# Patent

## Title
Antimicrobial peptides

## Abstract

Novel peptides having antimicrobial activity, and compositions containing the same.

## Background
The present invention relates to novel antimicrobial peptides, their derivatives, and compositions containing the same.
Several antimicrobial peptides or derivatives are currently in advanced clinical development, essentially in the form of topical drugs (Andrès, E., Dimarcq, J. -L. Cationic anti-microbial peptides: from innate immunity study to drug development. La revue de medicine interne (2004), 25, 629-635). However, very few of these molecules are suggested for intended use in aquaculture, e.g. penaeidins isolated from penaeid prawns (Penaeus vannamei). See, EP1000153.
This can be explained by the fact that several of these antimicrobial peptides (AMPs) are inhibited in the presence of salts, which is not the case of the peptides of the present invention, inasmuch as it was isolated from a deep-sea worm: Alvinella pompejana or the Pompeii worm.
This worm is also considered to be the one of the most heat-tolerant organisms known to science. In fact, it resists to temperatures from 10 to 80° C., which suggests that the molecules originated from this worm such as the peptide according to the present invention, are heat-stable and thus interesting in pharmacology.
The intensification of aquaculture productions has been made possible by the important use of antibiotics. Despite constraining legislations, the preventive intended use of antibiotics is widespread, in particular during the critical phases (e.g. early stage of life, metamorphosis, animal transfer), but also during animal growth. This massive use of antibiotics tends to promote the emergent evolution of antibiotic-resistant strains, which could decimate animal husbandry. Multi-antibiotic-resistant (MAR) strains appeared in the middle of the 80's. In addition to what hardly any new antibiotic families have been discovered during the last thirty years, offering too little new products against resistant strains.
An alternative solution would be to use AMPs. These molecules, which are naturally produced by animals and plants, have a broad-spectrum and fast antibiotic activity against Gram-Negative and Gram-Positive bacteria, fungi, and enveloped viruses. Moreover, these AMPs are not immunogenic and their action on the bacterial membrane would not favour the emergent evolution of antibiotic-resistant strains. The first AMP, developed in the 90's, is a derivative of the Magainin: the Pexiganan from Magainin Pharmaceuticals, Inc. In a phase III study, the Pexiganan was included in a cream, and showed the same effectiveness as an oral antibiotic therapy with Ofloxacin, for the treatment of superinfected cutaneous ulcerations amongst diabetics. Amongst the other AMPs in development, which may be of interest for the clinician in the near future, it should be mentioned Iseganan, developed by Intrabiotics Pharmaceuticals Inc., MBI peptides developed by Micrologix Biotech Inc., and derivative peptides of Histatin developed by Periondotix Inc. (Andres et al. previously cited). But also the above-mentioned penaeidins, which seem to have a greater molecular mass in comparison with the claimed peptide isolated from Alvinella pompejana. 
The peptides according to the invention could permit to diminish the use of antibiotics in aquaculture, by replacing a part of the latter. Due to the harmlessness of AMPs, their use would benefit brand image, but also health of the animals to be treated, customers and the environment, with no fear to favour the emergent evolution of antibiotic-resistant strains.
Thus, one of the aims of the invention is to provide new AMPs having broad-spectrum antibiotic activity.
The present invention relates to a novel peptide which comprises or consists of SEQ ID NO: 1, with the proviso that the aforesaid peptide does not consist of SEQ ID NOs: 2 to 5.
By the expression “peptide” is meant a contiguous amino acid chain. This contiguous amino acid chain can be called protein if it contains at least 50 amino acids. This contiguous amino acid chain can be either from natural origin or artificial (from chemical synthesis).
By the expression “contiguous amino acid chain” is meant amino acids touching or connected throughout in an unbroken sequence.
Because SEQ ID NO: 1 was issued from Alvinella pompejana, it has been called Alvinellacine. The Alvinellacine is the smallest active sequence against microorganisms. This peptide contains 4 cysteines that are involved in Cystine Bridges.
By the word “microorganisms” is meant organisms that are microscopic such as bacteria, fungi, and viruses.
The present invention also relates to a peptide which consists of SEQ ID NO: 6, or derives from SEQ ID NO: 1:

    
    
        by modification of its C-terminus, and/or N-terminus, and/or
        by substitution, and/or suppression, and/or addition of one or several amino acids in its peptide chain, and/or
        by modification of at least one —CO—NH— peptide linkage in its peptide chain, particularly by introduction of a retro or retro-inverso type linkage, and/or
        by substitution of at least one amino acid of its peptide chain, with a non-proteinogenic amino acid,

in particular, peptides or fragments of peptides having more than 80%, preferably more than 85%, preferably more than 90% and more preferably more than 95% of homology.

    
    


According to the invention, peptide deriving from SEQ ID NO:1 shares at least 80% identity with the amino acids 1 to 22 of SEQ ID NO:1 and have antimicrobial, antiviral and/or fungicide activity.
The SEQ ID NO: 6 corresponds to the prepropeptide of the Alvinellacine.
By the word “prepropeptide” is meant a precursor protein of the Alvinellacine, which includes an N-terminal signal peptide. This prepropeptide is subjected to a posttranslational modification in the Pompeii worm, and thus becomes the active peptide of SEQ ID NO: 1.
By the term “precursor protein” is meant an inactive protein, with no antimicrobial activity.
By the term “antimicrobial activity” is meant an action against bacteria, fungi, and viruses, leading to a decrease of their population and/or leading to the inhibition of microorganism growth.
By the term “signal peptide” is meant a fragment of the amino acid chain of SEQ ID NO: 6, which is a short sequence that directs the transport of said precursor protein in Alvinella pompejana. The signal peptide of SEQ ID NO: 6 goes from the 1st to the 19th amino acid residue.
By the term “posttranslational modification in the Pompeii worm” is meant the deletion process that occurs in Alvinella pompejana, corresponding to the cleavage of 186 contiguous amino acids from the N-terminal part of SEQ ID NO: 6, and leading to the obtaining of SEQ ID NO: 1.
By the term “modification of its C-terminus, and/or N-terminus” is meant the replacement of a group, e.g. —COOH or —NH2, located on the first or last amino acid of the peptide of SEQ ID NO: 1, whatever is the aim of the replacement, e.g. to coat the peptide to a support.
By the term “introduction of a retro type linkage” is meant the introduction of a retro amide linkage, which consists of a —NH—CO— peptide linkage in the SEQ ID NO: 1.
By the term “introduction of a retro-inverso type linkage” is meant the introduction of a retro inverso amide linkage, which consists of a —NH—CO— peptide linkage coupled with an inverse absolute configuration of the amino acid.
By the expression “non-proteinogenic amino acids”, it must be understood either amino acids not found in proteins (e.g. carnitine, L-canavanine, or L-DOPA), or not coded for in the standard genetic code (e.g. hydroxyproline and selenomethionine).
In an advantageous embodiment, the claimed peptide has at least 80% of identity with peptide of SEQ ID No: 1. Thus, the claimed peptide can have without limitation an amino acid sequence wherein one or more amino acids are substituted or suppressed everywhere inside said sequence or wherein one or more proteinogenic amino acids, i.e. natural amino acids, are added to said sequence, in particular a peptide that contains the four cysteines of SEQ ID NO: 1 which are involved in Cystine Bridges.
The present invention also relates to a peptide as defined above, characterized in that it is isolated and purified from Alvinella pompejana. 
By the expression “isolated and purified from Alvinella pompejana” is meant that the peptide is first extracted from Alvinella pompejana and then separated from the other organic debris, e.g. with the intended use of a commercial total protein extraction kit, followed by an affinity chromatography, or other well known technique commonly used by the man skilled in the art.
The present invention also relates to an isolated nucleic acid molecule encoding a peptide as defined above, in particular the nucleic acid molecule of SEQ ID NO: 7, with the proviso that it does not consist of SEQ ID NOs: 8 to 25.
The SEQ ID NO: 7 corresponds to a nucleic acid molecule encoding the above-mentioned prepropeptide of SEQ ID NO: 6.
The SEQ ID NOs: 8 to 25 correspond to disclosed nucleic acid molecules encoding the amino acid chains of SEQ ID NOs: 2 to 5.
By the expression “nucleic acid molecule” is meant a single or double DNA chain, or a single or double RNA chain, including DNA/RNA hybrid, which encode for the claimed peptide.
The present invention also relates to a recombinant nucleic acid construct comprising the above-mentioned nucleic acid molecule operably linked to an expression vector.
By the expression “operably linked” is meant that the aforesaid nucleic acid molecule is linked in a covalent way to an expression vector, permitting ribosome to translate the claimed nucleic acid molecule, or permitting RNA polymerase to produce a mRNA encoding the claimed peptide.
By the term “expression vector” is meant a molecule made of nucleic acids, in particular a plasmid, which comprises a promoter region and optionally an enhancer region. The expression vector can include other genes such as an antibiotic resistance gene, in order to select host cells that contain the nucleic acid molecule encoding the claimed peptide. Recombinant production techniques that use the aforementioned expression vector are well known by a man skilled in the art.
The present invention also relates to a host cell comprising the above-mentioned recombinant nucleic acid construct.
By the term “host cell” is meant any living cell, in particular eukaryotic cell such as fungous cell (e.g. Aspergillus niger), but also bacterial cell, or any non-living medium comprising transcriptional and/or translational machinery and optionally organelles from a cell, permitting to amplify the claimed nucleic acid construct and/or produce the claimed peptide.
The present invention further relates to peptides as defined above, for their use as drugs, in particular as antimicrobial, antiviral or fungicide agents.
Another aim of the invention is to provide a composition comprising at least one peptide as defined above.
Still another aim of the invention is to provide a composition as defined above, for its use as an antibacterial agent against Gram-negative and Gram-positive bacteria. A list of Gram − or Gram + bacteria can be found in the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen) or German Collection of Microorganisms and Cell Cultures catalogue.
Still another aim of the invention is to provide a composition as defined above, for its use in various domains like human and animal healthcare, agriculture and in aquaculture to avoid the development of infectious diseases in farming.
By the term “aquaculture” is meant the farming of aquatic organisms, in particular fish farming, algaculture and integrated multi-trophic aquaculture (IMTA).
By the term “aquatic organisms” is meant any freshwater or saltwater organism, including—without limitation—the following:

    
    
        fishes, such as carps, salmons and other cyprinids and salmonids;
        mollusks, such as oysters, clams, cockles, ark shells, scallops and mussels;
        crustaceans, such as crabs, lobsters, shrimps and prawns;
        aquatic plants, such as algae, and phytoplankton.
    
    


It is obvious for a man skilled in the art that the claimed peptides could be used in association with other techniques such as vaccines and physical methods intended to reduce the undesired microorganism populations.
The present invention further relates to a pharmaceutical composition comprising at least one of the claimed peptides, in association with a pharmaceutical acceptable vehicle.
By the expression “pharmaceutical acceptable vehicle” is meant any drug carrier chosen by a man skilled in the art.
In this context, they can be use in association with any pharmaceutically acceptable excipient, and in any pharmaceutically acceptable form such as for example caplets, hard gelatin capsules, other caps, soaps and lotions.
In another advantageous embodiment, the claimed pharmaceutical composition further comprise at least one another antimicrobial agent selected in the group comprising:

    
    
        tetracyclines, such as oxytetracycline or chlortetracycline;
        quinolones, such as oxolinic acid, flumequine or sarafloxacin;
        sulphonamides, optionally potentiated with trimethoprim or ormethoprim;
        nitrofurans, such as furazolidone;
        macrolides, such as erythromycin or spiramycin (E710);
        florfenicol;
        chloramphenicol.
    
    


Another aim of the invention is to provide the above-mentioned pharmaceutical composition for its use in animal and/or human antibiotic therapy.
The optimal administration route (e.g. oral route, topical route, bath treatment) should be determined by a man skilled in the art, as the optimal amount of peptide to use and duration of the treatment.
The present invention also relates to a dietary composition, in particular a food supplement further containing one or more nutritive ingredient, comprising a composition as defined above.
Another aim of the invention is to provide a composition as defined above, wherein said peptides are encapsulated, in particular nanoencapsulated. Encapsulation can bring several benefits, in particular permitting the delivery of the claimed peptides over time. Techniques for encapsulation and nanoencapsulation are well known by a man skilled in the art.
Still another aim of the present invention is to provide a disinfectant comprising a composition containing at least one of the claimed peptide.
By the term “disinfectant” is meant a mixture of compounds that provides an antimicrobial, antiviral and/or fungicide activity, and which is used for this purpose.
Amongst the other possible applications, it should be noted that the claimed peptides could be used for example in growth media, in particular media for insect and eukaryotic cells, in order to prevent the contamination of these media by microorganisms.



The following examples 1 to 3 and FIGS. 1 to 3 illustrate the invention.
FIG. 1 presents the chromatographic profile of the extract from Alvinella pompejana, obtained after its pre-purification on a Sep-Pak® 20 g column (WatersTM) and its purification by reversed-phase chromatography (RP-HPLC) using a C18 250×4.1 mm column (218TP54 VydacTM), as described in the experimental part. The two thicker lines indicate fractions that possess antimicrobial properties.
x-axis from left to right: Elution time (min);
y-axis (left side): optical density (O.D.) at 255 nm;
y-axis (right side): percentage of ACN in the eluant.
FIG. 2 presents the ability of the Alvinellacine to create pores in liposomes, herein used to mimic bacterial membranes.
x-axis from left to right: time (s);
y-axis (left side): fluorescence (arbitrary units).
The arrow shows the moment of addition of Alamethicin and the Alvinellacine.
The negative control shows the background noise, corresponding to the fluorescence measured without permeabilization of liposomes.
FIG. 3 presents the permeabilization of Gram-positive bacteria (Bacillus megaterium) and Gram-negative bacteria (Escherichia coli) after intended use of the Alvinellacine and two other AMPs (Cecropin originated from pork and Magainin originated from amphibian). These experiences were carried out at pH 7.4. The permeabilization of bacteria was measured 10 minutes after addition of each AMP for B. megaterium, while the same measurement was done after 120 minutes after addition of each AMP for E. coli. 
x-axis from left to right: concentration of Alvinellacine, Cecropin and Magainin (nM);
y-axis (left side): permeabilized bacteria (%).
Black symbols are for E. coli and white symbols are for B. megaterium. 



EXAMPLE 1
Extraction, Purification and Sequencing of the Endogenous Alvinellacine from Alvinella pompejana 
20 entire full-grown and sexually mature Pompeii worms were collected on the East Pacific Rise, by 3,000 meters deep. These adult worms were crushed, and the homogeneous resulting mixture was acidified to pH 3 by addition of HCl 1M. Then the solution was centrifuged at 10,000 g during 30 minutes. Proteins were thus concentrated in the centrifugation pellet and eliminated.
The supernatant containing the peptides was pre-purified on a pre-packed-with-methanol Sep-Pak® 20 g column (WatersTM), thereafter washed with HPLC grade water which was pre-acidified with a 0.05% trifluoroacetic acid (TFA) solution. Elution was carried out with a 2 to 60% gradient of acetonitrile (ACN), which was pre-acidified with a 0.05% trifluoroacetic acid (TFA) solution.
The fraction obtained with the 60% ACN eluant was then purified by reversed-phase chromatography (RP-HPLC) using a C18 250×4.1 mm column (218TP54 VydacTM). A 2 to 62% gradient of ACN was used for this purification. The optical density of each collected fraction was subsequently measured, in order to obtain a chromatographic profile (FIG. 1). Each peak on the chromatographic profile corresponds to a fraction that was dried, re-suspended in purified water and then tested for its antimicrobial activity.
The active fractions were further purified by successive RP-HPLCs using a C18 250×2.1 mm column (218TP52 VydacTM). The purity degree and the molecular mass of the active peptides were estimated by mass spectrometry.
The purified peptides were sequenced by Edman degradation (a well known technique for a man skilled in the art). The results show that one obtained sequence corresponds to an active peptide of SEQ ID NO: 1 that was totally unknown. Because of its origin, this peptide has been called Alvinellacine. The use of classical reverse genetics techniques permitted to obtain a nucleic acid molecule encoding the precursor protein of this peptide. The precursor protein of SEQ ID NO: 6 includes a signal peptide that goes from the 1st to the 19th amino acid residue of the sequence. The complementary DNA encoding the prepropeptide was further deduced from it and consists of SEQ ID NO: 7.
EXAMPLE 2
Evaluation of Mechanism of Antibacterial Activity
The mechanism of antibacterial activity was determined using liposomes filled with a fluorescent reagent. Liposomes consist of a bi-layer of phospholipids herein used to mimic the bacterial membrane. Permeabilization of the liposomes would result in a release of the internal compound, thus reacting with the medium and producing a fluorescent light that can be measured by spectrometry. The test is carried out with both negative and positive controls. Alamethicin, which is an antibiotic known to create pores in the bacterial membrane, is used as the positive control. The negative control is a solution of liposomes, intended for measurement of background noise (FIG. 2).
Results show that the Alvinellacine acts as the Alamethicin and permealibilizes the bi-layer of phospholipids.
This mode of action would not favour the emergent evolution of antibiotic-resistant strains.
EXAMPLE 3
Evaluation of Antibacterial Activity and Comparison with Other AMPs
The minimal inhibitory concentration (MIC) was determined according to the method of Hancock (Hancock, R. E. W. Sep. 19, 1999, posting date. [Online.] Hancock Laboratory Methods. Department of Microbiology and Immunology, University of British Columbia, British Columbia, Canada. [Antibiotics and Antimicrobial Peptides: MIC Determination by Microtitre Broth Dilution Method, last accessed September 21st.]). Permeabilization of bacterial membranes and pore-forming activity were assayed as previously described (Herbst, R., Ott, C., Jacobs, T., Marti, T., Marciano-Cabral, F., Leippe, M. Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. J. Biol. Chem. 2002. 277:22353-22360).
In liquid growth inhibition assay, the purified Alvinellacine was very active against the human pathogen Staphylococcus aureus (MIC 0.385-0.75 μM) and the clam pathogen Vibrio alginolyticus (MIC 0.006-0.012 μM).
To investigate the mode of action of Alvinellacine, we used the fluorescent dye SYTOX-Green® and either the Gram-positive Bacillus megaterium or the Gram-negative E. coli (FIG. 3). Results of this assay demonstrate that Alvinellacine rapidly permeabilizes bacterial membranes and is potently active against Gram-negatives and in a lesser extent against Gram-positives. Within the measurement period, the activity of Alvinellacine against the Gram-negative bacterium tested appeared to be over one order of magnitude higher than those for the well-known antimicrobial peptides Cecropin P1 and Magainin II. A lower activity was detected against the Gram-positive bacteria compared to the two positive controls. We also measured the pore-forming activity of Alvinellacine to further characterize its mode of action by using a minimalistic membrane system. More precisely, we monitored the dissipation of a membrane potential induced in liposomes composed of azolectin, a crude phospho lipid mixture from soy bean. Pore-forming activity was detected at final concentrations up to 0.5 nM, whereas the positive control Alamethicine gave a signal at 0.1 nM.

## Claims

1. A peptide consisting of SEQ ID NO: 1, said peptide having antibacterial activity, wherein said peptide is an isolated and purified peptide from Alvinella pompejana.

2. A method for treating gram-negative, gram-positive bacterial infections, comprising administering an effective amount of the peptide according to claim 1, to a patient in need thereof , wherein the bacterial infection is caused by a bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and combinations thereof.

3. A composition comprising said peptide according to claim 1.

4. A method for treating gram-negative, gram-positive bacterial infections, comprising administering an effective amount of the composition according to claim 3, to a patient in need thereof, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria and combinations thereof.

5. A pharmaceutical composition comprising said peptide according to claim 1 and a pharmaceutically acceptable vehicle.

6. The pharmaceutical composition according to claim 5, further comprising at least one other antimicrobial agent selected from the group consisting of:
tetracyclines;
quinolones;
sulphonamides;
nitrofurans;
macrolides;
florfenicol; and
chloramphenicol.

7. A method for treating gram-negative, gram-positive bacterial infections, comprising administering an effective amount of the pharmaceutical composition according to claim 6, to a patient in need thereof, wherein the bacterial infection is caused by a bacteria selected from the group consisting of Gram-negative and Gram-positive.

8. A dietary composition comprising a composition according to claim 3; and one or more nutritive ingredients.

9. The composition according to claim 3, wherein said peptide is encapsulated.

10. A disinfectant comprising a composition according to claim 3.