mls of 10% heat inactivated horse serum was 
utilized; TNF Assay Media was the same as RAW cell media except 4 .mu.g/ml 
Actinomycin D was added.) 
The plates were incubated at 37.degree. C. at 5% CO.sub.2 for 2 days. The 
media was then aspirated and replaced with 100 .mu.l of the dye MTT (0.5 
mg/ml) in modified Eagle Medium without phenol red. The plates were then 
incubated at 37.degree. C. at 5% CO.sub.2 for 3 hours. The dye was then 
removed and replaced with 100 .mu.l of absolute ethanol. The plates were 
left at room temperature for 10-15 minutes to dissolve the formazan dye 
crystals. 
The plates were read at 570 nm in a ELISA plate reader with 690 nm 
reference filter. One unit of TNF activity is defined as the amount 
required to kill 50% of the L929 cells. The TNF level in Units per ml 
therefore was the reciprocal of the dilution which led to a 50% killing of 
L929 cells. 
FIGS. 3 and 4 show levels of TNF (U/ml) after a 6 hour treatment with 
increasing amounts (0, 5, 10, 20, 30, 40 or 50 .mu.g) of either CEME (ME), 
CEMA (MA) or MBI 29 (SEQ ID NO:1) peptide and 100 ng of LPS. TNF levels 
were measured six hours after the addition of E. coli 0111:B4 or E. coli 
Bort LPS (FIGS. 3a, 3b, 3c and FIG. 4 as labeled). The data shows the 
results of 2 separate experiments and indicate that both peptides 
efficiently reduce the level of LPS-induced TNF in the culture with two 
distinct LPS samples at concentrations of peptides as low as 5 .mu.g/ml. 
EXAMPLE 3 
Cationic Peptide Reduction of LPS-Induced TNF 
In order to determine how rapidly the cationic peptides reduced LPS-induced 
TNF production, E. coli 0111:B4 was added at time 0 to RAW macrophages. 
MEBI 29 or Polymyxin B was added at time 0, 30 and 60 minutes. Levels of 
TNF were measured after 6 hours. The results are shown in FIGS. 5a and 
5b(PMB, Polymyxin B; 29, MBI 29 (SEQ ID NO:1)). The results show that MBI 
29 inhibited TNF induction by LPS in a similar manner to polymyxin B. 
Furthermore, MBI 29 was effective at reducing the ability of LPS to induce 
TNF in RAW cell lines even when added 60 minutes after the addition of 
LPS. MBI 29 demonstrated a distinct and reproducible advantage over 
polymyxin B when added 60 minutes after the addition of LPS. To confirm 
that MBI 29 was acting on LPS rather than directly upon macrophage cell 
lines, 20 .mu.g of MBI 29 was added to RAW cells and incubated for 60 
minutes prior to aspiration of the medium and washing the cells 3 times 
with HBSS (Hanks Buffered Salt Solution). Addition of 10 ng or 100 ng of 
LPS to the washed RAW cells resulted in a high level of TNF induction 
(14,000-20,000 Units of TNF per ml), suggesting that the MBI 29 had not 
permanently depressed the ability of RAW cells to induce TNF in response 
to LPS addition. In contrast, the aspirated medium containing MBI 29 could 
depress the ability of fresh RAW cells to induce TNF in response to 10 n 
or 100 ng of E. coli LPS by 98.5% and 75% respectively. Up to 50 .mu.g of 
MBI 29 caused no apparent decrease in RAW cell viability as judged by 
Trypan blue exclusion. 
EXAMPLE 4 
Protection From Lethal LPS Endotoxocity in a Mouse Endotoxic Shock Model 
The ability of MBI 29 and MBI 26 to protect against LPS-induced endotoxemia 
is assessed in vivo. Mice (8-10 weeks old) are injected intraperitoneally 
with 20 .mu.g D-galactosamine (Dgal) to sensitize them to LPS according to 
the method of Galanos (Galanos, et al., Proc. Natl. Acad. Sci., USA, 
76:5939-5943, 1979), followed by 0, 50, 100, or 200 .mu.g MBI 29 or MBI 26 
in 100 .mu.l. Immediately afterwards LPS (10 or 20 .mu.g) in 100 .mu.l is 
injected. The mice are observed at 24 and 48 hours after injections and 
survivors noted. 
To demonstrate that survival is associated with a reduction in TNF levels, 
10 .mu.g of LPS and 20 mg of Dgal are injected at time 0. Thirty minutes 
later, the mice are sacrificed and the blood is taken and centrifuged to 
separate the serum which was used in the cell cytotoxic L929 assay. The 
results suggest that the bactolysins have potential in therapy against 
endotoxin-associated disorders. 
Although the invention has been described with reference to the presently 
preferred embodiment, it should be understood that various modifications 
can be made without departing from the spirit of the invention. 
Accordingly, the invention is limited only by the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 21 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LysTrpLysSerPheIleLysLysLeuThrThrAlaValLysLysVal 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LysTrpLysSerPheIleLysLysLeuThrSerAlaAlaLysLysVal 
151015 
ValThrThrAlaLysProLeuIleSerSer 
2025 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LysTrpLysSerPheIleLysAsnLeuThrLysGlyGlySerLysIle 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LysTrpLysLysPheIleLysAsnLeuThrLysGlyGlySerLysIle 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LysTrpLysSerPheIleLysAsnLeuGluLysValLeuLysProGly 
151015 
GlyLeuLeuSerAsnIleValThrSerLeu 
2025 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LysTrpLysSerPheIleLysAsnLeuGluLysValLeuLysLysGly 
151015 
ProIleLeuAlaAsnLeuValSerIleVal 
2025 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4, 7, 8, 10, 11, 14, 15 
(D) OTHER INFORMATION: where Xaa at positions 4, 7, 8, 10, 11, 
14, or 15 is valine, leucine, isoleucine, phenylalanine, 
tyrosine, tryptophan, methionine, alanine, cysteine, 
glycine, proline or norleucine acid 
(B) LOCATION: 5, 6, 9, 12, or 13 
(D) OTHER INFORMATION: where Xaa at positions 5, 6, 9, 12, or 
13 is serine, threonine, asparagine, glutamine, aspartic, 
glutami, lysine, arginine or histidine acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LysTrpLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaVal 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4, 7, 8, 10, 11, 14, 15 
(D) OTHER INFORMATION: where Xaa at positions 4, 7, 8, 10, 11, 
14, or 15 is valine, leucine, isoleucine, phenylalanine, 
tyrosine, tryptophan, methionine, alanine, cysteine, 
glycine, proline or norleucine acid 
(B) LOCATION: 5, 6, 9, 12, or 13 
(D) OTHER INFORMATION: where Xaa at positions 5, 6, 9, 12, or 
13 is serine, threonine, asparagine, glutamine, aspartic, 
glutami, lysine, arginine or histidine acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LysTrpLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaVal 
151015 
ValThrThrAlaLysProLeuIleSerSer 
2025 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4, 7, 8, 10, 11, 14, 15 
(D) OTHER INFORMATION: where Xaa at positions 4, 7, 8, 10, 11, 
14, or 15 is valine, leucine, isoleucine, phenylalanine, 
tyrosine, tryptophan, methionine, alanine, cysteine, 
glycine, proline or norleucine acid 
(B) LOCATION: 5, 6, 9, 12, or 13 
(D) OTHER INFORMATION: where Xaa at positions 5, 6, 9, 12, or 
13 is serine, threonine, asparagine, glutamine, aspartic, 
glutami, lysine, arginine or histidine acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
LysTrpLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaIle 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4, 7, 8, 10, 11, 14, 15 
(D) OTHER INFORMATION: where Xaa at positions 4, 7, 8, 10, 11, 
14, or 15 is valine, leucine, isoleucine, phenylalanine, 
tyrosine, tryptophan, methionine, alanine, cysteine, 
glycine, proline or norleucine acid 
(B) LOCATION: 5, 6, 9, 12, or 13 
(D) OTHER INFORMATION: where Xaa at positions 5, 6, 9, 12, or 
13 is serine, threonine, asparagine, glutamine, aspartic, 
glutami, lysine, arginine or histidine acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
LysTrpLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaGly 
151015 
GlyLeuLeuSerAsnIleValThrSerLeu 
2025 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4, 7, 8, 10, 11, 14, 15 
(D) OTHER INFORMATION: where Xaa at positions 4, 7, 8, 10, 11, 
14, or 15 is valine, leucine, isoleucine, phenylalanine, 
tyrosine, tryptophan, methionine, alanine, cysteine, 
glycine, proline or norleucine acid 
(B) LOCATION: 5, 6, 9, 12, or 13 
(D) OTHER INFORMATION: where Xaa at positions 5, 6, 9, 12, or 
13 is serine, threonine, asparagine, glutamine, aspartic, 
glutami, lysine, arginine or histidine acid 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
LysTrpLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaGly 
151015 
ProIleLeuAlaAsnLeuValSerIleVal 
2025 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 4...4 
(D) OTHER INFORMATION: where Xaa at position 4 is S, K, or E 
(B) LOCATION: 8...8 
(D) OTHER INFORMATION: where Xaa at position 8 is K, N, or E 
(B) LOCATION: 10...10 
(D) OTHER INFORMATION: where Xaa at position 10 is T, E, or Q 
(B) LOCATION: 11...11 
(D) OTHER INFORMATION: where Xaa at position 11 is K, T, S, or 
(B) LOCATION: 12...12 
(D) OTHER INFORMATION: where Xaa at position 12 is V, A, or G 
(B) LOCATION: 13...13 
(D) OTHER INFORMATION: where Xaa at position 13 is V, L, A, or 
G 
(B) LOCATION: 14...14 
(D) OTHER INFORMATION: where Xaa at position 14 is K, S, or A 
(B) LOCATION: 15...15 
(D) OTHER INFORMATION: where Xaa at position 15 is K, or P 
(B) LOCATION: 16...16 
(D) OTHER INFORMATION: where Xaa at position 16 is V, G, or I 
(B) LOCATION: 17...17 
(D) OTHER INFORMATION: where Xaa at position 17 is L, V, G, or 
P 
(B) LOCATION: 18...18 
(D) OTHER INFORMATION: where Xaa at position 18 is T, L, or I 
(B) LOCATION: 19...19 
(D) OTHER INFORMATION: where Xaa at position 19 is T, or L 
(B) LOCATION: 20...20 
(D) OTHER INFORMATION: where Xaa at position 20 is G, A, or S 
(B) LOCATION: 21...21 
(D) OTHER INFORMATION: where Xaa at position 21 is L, K, or N 
(B) LOCATION: 22...22 
(D) OTHER INFORMATION: where Xaa at position 22 is P, I, or L 
(B) LOCATION: 23...23 
(D) OTHER INFORMATION: where Xaa at position 23 is A, V, or L 
(B) LOCATION: 24...24 
(D) OTHER INFORMATION: where Xaa at position 24 is L, I, T, or 
S 
(B) LOCATION: 25...25 
(D) OTHER INFORMATION: where Xaa at position 25 is I, or S 
(B) LOCATION: 26...26 
(D) OTHER INFORMATION: where Xaa at position 26 is S, L, or V 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
LysTrpLysXaaPheIleLysXaaLeuXaaXaaXaaXaaXaaXaaXaa 
151015 
XaaXaaXaaXaaXaaXaaXaaXaaXaaXaa 
2025 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(B) LOCATION: 8...8 
(D) OTHER INFORMATION: where Xaa at position 8 is K, or N 
(B) LOCATION: 11...11 
(D) OTHER INFORMATION: where Xaa at position 11 is K, or T 
(B) LOCATION: 12...12 
(D) OTHER INFORMATION: where Xaa at position 12 is V, A, or G 
(B) LOCATION: 13...13 
(D) OTHER INFORMATION: where Xaa at position 13 is V, or L 
(B) LOCATION: 14...14 
(D) OTHER INFORMATION: where Xaa at position 14 is K, or S 
(B) LOCATION: 15...15 
(D) OTHER INFORMATION: where Xaa at position 15 is K, or P 
(B) LOCATION: 16...16 
(D) OTHER INFORMATION: where Xaa at position 16 is V, or G 
(B) LOCATION: 17...17 
(D) OTHER INFORMATION: where Xaa at position 17 is L, V, or G 
(B) LOCATION: 18...18 
(D) OTHER INFORMATION: where Xaa at position 18 is T, or L 
(B) LOCATION: 19...19 
(D) OTHER INFORMATION: where Xaa at position 19 is T, or L 
(B) LOCATION: 20...20 
(D) OTHER INFORMATION: where Xaa at position 20 is G, A, or S 
(B) LOCATION: 21...21 
(D) OTHER INFORMATION: where Xaa at position 21 is L, K, or N 
(B) LOCATION: 22...22 
(D) OTHER INFORMATION: where Xaa at position 22 is P, I, or L 
(B) LOCATION: 23...23 
(D) OTHER INFORMATION: where Xaa at position 23 is A, V, or L 
(B) LOCATION: 24...24 
(D) OTHER INFORMATION: where Xaa at position 24 is L, or I 
(B) LOCATION: 25...25 
(D) OTHER INFORMATION: where Xaa at position 25 is I, or S 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
LysTrpLysSerPheIleLysXaaLeuThrXaaXaaXaaXaaXaaXaa 
151015 
XaaXaaXaaXaaXaaXaaXaaXaaXaaSer 
2025 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LysTrpLysLeuPheLysLysIleGlyIleGlyAlaValLeuLysVal 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
LysTrpLysLeuPheLysLysIleGlyIleGlyAlaValLeuLysVal 
151015 
LeuThrThrGlyLeuProAlaLeuLysLeuThrLys 
2025 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
LysTrpLysGluPheIleLysLysLeuThrThrAlaValLysLysVal 
151015 
LeuThrThrGlyLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
LysTrpLysLysPheIleLysGluLeuGlnLysValLeuAlaProGly 
151015 
GlyLeuLeuSerAsnIleValThrSerLeu 
2025 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
LysTrpLysSerPheIleLysLysLeuThrSerValLeuLysLysVal 
151015 
ValThrThrAlaLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
LysTrpLysSerPheIleLysAsnLeuThrLysValLeuLysLysVal 
151015 
ValThrThrAlaLeuProAlaLeuIleSer 
2025 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
LysTrpLysSerPheIleLysLysLeuThrSerValLeuLysLysVal 
151015 
ValThrThrAlaLysProLeuIleSerSer 
2025 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
LysTrpLysLysPheIleLysGluLeuGlnLysValLeuLysProGly 
151015 
GlyLeuLeuSerAsnIleValThrSerLeu 
2025 
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