WO2013028658A1 - Method for preventing or treating a bacterial infection - Google Patents

Abstract

The present invention is method for preventing or treating a gram-positive bacterial infection using an antibacterial peptide derived from influenza A virus polymerase basic 1, frame 2 (PB1-F2) peptide.

Description

Method for Preventing or Treating a Bacterial Infection Introduction

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 61/526,784 filed August 24, 2011, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant number AI-66349 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background of the Invention

[0003] Influenza A viruses (IAVs) of the H3N2 subtype have been an important public health threat since the pandemic of 1968 (Kilbourne (1977) JAMA 237:1225-8). The pandemic strain was a reassortant, composed of 6 gene segments from the previously circulating H2N2 viruses, but with hemagglutinin (HA) and polymerase basic protein 1 (PB1) segments derived from an avian source (Kawaoka, et al . (1989) J. Virol. 63:4603-8). It infected about 40% of the human population causing more than one million deaths worldwide (Doshi (2008) Am. J. Public health 98:939-45). Since that time, H3N2 viruses have caused severe seasonal influenza every two to three seasons, on average. However, although regional differences exist, influenza-like illness attributed to H3N2 strains has generally declined over the decades during which this virus adapted to humans (Fleming & Elliot (2008) Epidemiol. Infect. 136:866-75), paralleling a decline in attributable all cause and excess mortality (Reichert, et al. (2004) Am. J. Epidemiol. 160:492-502; Doshi (2008) supra). The mechanisms by which H3N2 viruses become more or less virulent in humans are not clear. [0004] IAVs contain an eight segment, negative-strand, RNA genome. In addition to PB1, segment 2 of most IAV strains encodes a small (up to 90 amino acids) accessory protein in the +1 open reading frame (ORF; Chen, et al . (2001) Nat. Med. 7:1306-12). A third product, Ν40, can also be produced from an upstream start site in the PB1 ORF (Wise, et al.

(2009) J. Virol. 83:8021-31). Since its discovery in 2001 (Chen, et al. (2001) supra), PB1-F2 (polymerase basic 1, frame 2) has been studied as a potentially important viral virulence factor because of a link to the pathogenicity of strains including highly pathogenic avian influenza viruses of the Η5Ν1 subtype, and the 1918 (Η1Ν1) , 1957 (Η2Ν2) and 1968 (Η3Ν2) pandemic viruses (Glaser, et al. (2005) J. Virol. 79:11533-6; Conenello, et al. (2007) PLoS Pathog. 3:1414-21; McAuley, et al . (2007) Cell Host Microbe 2:240- 9; McAuley, et al . (2010) PLoS Pathog. 6:el001014). Several mechanisms for the activity of PB1-F2 have been proposed. Select laboratory-adapted strains can cause apoptotic cell death in vitro through a mechanism dependent on interactions with mitochondrial proteins, but this function may not be generalizable (Chen, et al. (2001) supra; Zamarin, et al. (2005) PLoS Pathog. I:e4; McAuley, et al .

(2010) supra) . PB1-F2 expression may also alter polymerase activity or replication kinetics in vitro, although the effects are cell-type and virus-specific and have not been shown to translate into changes in pathogenesis (Mazur, et al. (2008) Cell Microbiol. 10:1140-52; McAuley, et al . (2010) J. Virol. 84:558-64). In vivo, effects of PB1-F2 appear to be largely mediated through interactions of this accessory protein with the immune system, either through potentiation of inflammatory responses (McAuley, et al. (2007) Cell Host Microbe 2:240-9; McAuley, et al. (2010) supra) or blockade of early type I interferon pathways (Conenello, et al. (2011) J. Virol. 85:652-62).

[0005] The ability of IAVs to generate excessive inflammation in the lungs appears to be a hallmark of virulent pandemic strains ( ash, et al. (2006) Nature 443:578-81). In vitro and in mouse models, the PB1-F2 protein plays an important role in the development of IAV- related immunopatholo.gical consequences. It has been demonstrated that PB1-F2 proteins from the pandemic viruses of 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2) and from highly pathogenic H5N1 strains of avian origin enhance viral- mediated lung inflammation via an elevation of cytokines in broncho-alveolar lavage fluid and a significant influx of neutrophils and macrophages into the lungs (McAuley, et al . (2010) supra) . These enhanced inflammatory responses contributed to morbidity and mortality due to both primary viral and secondary bacterial infections (McAuley, et al . (2007) supra; McAuley, et al. (2010) supra). However, this ability was lost during adaptation into seasonal strains either through truncation (in the HlNl lineage) or mutation (in the H3N2 lineage) .

Summary of the Invention

[0006] The present invention features a method of preventing or treating a gram-positive bacterial infection in a subject by administering to a subject an effective amount of an antibacterial PB1-F2 peptide of SEQ ID NO: 3, or a pharmaceutically acceptable salt, amide, ester, or prodrug thereof. In some embodiments, the peptide is administered orally, topically, or on the surface of a medical device. In other embodiments, the gram-positive bacterium is selected from the genus of Staphylococcus, Enterococcus, and Streptococcus (e.g., Streptococcus pneumoniae, Streptococcus pyogenes) . In yet other embodiments, the gram-positive bacterium is multi-drug resistant. A disinfectant composition containing the antibacterial PB1-F2 peptide in a solvent is also provided.

Detailed Description of the Invention

[0007] It has been demonstrated that PB1-F2 proteins from the 1968 pandemic strain (A/Hong Kong/1/68; HK68) exhibit an inflammatory potential, whereas the PB1-F2 from the uh95 (A/Wuhan/359/95; Wuh95) strain, which is descended from HK68, does not. It has now been shown that amino acid residues leucine 62 (L62) , arginine 75 (R75) , arginine 79

(R79), and leucine 82 (L82) are sufficient in the H3N2 background for generation of inflammatory responses to the C-terminus of PB1-F2 including elevated pro-inflammatory cytokines and an influx of jieutrophils and macrophages into the lungs and airways, with resulting pathologic damage, weight loss, and death. Changes at these four positions

(L62P; R75H; R79Q and L82S) were sufficient to abrogate such pro-inflammatory activity in HK68. Moreover, it was observed that the non-inflammatory uh95 PB1-F2 possesses anti-bacterial activity mediated by these specific amino acid residues. Specifically, it was found that the uh95 PB1-F2 peptide kills gram-positive bacteria, including medically important bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes without causing inflammation.

[0008] Not wishing to be bound by theory, it is believed that the Wuh95 PB1-F2 peptide may disrupt the bacterial cell wall thereby killing bacteria. Indeed, the C-terminal portion of PB1-F2, i.e., residues 55 to 85, is proposed to form an alpha-helix in a membrane-associated environment

(Bruns, et al . (2007) J. Biol. Chem. 282:353-63) and is highly cationically charged (Chanturiya, et al. (2004) J. Virol. 78:6304-12), similar to other small peptides with anti-microbial activity.

[0009] Accordingly, the present invention is an isolated and optionally purified influenza virus-derived antibacterial PB1-F2 peptide for use in killing gram- positive bacteria and in the prevention and treatment of bacterial infection. By "isolated" it is meant separated from other proteins produced by influenza A virus. In some embodiments, the antibacterial PB1-F2 peptide is derived from wild-type PB1-F2 peptides from influenza A virus. Such wild-type proteins are known in the art as 87-90 amino-acid residue peptides encoded by an alternate reading frame within the PBl gene (Chen, et al . (2003) supra). The amino acid sequence and corresponding nucleotide sequence for exemplary wild-type PB1-F2 proteins of selected influenza type A strains are listed in Table 1.

TABLE 1

H2N3 A/mallard/Wisconsin/080S2841/ ADU20403 CY079482

2008

A/duck/OH/492493/2007 ADX99442 JF327333

[00010] For the purposes of the present invention, an antibacterial PB1-F2 peptide is a 15 to 90 amino acid residue peptide comprising or consisting of the 15-40 C- terminal amino acid residues of a wild-type PB1-F2 peptide from influenza A virus and having a proline at position 62, histidine at position 75, glutamine at position 79 and serine at position 82 relative to SEQ ID NO:l. Such PB1-F2 peptides, e.g., composed of residues 50-87, have been generated and shown to be stable and generate an elevated membrane conductance (Henkel, et al. (2010) PLoS One 5:ellll2) .

[00011] More particularly, an antibacterial PB1-F2 peptide of the invention has the following structure: R_x-Pro-

(12)Xaa-His- (3)Xaa-Gln- (2 ) Xaa-Ser-R₂ (SEQ ID NO:3). In accordance with this structure Ri is between 1 and 61 consecutive amino acid residues of the N-terminus of an influenza A virus PB1-F2 peptide. In particular embodiments, the amino acid residues of Ri are those immediately preceding the amino acid residue at position 62 of PB1-F2. In this respect, Ri can include residues 5-61, 10-61, 20-61, 30-61, 40-61, 50-61 or 55-61 of a wild-type PB1-F2. In some embodiments, Ri includes at least the amino acid residues located at positions 55-61 or positions 50-61 of a wild-type PB1-F2. By way of illustration, Table 2 lists peptides suitable for use as Ri in the antibacterial PB1-F2 peptide of the present invention. TABLE 2

* Location is where the residues are in the source PB1-F2 peptide sequence.

Udo72 = A/Udorn/307/72, Mem74 = A/Memphis/101/74 , Alb78 = A/Albany/14/78, HK87 = A/Hong Kong/7/87, Pan99 A/Panama/2007/99, NY10 = A/New York/20343/10, Pr8 A/Puerto Rico/8/34, Sf = A/Brevig Mission/1/1918, and Bf = consensus sequence of PB1-F2 derived from 13 different bird flu virus strains (Henkel, et al . (2010) supra) .

[00012] In embodiments where Ri includes at least the amino acid residues located at positions 55-61 of a wild-type PB1-F2, desirably residues 55-61 have the sequence

(Thr/Ile) -Val- (Ser/Phe/Tyr) -Trp- (Arg/Lys) - (Pro/Leu/Gin) -Trp

(SEQ ID NO: 16). In embodiments where Ri includes at least the amino acid residues located at positions 50-61 of a wild-type PB1-F2, desirably residues 50-61 have the sequence (Asp/Val/Gly) -Met- (His/Pro) -Lys-Gln- (Thr/Ile) -Val-

(Ser/Phe/Tyr) -Trp- (Arg/Lys) - (Pro/Leu/Gin) -Trp (SEQ ID

NO:17) .

[00013] The (12)Xaa portion of the antibacterial PB1-F2 peptide of the invention is the 12 consecutive amino acid residues located between the amino acid residues at positions 62 and 75 of a wild-type PB1-F2, i.e., residues 63-74 of SEQ ID NO:l. By way of illustration, Table 3 lists amino acid residues suitable for use as the (12)Xaa portion of the antibacterial PB1-F2 peptide of the present invention .

TABLE 3

Udo72 = A/Udorn/307/72, Mem74 = A/Memphis/101/74, Alb78 = A/Albany/14/78, HK87 = A/Hong Kong/7/87, Pan99 A/Panama/2007/99, NY10 = A/New York/20343/10, Pr8 A/Puerto Rico/8/34, Sf = A/Brevig Mission/1/1918, and Bf = consensus sequence of PB1-F2 derived from 13 different bird flu virus strains (Henkel, et al . (2010) supra).

[00014] In certain embodiments, (12)Xaa has the sequence Ser-Leu- (Lys/Arg) - (Asn/Ser) -Pro- (Thr/Ile) - (Gin/Leu/Pro) -

(Gly/Val) - (Ser/Phe) -Leu- (Arg/Lys) -Thr (SEQ ID NO:24).

[00015] The (3)Xaa portion of the antibacterial PB1-F2 peptide of the invention is the three consecutive amino acid residues located between the amino acid residues at positions 75 and 79 of a wild-type PB1-F2, i.e., residues 76-78 of SEQ ID NO:l. By way of illustration, Table 4 lists amino acid residues suitable for use as the (3)Xaa portion of the antibacterial PB1-F2 peptide of the present invention.

TABLE 4

Pan99 ALK

NY10 ALK

Pr8 VLK

Sf VLK

Bf VLK

Udo72 = A/Udorn/307/72, Mem74 = A/Memphis/101/74 , Alb78 = A/Albany/14/78, HK87 = A/Hong Kong/7/87, Pan99 A/Panama/2007/99, NY10 = A/New York/20343/10, Pr8 A/Puerto Rico/8/34, Sf = A/Brevig Mission/1/1918, and Bf = consensus sequence of PB1-F2 derived from 13 different bird flu virus strains (Henkel, et al . (2010) supra).

[00016] In certain embodiments, (3)Xaa has the sequence (Val/Ala) -Leu-Lys .

[00017] The (2)Xaa portion of the antibacterial PB1-F2 peptide of the invention is the two consecutive amino acid residues located between the amino acid residues at positions 79 and 82 of a wild-type PB1-F2, i.e., residues 80-81 of SEQ ID NO:l. By way of illustration, Table 5 lists amino acid residues suitable for use as the (2)Xaa portion of the antibacterial PB1-F2 peptide of the present invention.

TABLE 5

Udo72 = A/Udorn/307/72, Mem74 = A/Memphis/101/74 , Alb78 = A/Albany/14/78, HK87 = A/Hong Kong/7/87, Pan99 A/Panama/2007/99, NY10 = A/New York/20343/10, Pr8 A/Puerto Rico/8/34, Sf = A/Brevig Mission/1/1918, and Bf = consensus sequence of PB1-F2 derived from 13 different bird flu virus strains (Henkel, et al. (2010) supra). [00018] In certain embodiments, (2)Xaa has the sequence (Trp/Leu) - (Lys/Arg) .

[00019] In accordance with an antibacterial PB1-F2 peptide of the invention R₂ is between 1 and 8 consecutive amino acid residues of the C-terminus of an influenza A virus PB1-F2 peptide. In particular embodiments, the amino acid residues of R2 are those immediately following the amino acid residue at position 82 of PB1-F2. In this respect, R2 can include residues 83-85, 83-86, 83-87, 83-88, 83-89 or 83-90 of a wild-type PB1-F2. In some embodiments, R₂ includes at least the amino acid residues located at positions 83-85 or positions 83-87 of a wild-type PB1-F2. By way of illustration, Table 6 lists peptides suitable for use as R₂ in the antibacterial PB1-F2 peptide of the present invention .

TABLE 6

* Location is where the residues are in the source PB1-F2 peptide sequence.

Udo72 = A/Udorn/307/72, Mem74 = A/Memphis/101/74 , Alb78 = A/Albany/14/78, HK87 = A/Hong Kong/7/87, Pan99 A/Panama/2007/99, NY10 = A/New York/20343/10, Pr8 A/Puerto Rico/8/34, Sf = A/Brevig Mission/1/1918, and Bf = consensus sequence of PB1-F2 derived from 13 different bird flu virus strains (Henkel, et al. (2010) supra). [00020] Modifications to the peptides specifically disclosed herein are also contemplated as long as the essential structure and function of the peptide is maintained.

[00021] The instant peptide can be produced by any conventional method including recombinant expression, chemical synthesis or a combination thereof. When recombinantly produced, a peptide of the invention can be derived from a wild-type PB1-F2 sequence, wherein residues at positions 62, 75, 79 and 82 (with respect to SEQ ID NO:l) have been substituted with proline, histidine, glutamine, and serine, respectively. The preparation of peptides with amino acid substitutions is routinely carried out in the art and can be performed with commercially available reagents and/or kits, e.g., QUICKCHANGE Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA) . In addition, the instant peptide can be produced with one or more tags to facilitate isolation and/or purification of the peptide to homogeneity.

[00022] The present invention also includes vectors harboring nucleic acids encoding the antibacterial PB1-F2 peptide of the invention. Such vectors are known or can be constructed by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the PB1-F2 peptide sequences. Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteriophage vectors. Examples of other vectors include viruses such as bacteriophages, baculoviruses, and retroviruses, DNA viruses, cosmids, plasmids, liposomes, and other recombination vectors. The vectors can also contain elements for use in either prokaryotic or eukaryotic host systems. One of ordinary skill in the art will know which host systems are compatible with a particular vector, how to introduce said vectors into the host system, and isolate and/or purify the antibacterial PB1-F2 peptide for use in the methods disclosed herein.

[00023] Having demonstrated that the instant PB1-F2 peptide exhibits antibacterial activity toward gram-positive bacteria, the peptide of the invention is of use in killing gram-positive bacteria including medically important bacteria from the genera Bacillus (e.g., B. anthracis, B. cereus) , Streptococcus [e.g., S. pneumoniae, S. pyogenes (Group A) , S. nutans, S. agalactiae (Group B) ) , Listeria (e.g. L. monocytogenes), Staphylococcus (e.g., S. aureus, S. epidermidis, S. saprophyticus, S. lugdunensis, S. schleiferi, S. caprae) , Enterococcus (e.g., E. casseliflavus, E. gallinaru , E. raffinosus) and Clostridium (e.g., C. botulinum, C. perfringens, C. tetani, C. sordellii) , as well as in the prevention and/or treatment of infections by said gram-positive bacteria, including drug resistant bacteria such as certain strains of S. aureus which are known to be multiple drug resistant.

[00024] Accordingly, the present invention encompasses preventing or treating a gram-positive bacterial infection or disease in a subject by administering to a subject having, or at risk of having, a gram-positive bacterial infection or disease an effective amount of an antibacterial PB1-F2 peptide of this invention. As used herein, an effective amount of an antibacterial PB1-F2 peptide is an amount which prevents, eliminates or alleviates at least one sign or symptom of a bacterial infection or disease. Signs or symptoms associated with a bacterial infection or disease include, but are not limited to, bacterial colonization, sepsis, meningitis, or pneumonia. The amount of antibacterial PB1-F2 peptide required to achieve the desired outcome of preventing, eliminating or alleviating a sign or symptom of a bacterial infection or disease can be dependent on the pharmaceutical composition of the antibacterial PB1-F2 peptide, the patient and the condition of the patient, and the mode of administration. Subjects benefiting from treatment with the instant peptide include human and livestock (e.g., chickens, turkeys, cows and pigs) as well as any other animal with or at risk of having a bacterial infection.

[00025] The activity and dose of an antibacterial PB1-F2 peptide can be determined in vivo by administering to a test animal the antibacterial PB1-F2 peptide and determining whether said peptide provides protection or treatment against an infection.

[00026] A pharmaceutical composition is one that contains the antibacterial PB1-F2 peptide and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is a material useful for the purpose of administering the medicament, which is preferably sterile and non-toxic, and can be solid, liquid, or gaseous materials, which is otherwise inert and medically acceptable, and is compatible with the active ingredients. A generally recognized compendium of methods and ingredients of pharmaceutical compositions is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2000.

[00027 ] For the purpose of the present invention, pharmaceutically acceptable salts, esters, amides, and prodrugs refer to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the peptides of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of peptides of the present invention. These salts can be prepared in situ during the final isolation and purification of the peptide or by separately reacting the purified peptide in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. See, for example, Barge, et al. (1977) J. Pharm. Sci. 66:1-19.

[00028] Examples of pharmaceutically acceptable, non-toxic esters of the peptides of this invention include C1-C6 alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-C7 cycloalkyl esters as well as arylalkyl esters such as, but not limited to benzyl. C1-C4 alkyl esters are preferred. Esters of the peptides of the present invention may be prepared according to conventional methods .

[00029] Examples of pharmaceutically acceptable, non-toxic amides of the peptides of this invention include amides derived from ammonia, primary C1-C6 alkyl amines and secondary C1-C6 dialkyl amines wherein the alkyl groups are straight or branched chain. In the case of secondary amines, the amine may also be in the form of a 5- or 6- membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C1-C3 alkyl primary amines, and C1-C2 dialkyl secondary amines are preferred. Amides of the compounds of the invention may be prepared according to conventional methods.

[00030] The term "prodrug" refers to peptides that are rapidly transformed in vivo to yield the parent peptide, for example, by hydrolysis in blood.

[00031] In addition, the peptides of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[00032] For oral prophylaxis, the instant peptide may be incorporated with excipients and used in the form of non- ingestible mouthwashes, dentifrices or chewing-type gums. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution) . Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in an effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. [00033] The active antibacterial PB1-F2 peptide may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or may be incorporated directly with the food of the diet. For oral therapeutic administration, the antibacterial PB1-F2 peptide may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active peptide in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[00034] The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the- above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[00035] The instant antibacterial PB1-F2 peptide may also be administered parenterally, e.g., formulated for intravenous, intramuscular, or subcutaneous injection. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose . Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[00036] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol , phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00037] Sterile injectable solutions are prepared by incorporating the active peptide in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00038] Dosage forms for topical administration of a peptide of this invention include ointments, powders, sprays, and inhalants. The active peptide is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.

[00039] Intravascular devices, such as catheters, have become indispensable tools in the care of seriously ill patients. It is estimated that in the United States alone, 150 million catheters are purchased each year. However, due to the morbidity and mortality resulting from catheter- related infections and the high cost of managing such complications, the benefit derived from these devices may be offset. It has been shown that bloodstream infection due to the use of intravascular catheters (IVC) increased dramatically during the last ten years. From 1975 to 1977, an estimated 3% infection occurred among IVC users, while in 1992 to 1993, this rate increased to 19%. The death rate from such infection is about 8000 to 16000 per year, exceeding the death rate for AIDS. The cost for treating IVC-related infections is approximately 132 to 1600 million per year.

[00040] Most infections came from the human skin and the hub of the catheter. Among the infectious bacteria, 40% are coagulase-negative staphylococci, such as S. epidermidis, and 14% are coagulase-positive S. aureus. The remaining are mostly other gram-positive bacteria such as bacilli and enterococci .

[00041] Prophylactic methods have been developed to prevent IVC-related infections. The first line of treatment is to sterilize the insertion site with iodine and 70% ethanol . However, compliance with the written protocol is low; only 23% operations follow the protocol. Another preventative measure is the use of catheters impregnated with antibiotics or antiseptic agents such as chlorhexidine and silver sulfadiazine. In clinical trials, mixed results were obtained using such catheters. In addition to the problem of drug resistance by the infecting bacteria, the antibiotics coating the catheters can also be washed away by body fluid, as the attachment of antibiotics to the catheter surface is mainly through ionic interactions. [00042] Given its activity, the instant peptide is of particular use in the prevention of IVC-related infections. The instant antibacterial PB1-F2 peptide can be coated on the surface of catheters or any other intravascular device and/or linked to polymers used in the manufacture of these devices thereby preventing infection caused by intravascular devices. Such devices include indwelling tubes or catheters, artificial valves, pacemakers, implantable devices, etc. The instant peptide can be utilized alone, or in combination with at least one other entity, such as linked to a polymer, and incorporated, coated, or otherwise combined with the materials used in producing the patient contact portions of the medical devices. The polymer can be a hydrophobic material or matrix that can be attached to an indwelling device such as a catheter through hydrophobic bonding or can be tethered to the indwelling device through a molecular linker. By combining and/or incorporating the antibacterial peptide of the present invention into medical devices, both active and passive infection control can be achieved.

[00043] In addition to therapeutic applications, the instant antibacterial PB1-F2 peptide can be used as a disinfectant. Accordingly, the present invention also includes a disinfectant composition containing the instant antibacterial PB1-F2 peptide of SEQ ID NO: 3 in a solvent. In some embodiments, the solvent is a detergent or cleanser solution (e.g., sodium lauryl sulfate), an alcohol (e.g., ethanol, methanol, or isopropanol) , acetic acid or water. In addition to the instant peptide, the disinfectant composition can include dyes, fragrances, other antibacterials , surfactants and/or demulsifiers . The instant disinfectant composition can be sprayed, wiped, or applied to a surface to eliminate harmful bacteria. For example, the instant peptide can be used in a liquid denture cleanser or toothbrush cleanser to reduce or eliminate harmful bacteria. In addition, the instant composition finds application as a topical disinfectant, wherein a user may clean surfaces such as those found in medical offices, hospitals, bathrooms or restaurants for the reduction or elimination of harmful bacteria.

[00044] The invention is described in greater detail by the following non-limiting examples.

Example 1 Materials and Methods

[00045] Peptides. Using the predicted HK68 (A/Hong Kong/1/1968) and Wuh95 (A/Wuhan/359/1995) PB1-F2 protein sequences (SEQ ID NO:l and SEQ ID NO:2, respectively), peptides from the C-terminal ends (residues 61 through 87; SEQ ID NOs:35 and 36) with no, single, or multiple substitutions at positions 62, 73, 75, 79 and 82 (using PB1-F2 amino acid numbering) were synthesized by GenScript, Corp. (Piscataway, NJ) .

HK68, full length

MEQEQDTPWT QSTEHINIQK KGSGQQTRKL ERPNLTQLMD HYLRIMSQVD MHKQTVSWKQ WLSLKNPTQG SLKTRVLKRW KLFNKQGWTD (SEQ ID NO:l)

WUH95, full length

MEQEQDTPWT QSTEHTNIQK KGSGRQTQKL GHPNSTRLMD HYLRIMSQVD MHKQTVSWRP WPSLKNPTQG SLRTHVLKQW KSFNKQGWTN (SEQ ID NO: 2)

HK68, residues 61-87

WLSLKNPTQG SLKTRVLKRW KLFNKQG (SEQ ID NO: 35)

WUH95, residues 61-87

WPSLKNPTQG SLRTHVLKQW KSFNKQG (SEQ ID NO: 36) [00046] Mutations to the Wuh95 PB1-F2 peptide (Pro62 to Leu [P62L]; Arg73 to Lys [R73K] ; His75 to Arg [H75R] ; Gln79 to Arg [Q79R]; and Ser82 to Leu [S82L] ) were made to emulate the proposed pro-inflammatory residues found naturally in HK68. A peptide with the four key mutations P62L, H75R, Q79R, and S82L was designated Wuh95+4. Similarly, peptides on a HK68 background were made with reciprocal mutations (Leu62 to Pro [L62P] ; Lys73 to Arg [K73R] ; Arg75 to His [R75H] ; Arg79 to Gin [R79Q] ; and Leu82 to Ser [L82S] ) . A peptide with the inverse four mutations L62P, R75H, R79Q, and L82S in the HK68 background was designated HK68-4. Peptides, provided as a lyophilized powder, were solubilized in cell culture media or phosphate-buffered saline (PBS) pH 7.2 before use in in vitro and in vivo experiments, respectively.

[00047] Cell Cultures. MDCK (Madin-Darby Canine Kidney) epithelium cells were grown in IX minimum essential medium

(MEM) that contained 5% fetal bovine serum. The 293T (human kidney epithelium) cells and J774 (mouse macrophage) cells, were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum.

[00048] Infectious Agents. Chimeric influenza viruses uh95/PR8 and uh95+3/PR8 were generated by reverse genetics based on a 6:2 reassortment (Hoffmann, et al.

(2002) Vaccine 20:3165-70), containing the six internal gene segments of Wuh95 and the HA and neuraminidase (NA) gene segments of PR8. Before virus rescue, the PBl gene segment of Wuh95 was modified using site-directed mutagenesis (QUIKCHANGE; Stratagene, La Jolla, CA) by previously described methods (McAuley, et al. (2007) supra) to generate a virus variant possessing P62L, H75R, and S82L mutations in the PB1-F2 ORF (Wuh95+3/PR8 ) . The rescued chimeric viruses were amplified once in MDCK cells for stocks and PB1 gene was fully sequenced to demonstrate that they were free of mutations other than those described. Infectivity of the rescued viruses was determined using plaque assays in MDCK cells, according to known methods.

[00049] S. pneumoniae strain A66.1 (type 3) was grown in Todd Hewitt broth (DIFCO Laboratories, Detroit, MI) to an OD₆2o of approximately 0.4, and then frozen at -80 °C mixed 2:1 with 5% sterile glycerol. The titers of the frozen stocks were quantitated on tryptic soy agar (DIFCO Laboratories, Detroit, MI) supplemented with 3% v/v sheep erythrocytes .

[00050] Cell Culture Assays. To determine cytokine concentrations, J774 cells in 6-well plates were treated with a panel of H3N2 PB1-F2 peptides at doses ranging from 100 to 500 μΜ in DMEM containing 3% bovine serum albumin. Culture supernatant fluids were collected 4 hours after infection and frozen before use.

[00051] Mice. Experiments using 8-week-old female BALB/c or DBA/2 mice (Jackson Laboratories, Bar Harbor, ME) were performed in a Biosafety Level 2 facility. Animals were given general anesthesia composed of 2.5% inhaled isoflurane (Baxter Healthcare Corporation, Deerfield, IL) prior to all interventions, and all studies were approved by the Animal Care and Use Committee.

[00052] Peptides Studies With Mice. All studies involving peptides were performed in BALB/c mice. To determine the pathogenicity of H3N2 PB1-F2 peptides, mice (10 per group) were given 60 mg of uh95, Wuh95+4, HK68 or HK68-4 PB1-F2 peptides once, in a volume of 100 μΐ intranasally (IN) , and monitored for weight loss and survival. For lung pathology, mice (3 per group) were administered 40 mg of PB1-F2 peptides, or PBS, and euthanized 3 days later. To analyze cellular immune response and levels of cytokines, the bronchoalveolar lavage (BAL) fluids from at least 5 mice per group were collected at day 3 after administration of 40 mg of H3N2 PB1-F2 peptides or PBS.

[00053] In a bacterial super-infection model, mice were exposed to 40 mg of peptides, and 1 day later challenged with 2000 colony-forming units (CFU) of S. pneumoniae per mouse. Control exposed animals received PBS instead of peptide. Weight changes (calculated for each mouse as a percentage of its weight on day 0 before peptide administration) and survival of mice were monitored for 21 days after the bacterial challenge. For bacterial titers, lungs of 3-5 mice per group were collected on days 1, 3, and 5 after bacterial challenge.

[00054] Histopathologic Analysis. Lungs were removed immediately following euthanasia, sufflated, and fixed in 10% neutral buffered formalin overnight. The lungs were processed routinely, embedded in paraffin, sectioned at 5 mm, stained with hematoxylin and eosin, and examined microscopically for histopathologic alterations. The lungs were assigned a grade 0-3 based on the histologic character of the lesions. A score of 0 was given when no pathologic changes could be detected. A score of 1 was given to mild findings including minimal infiltrates of lymphocytes and plasma cells around airways and vessels, minimal epithelial hyperplasia, minimal leukocyte infiltration of alveolar spaces, and <10% of the lung affected. A score of 2 was given for moderate findings including moderate infiltrates of lymphocytes and plasma cells around airways and vessels, moderate epithelial hyperplasia with focal necrosis, focally extensive infiltration of the alveolar spaces by leukocytes with some consolidation, focal pleuritis, and >10% but <30% of the lung affected. A score of 3 was given for more severe findings including extensive necrosis of airway epithelium and the interstitium, extensive leukocyte infiltration and consolidation, severe pleuritis, and lobar involvement. Grading and description of pathology were performed by an experienced veterinary pathologist single- blinded to the purpose of the study and the composition of the groups .

[00055] Virus Studies With Mice. All studies involving chimeric Wuh95/PR8 and Wuh95+3/PR8 viruses were performed in DBA/2 mice. To determine the pathogenicity of Wuh95/PR8 and Wuh95+3/PR8, mice (10 per group) were infected IN with viruses at doses ranging from 10⁴ to 10⁶ plaque-forming units (PFU) per mouse in 100 μΐ of sterile PBS, and observed for 21 days for weight loss, length of survival, and death.

[00056] To compare the growth of chimeric viruses, histopathologic changes in the lungs, and cellular immune response and cytokines in the BAL fluids, mice were infected IN with a dose of 10^4-5 PFU per mouse. At 6, 12, and 18 hours, and 1, 3, 5, 7, 9 or 11 days after infection, lungs from three or more mice from each group were harvested for virus titers. Histopathologic changes in the mouse lungs (3 per group) were determined at day 3 and 7 post-infection (p.i). Cellular immune response in the BAL fluids from at least 5 mice per group was analyzed at 3 and 9 days p.i.

[00057] In a secondary bacterial pneumonia model, mice were infected with 10³'⁵ PFU of Wuh95/PR8 and Wuh95+3/PR8 per mouse and challenged 7 days later with 100 CFU of S. pneumoniae per mouse. Control-infected animals received PBS initially instead of virus. Weight changes (calculated for each mouse as a percentage of its weight on day 0 before virus infection) of mice were monitored for 21 days after the bacterial challenge. Viral and bacterial titers in the mouse lungs (at least 5 per group) as well as cellular immune response and levels of cytokines in the BAL fluids were determined at day 2 after bacterial challenge.

[00058] Lung Viral and Bacterial Titers. The lungs were removed under sterile conditions, washed three times with PBS, homogenized, and suspended in PBS (total volume, 1 ml) . The suspensions for virus titration were centrifuged at 2,000 x g for 10 minutes to clear cellular debris. Virus titers were determined by plaque assays in MDCK cells. Lung homogenates were used directly for bacterial cultures prior to centrifugation. Pneumococcal colony counts were done by using 10-fold dilutions on tryptic soy agar plates (DIFCO) supplemented with 3% (vol/vol) sheep erythrocytes and 400 pg/ml of kanamycin.

[00059] Analysis of Cellular Immune Response in BAL Fluid. Following euthanasia by C0₂ inhalation, the trachea was exposed and cannulated with a 24-gauge plastic catheter

(Becton Dickinson Infusion Therapy Systems, Inc., Sandy, UT) . Lungs were lavaged twice with 1.5 ml of cold, sterile Hank's buffered salt solution supplemented with 0.05 mM EDTA. The number of white blood cells (WBC) per ml of the resulting suspension was then determined on a Hemavet 3700

(Drew Scientific, Dallas, TX) using a 100 μΐ aliquot. Flow cytometry (LSRII, Becton Dickinson, San Jose, CA) was performed on the BAL fluid suspension after incubation of cells with 75 μΐ of 1:200 dilution of Fc block (anti-mouse CD16/CD32, BD Bioscience Inc., San Jose, CA) on ice via staining (1 μ1/10⁶ cells) with anti-mouse antibodies: CDllc

(eFluor 450), F4/80 (FITC) , HC II (PE) , Ly6G (PerCp- Cy5.5), Ly6C (APC) , and CDllb (APC-EFLUOR 780) (eBioscience Inc., San Diego, CA) . The data were analyzed by FlowJo 8.8.6 (Tree Star, Ashland, OR), where viable cells were gated from FSC and SSC plot, then different inflammatory cell subsets were gated using markers: neutrophils (CDllb ¹ /CDllc¹⁰ F4-80^lo/Ly6G^hiLy6C^int) , exudates macrophages

(CDllb^hi/CDllc^l0_int F4-80^lo_int/Ly6C^hi ly6G^int) , and inflammatory dendritic cells (CDllb^hi/Ly6G^l0"int/CDllc^int"hi MHC II^hi) . Analysis of BAL fluid cell composition was based upon the proportion of viable events analyzed by flow cytometry as related to the number of white blood cells (WBC)/ml.

[00060] Analysis of Cytokines and Chemokines . Cytokines were determined in culture supernatants from J774 cells and BAL fluids of mice. Before measurement, BAL fluids were centrifuged at 10, 000 x g for 5 minutes and the supernatants were frozen. The concentrations of interleukin (IL)-6, and chemokines KC, granulocyte colony stimulating factor (G-CSF) , monocyte chemotactic protein (MCP)-I, macrophage inflammatory protein ( ΙΡ)-Ια, and tumor necrosis factor (TNF) -a were measured by using the "mouse 22-plex" cytokine assay (Millipore Co., Billerica, MA) and read on a LUMINEX X PONENT 3.1 (BIO-RAD Laboratories, Austin, TX) according to the manufacturer's procedure. Samples were diluted 1:2 and 1:4, and run in triplicate in all assays with appropriate internal controls.

[00061] Statistical Analyses. Comparison of survival between groups of mice was done by using the log-rank chi-square test to analyze the Kaplan-Meier survival data over the period of 21 days. The mean number of days until death was estimated as the number of days that the mice survived after peptide administration, or viral, or bacterial infection. If no death occurred during the observation period, the mean days to death was considered to be 21 days. Analysis of variance (ANOVA) with Dunn's correction was used to estimate and compare the viral and bacterial titers, weight loss, cell counts, and cytokine levels. Paired Student's t-test was used to compare matched groups of mice for weight loss. A P value <0.05 was considered significant for these comparisons. SIGMASTAT for WINDOWS (SysStat Software, Inc., V 3.11) was used for all statistical analyses.

Example 2 : Mapping of the H3N2 PB1-F2 Proposed Pro- Inflammatory Sequence

[00062] The sequences of the HK68 and Wuh95 PB1-F2 proteins differ at five positions (residues 62, 73, 75, 79 and 82) in the C-terminal third (residues 61-87) that had previously been demonstrated sufficient to induce inflammatory responses (McAuley, et al. (2007) supra; McAuley, et al. (2010) supra). To establish the role of these residues individually in PB1-F2 pathogenicity, HK68 or Wuh95 PBl-F2-derived peptides possessing single or multiple substitutions at these positions were synthesized. These peptides were then administered a single time to groups of BALB/c mice (n=10) IN at a dosage of 60 mg per mouse .

[00063] Mice exposed to the H 68 PBl-F2-derived peptide lost more than 20% of their initial weight and 50% of them died within 21 days of peptide administration (Table 7). In contrast, exposure to Wuh95 PB1-F2 caused no weight loss or mortality in mice (Table 8). Single amino acid substitutions at residues 62, 75, and 82 on HK68 and residues 75, 79 and 82 on Wuh95 PB1-F2 backgrounds significantly altered the properties of the natural peptides (reflected by either weight loss or survival; P < 0.05). Mutation at amino acid residue 73 in either background did not cause a statistically significant effect. The most prominent alterations in weight and mortality in mice were observed with Wuh95 and HK68 PB1-F2 peptides possessing quadruple mutations at residues 62, 75, 79, and 82 (hereafter referred to as Wuh95+4 and to as HK68-4, respectively) ; the combination of these four changes was sufficient in either background to fully revert the phenotype (Table 7 and Table 8) .

TABLE 7

*Number of mice that survived exposure to peptide with mutations differed significantly from that with naturally occurring peptide, as compared by the Kaplan-Meier method followed by log-rank test.

TABLE 8

Example 3 : Inflammatory Responses in Mice Caused by H3N2 PB1-F2 Peptides

[00064] To determine the role of residues 62, 75, 79, and 82 in PB1-F2-mediated inflammation, the lungs of mice were examined for histopathologic changes at day 3 after peptide administration. Extensive lung consolidation with dense neutrophilic infiltrates in a peri-bronchiolar distribution, and abundant monocytes and macrophages were observed in the parenchyma in lungs of mice exposed to the Wuh95+4 or HK68 PB1-F2 peptides containing L62, R75, R79, and L82 residues. There were no signs of lung inflammation in the control group, which received PBS only or in the groups of mice exposed to the Wuh95 and HK68-4 PB1-F2 peptides. Quantification of the immune cells and cytokines present in BAL fluid showed that the extensive pulmonary inflammation observed in mice exposed to Wuh95+4 and HK68 PB1-F2 peptides resulted from an influx of WBCs into the BAL fluid, characterized by a significant increase in levels^' of neutrophils, macrophages, and cytokines including IL-6, KC, MCP-1, ΜΙΡ-Ι and G-CSF as compared to those in PBS or in Wuh95 or HK68-4 treated groups (P < 0.05).

[00065] Cytokine responses measured in J774 cells treated with H3N2 PB1-F2 peptides for 4 hours at dosages ranging from 100· μΜ to 500 μΜ revealed significant increases in the levels of TNF in culture supernatant fluids in cells treated with 250 μΜ or 500 μΜ of Wuh95+4 and HK68 as compared to those treated with Wuh95 and HK68-4 PB1-F2 peptides (P<0.05). It was concluded from these data that the presence of the HK68 amino acid motif at positions 62, 75, 79, and 82 was sufficient to allow PB1-F2 derived peptides to cause inflammation, and that these inflammatory responses could cause morbidity and mortality in mice independent of other virulence factors expressed by the virus . Example 4: The Inflammatory Motif of the H3N2 PB1-F2 Peptide Primes for Bacterial Pneumonia

[00066] It has been demonstrated that PB1-F2 can accelerate development of secondary bacterial pneumonia in mice

(McAuley, et al . (2007) supra; Iverson, et al. (2011) J. Infect. Dis. 203:880-8). Whether this was dependent on the inflammatory activity of the protein was unclear. To establish the role of the H3N2 PB1-F2 inflammatory motif in priming for bacterial pneumonia, mice were treated with 40 mg of a panel of PB1-F2 peptides and 1 day later challenged with 2000 CFU of S. pneumoniae. Weight loss and survival of mice were monitored for 21 days after the bacterial challenge.

[00067] Mice exposed to the inflammatory HK68 and Wuh95+4 PB1-F2 peptides lost about 15% of their initial weight after peptide administration. Secondary challenge with S. pneumoniae enhanced this weight loss and had a significant effect on mortality. All mice exposed to the HK68 peptide and challenged with PBS (instead of bacteria) survived, while 40% died by day 3 when super-infected. In the group of mice exposed to Wuh95+4 peptide and challenged with PBS 20% mortality was observed, and all mice in the Wuh95+4 group, which were challenged with bacteria, died by day 5 after peptide administration. The most striking differences in weight loss and survival were seen when comparing the groups of mice exposed to the H3N2 PB1-F2 peptides with (Wuh95+4 and HK68) and without (Wuh95 and HK68-4) inflammatory motifs in the bacterial super-infection model. Mice in the latter two groups lost an average of only 5% of their initial weight after peptide administration, did not lose further weight upon bacterial challenge, and did not die. Interestingly, the weight loss of mice exposed to PBS (instead of peptide) and bacteria was higher by day 7 after peptide administration than that in the groups of mice exposed to non-inflammatory peptides (Wuh95 and HK68-4) and bacteria .

[00068] Examination of the mouse lung bacterial titers at day 1, 3, and 5 after pneumococcal challenge indicated that the excessive weight loss and mortality observed in groups of mice exposed to the inflammatory peptides (Wuh95+4 and HK68) were due to a significant increase (up to 70-fold) in bacteria as compared to groups of mice treated with the corresponding non-inflammatory versions (Wuh95 and HK68+4; P < 0.05). Unexpectedly, the bacterial lung titers in PBS- treated group were significantly higher than those of groups of mice exposed to uh95 and HK68-4, indicating that the non-inflammatory variants of the H3N2 PB1-F2 peptides had anti-bacterial properties (P < 0.05). This antibacterial effect correlated with the modest differences in weight loss and survival observed when comparing PBS and bacteria groups to non-inflammatory peptide and bacteria groups .

[00069] The results from these experiments with H3N2 PB1-F2 derived peptides indicate that the presence of amino acid residues L62, R75, R79, and L82 caused significant pulmonary inflammation and accelerated development of pneumococcal infection. The presence of amino acid residues P62, H75, Q79, and S82 on H3N2 PB1-F2 did not induce inflammation but had an apparent bactericidal effect on S. pneumoniae . Example 5: Role of the H3N2 PB1-F2 Inflammatory Motif in Virus Pathogenicity

[00070] To determine the role of residues 62, 75, 79, and 82 in virus pathogenicity in mice,- an attempt was made to rescue chimeric viruses carrying six genes from Wuh95 and the HA and NA genes from PR8 without or with mutations in the Wuh95 PB1-F2. However, in case of the 79 amino acid residue the primary PBl amino acid sequence would have been altered and this virus could not be rescued. Thus, studies were performed with the chimeric Wuh95/PR8 strain (P62, H75 and S82 amino acid sequence with respect to the PB1-F2 ORF) and a virus possessing triple mutations of P62L, H75R and S82L on uh95 PB1-F2 ( uh95+3/PR8 ) . DBA/2 mice were than infected IN with doses ranging from 10⁴ to 10⁶ pFU per mouse and weight loss and mortality were monitored for 21 days

(Table 9) . Control mice were treated only with PBS.

TABLE 9

[00071] The data indicated that, as a trend, the chimeric Wuh95+3/PR8 virus possessing the inflammatory P62L, H75R and S82L mutations on PB1-F2 caused greater weight loss and mortality in mice as compared to those infected with Wuh95/PR8. The clearest differences between the two viruses were observed at a dose of 10⁴'⁵ PFU per mouse. The mice infected with Wuh95+3/PR8 lost an average of 16.3% of their initial weight by day 7 p.i. and 40% of them died by day 10 p.i., versus 10.2% of weight loss by day 7 p.i. and 10% mortality observed for Wuh95/PR8. Differences in weight loss reached statistical significance by day 7 p.i. for groups infected with lO^4-0 and 10⁴'⁵ PFU per mouse.

[00072] Examination of the growth kinetics of Wuh95/PR8 and Wuh95+3/PR8 in the lungs of mice infected with 10^4-5 PFU per mouse showed that, despite greater weight loss and mortality, the growth of Wuh95+3/PR8 in mouse lungs was similar or modestly lower than that of Wuh95/PR8 up to day 3 p.i. Differences in lung virus titers of 5- to 10-fold between the two groups were observed, with the greatest differences observed at 12 hours after infection. Thus, improved replication was not responsible for the observed pathogenic phenotype. In addition, examination of the levels of WBC, macrophages, neutrophils, and inflammatory dendritic cells, histopathology, and the levels of proinflammatory cytokines in the BAL fluid of mice infected with 10⁴·⁵ PFU per mouse of Wuh95/PR8 and Wuh95+3/PR8 at day 3, 7 and 9 p.i. did not reveal significant differences in these parameters between the groups at these time-points.

Example 6: The Inflammatory Motif of the 1968 H3N2 PB1-F2 Primes for Secondary Bacterial Pneumonia in the Context of the Full Virus

[00073] To determine the role of the Wuh95+3/PR8 PB1-F2 inflammatory residues in promoting secondary bacterial pneumonia, a nonlethal synergistic model between chimera viruses and S. pneumoniae was developed by infecting groups of 10 mice with a single dose of 10^3,5 PFU of Wuh95/PR8 or Wuh95+3/PR8 per mouse, and 7 days later challenging with 100 CFU of bacteria per mouse. The differences between the two chimeric viruses were evaluated by measuring weight loss, and bacterial and virus lung titers. Infecting mice with either virus at the chosen dose did not result in significant weight loss during observation (99.0% and 98.8% of starting weight at day 7 for the Wuh95/PR8 and Wuh95+3/PR8 groups, respectively, P>0.1). Viral lung titers of Wuh95/PR8 and Wuh95+3/PR8 were similar (ΐο³·^5±0·⁵ PFU/ml and ιο³·^8±0·⁴ PFU/ml, respectively) at the time of secondary bacterial challenge (day 7 after viral infection) . Bacterial super-infection notably increased the weight loss only in the group of mice infected with Wuh95+3/PR8 (96.0% of starting weight vs. 90.5% for the uh95/PR8 and Wuh95+3/PR8 groups, respectively, P<0.05). No virus was present in the lungs in either group at day 2 after the bacterial challenge (day 9 after viral infection) . Analysis of bacterial lung titers at this time point showed no detectable titers in the groups of mice infected with Wuh95/PR8 or exposed to PBS (instead of virus) prior to bacterial super-infection. In contrast, the mean level of bacterial titers in the group of mice infected with Wuh95+3/PR8 was ιο⁶·^8±0·⁵ CFU/mL (P<0.05). This result was similar to the data obtained with Wuh95 and Wuh95+3 PB1-F2 peptides in the bacterial pneumonia model, indicating that the Wuh95 PB1-F2 has bactericidal properties. It was concluded from these data that, in the context of the full virus, the pathogenic effects of H3N2 PB1-F2s are manifest primarily through interactions with bacteria. The proinflammatory properties of the 1968 pandemic strain PB1-F2 support bacterial super-infection, while anti-bacterial properties of the uh95 PB1-F2 suppress bacterial growth in the lung.

Claims

What is claimed is :

1. A method of preventing or treating a gram-positive bacterial infection in a subject comprising administering to a subject an effective amount of an antibacterial polymerase basic 1, frame 2 (PB1-F2) peptide of SEQ ID NO: 3, or a pharmaceutically acceptable salt, amide, ester, or prodrug thereof.

2. The method of claim 1, wherein the peptide is administered orally, topically, or on the surface of a medical device.

3. The method of claim 1, wherein the gram-positive bacterium is selected from the genus of Bacillus, Listeria , Staphylococcus, Enterococcus, Clostridium and Streptococcus .

4. The method of claim 1, wherein the gram-positive bacterium is multi-drug resistant.

5. The method of claim 3, wherein the gram-positive bacterium is a Streptococcus pneumoniae.

6. A disinfectant composition comprising an antibacterial polymerase basic 1, frame 2 (PB1-F2) peptide of SEQ ID NO: 3 in a solvent.