CA2349837A1 - Lysozyme fusion proteins in infections - Google Patents

Abstract

A method and composition for prophylaxis and/or therapeutic treatment of bacterial infections, particularly respiratory bacterial infections. A fusion protein of lysozyme and the carboxyl terminal propeptide of surfactant protein-B (SP-B) with the preceding ten amino acids of the mature SP-B peptide, or recombinant lysozyme alone, is administered in a pharmaceutically acceptable medium to an individual. The fusion protein or recombinant lysozyme may be selected so as to deliver it to a target infection site, such as the lungs or gastrointestinal tract. The method and composition eliminates problems associated with conventional antibiotic treatments, such as inefficacy and promotion of antibiotic resistant bacterial strains.

Description

LYSOZY~/IE FUSION PROTEINS IN INFECTIONS
Related Applications This application is a Continuation-In-Part of U.S. Application Serial No. 09/193,877 filed November 18, 1998, now pending.
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant Nos. R01-HL56285 and HL56285S awairded by the National Institutes of Health.
Field of the Inventioy The invention relates to prophylactic and therapeutic uses of recombinant lysozyrne and a lysozyme/surfactant protein-B fusion protein in bacterial infections.
Background of the in n i n Bacterial infections remain a leading cause of worldwide morbidity and mortality. While antibiotics administered to treat bacterial infections are often safe and efficacious, there is widespread concern over bacterial strains -z-that have become rEaistant to classic antibiotic treatment regimens. In individuals infected with resistant strains, antibiotic administration results in incomplete and ineffecaive treatment, necessitating additional treatment along with propagation of the resistant strains. Preventative measures and alternative treatments that do not rely on antibiotics are therefore desirable.
In certain individuals such as those who are immunocompromised, who are in less than optimal health, who tack fully functional immune systems such as neonates or geriatric patients, or who suffer from a disease affecting the respiratory tract such as cystic fibrosis or the gastrointestinal tract such as ulcerative colitis or sprue, bacterial infections may have severe consequences leading to serious illness or even death. For example, production of altered mucus in cystic fibrosis patients leads to dilation of the exocrine ducts, destruction of acinar tissue, and replacement of the destroyed tissue by fibrous connective tissue. Involvement of the lungs leads to pneumonia and bronchiectasis. The paucity of systemic dissemination of infection in these patients, even in the presence of substantial bacterial colonization of the lungs, indicates that their systemic immunity is essentially intact, yet they are susceptible to pulmonary infections. These patients often die in their teens or early twenties from terminal lung infections in spite of aggressive antibiotic 'therapy.
The respiratory and gastrointestinal tracts are frequent sites of bacterial infections in normal individuals. The normal respiratory tract has natural clearance mechanisms that help to prevent bacterial colonization.
These mechanisms include the presence of a mucus gel that acts as a barrier .. g-to bacterial invasion, the propulsive forces of the cilia on the epithelial lining of the airways, and the secretion of antibacterial humoral factors such as the secretory immunoglobulins IgA and IgM, the proteins lactoferrin, betalysin and fibronectin, complement components and the enzyme lysozyme. Of these antimicrobial factors, Ilysozyme is the best established antimicrobial substance in airway secretions. -Human lysozyme is a naturally occurring enzyme that is known to exhibit bactericidal activity in vitro and thus would appear to be a promising way to prevent and/or treat bacterial infections. Lysozyme is a small ( 15 kilodaltonsl, basic protein that is produced in most tissues. It is secreted and is present in most body secretions such as mucus. In the lungs, immunohistochemical methods have localized iysozyme to the bronchial serous submucosal glands, alveolar macrophages and lamellar bodies in Type II
alveolar epithelial cellls. Approximately 80% of fysozyme secreted into the airway comes from the mucosal layer of the upper airways.
In vitro, liysozyme has been demonstrated to act independently to cause bacterial death. It is known that one way lysozyme kills bacteria is by hydrolyzing the giycosidic bond between C-1 of N-acetylmuramic acid and C-4 of N-acetylgiucosamine in the bacterial polysaccharide cell wall. Lysozyme can also kill bacteria by acting synergistically with other proteins such as complement or antibody to lyse bacterial cells. Lysozyme, produced by polymorphonuclear leukocytes such as neutrophiis, inhibits chemotaxis of polymorphonuclear leukocytes and limits the production of oxygen free radicals following an infection. This limits the degree of inflammation, while at the same time enhances phagocytosis by these cells. Lysozyme is also probably involved in the response of airway tissue to injury.
While the antibacterial effects of lysozyme in vitro have been well documented, there has heretofore been no way to exploit these effects of lysozyme for in vivo use. Previous reports furthermore implied that sustained lysozyme administration. would be harmful.
Pulmonary surfactant, a complex mixture of phospholipids and proteins, is synthesized and secreted by alveolar type II epithelial cells, a specialized exocrine cell. Surfactant protein-B (SP-B) is one of the protein components of pulmonary surfactant. Normal respiratory function requires pulmonary surfactanl: for maintenance of alveolar patency.
A method and composition for the prophylaxis and/or therapeutic treatment of bacterial infections, particularly respiratory bacterial infections as frequently occurs in patients with cystic fibrosis, is thus desirable.
Summaryr of t Inve~ ti n This invention is directed to a composition for the prophylaxis or therapeutic treatment of a bacterial infection in a mammal. The composition is either a lysozymeisurfactant protein-B (SP-B) fusion protein SEQ ID N0:3 (rat lysozyme SEQ ID N0:1 fused with the carboxyl terminal SP-B propeptide and the preceding ten amino acids of the mature SP-B peptide SEQ ID N0:4), or a IysozymeiSP-B fusion protein SEQ ID NO:fi (human lysozyme SEQ ID
N0:5 fused with the carboxyl terminal SP-B propeptide and the preceding ten amino acids of the mature SP-B peptide SEQ ID N0:4), or recombinant lysozyme alone SEQ ID N0:1. The composition prevents or treats a respiratory infection such as occurs frequently in individuals with cystic fibrosis, or may occur in individuals having other types of respiratory diseases, or a gastrointestinal infection. The invention's use of recombinant lysozyme reduces mortality and enhances in vivo clearance of bacteria such as Pseudomonas aeruginosa, which is the major airway pathogen in patients with cystic fibrosis. Furthermore, the elevated lysozyme activity in bronchoalveolar lavage fluid resulting from administration of lysozyme is not associated with altered lung function or structure, or with chronic inflammatory disease.
The invention is also directed to a method of preventing or treating a bacterial infection in a mammal by administering a lysozyme/SP-B
fusion protein SEQ ID N0:3 or SEQ ID N0:6 in a pharmaceutically acceptable composition in a dosing regimen sufficient to prevent or treat the infection.
The route of administration may be parenteral, for example by inhalation, or nonparenteral.
The invE:ntion is still further directed to a fusion protein SEQ ID
N0:3, comprising a rat lysozyme SEQ ID N0:1 and a carboxyl terminal SP-B
propeptide with the 'ten terminal amino acids of the mature peptide SEQ ID
N0:4, having antibacterial activity in a mammal.
The invE:ntion is still further directed to a fusion protein SEQ ID
N0:6, comprising a human lysozyme SEQ ID N0:5 and a carboxyl terminal SP-B propeptide with the: ten terminal amino acids of the mature peptide SEQ ID
N0:4, having antibacterial activity in a mammal.
The invention is additionally directed to a method of treating a bacterial respiratory infection in an individual having cystic fibrosis by administering a lysozyme/SP-B fusion protein SEQ ID N0:3 or SEQ ID N0:6 to the individual. The fusion protein may be administered by aerosol installation and/or inhalation.
The invention is also directed to a method of preventing or treating a bacterial infection in a mammal by administering recombinant lysozyme SEQ ID N0:1 in a pharmaceutically acceptable composition at a dose sufficient to prevent or treat the infection, such as treating a bacterial infection in a patient with cystic fibrosis.
The invention is additionally directed to a composition comprising recombinant lysozyme SEQ ID N0:1 having antibacterial activity.
These and other methods and compositions will be apparent in light of the following figures and detailed description.
Brief Description of..the Figures FIG. 1 i;s a histogram showing bacterial clearance from lungs of transgenic (lysozyme:/surfactant protein-B fusion protein) and control mice.
FIG. 2A is a photograph showing expression of recombinant lysozyme SEQ ID N0:1 in a transgenic mouse line probed with mouse lysozyme complementary DNA (cDNA), and FIG 2B is a photograph showing expression of recombinant lysozyme SEQ ID N0:1 in the same transgenic mouse line probed with rat cDNA.
FIG. ;3 is a photograph showing analysis of lysozyme protein expression.
FIG. 4 is a histogram of lysozyme activity in bronchoalveolar lavage (BAL) fluid.

_7_ FIG. 5A ins a photograph showing lysozyme cellular localization in wild type mice, and FIG. 5B is a photograph showing lysozyme cellular localization in transgenic mice.
FIG. 6A is a photograph showing lung structure in transgenic mice that overexpress lysozyme, and FIG. 6B is a photograph showing lung structure in wild type mice.
FIG. 7 is a histogram illustrating the cellular composition of BAL
fluid.
FIG 8 is a histogram showing clearance of Group B Streptococcus (GBS) from the lungs at six hours post-infection.
FIG 9 is a histogram showing clearance of Pseudomonas aeruginosa from the lungs at twenty-four hours post infection.
pg~,i g Description czf the Preferred Embodiment Preparation of Recombinant Lysozyme and SP-B and Fusion Protein Rat lysozyme is a hydrophobic peptide of 148 amino acids SEQ
ID N0:1 . Human lysozyme SEQ ID N0:5 also has 148 amino acids and has 69% homology with rat lysozyme.
Surfactant protein-B (SP-B) is a hydrophobic peptide of 79 amino acids that avidly associates with surfactant phospholipids in the alveolar airspace. Human SP'-B is synthesized by alveolar type II epithelial cells as a prepropeptide of 381 amino acids SEQ ID N0:2. Mature SP-B is generated by sequential cleavage of a 23 amino acid signal peptide, an amino terminal (N-terminal) propeptide of 177 amino acids, and a carboxyl-terminal (C-terminal) propeptide of 102 amino acids. The C-terminal propeptide has been shown to _g_ function in maintenance of the size of lamellar bodies which store SP-B and in determining the intracellular surfactant pool size.
Complementary DNA (cDNA) corresponding to rat lysozyme and human SP-B was syrnthesized. The protocol used for synthesis was that described in Akinbi E;t al., J. Biol. Chem. 1997; 272: 9640-9647, which is expressly incorporated by reference herein in its entirety. Complementary DNA
to rat lysozyme was. generated as follows. Alveolar Type II epithelial cells were isolated from adult rat lung as described by Dobbs, et al., An irrt~roved method for isolatincLype II cells in hiah xield and I uritv, Am. Rev. Respir.
Dis.
134:140-145, 198Ei1. Total RNA was isolated from Type II cells by the method of Chirgwin, et al., The isolation of biologicaIIK active ribonucleic acid from sources enriched in ribonucle~se, Biochemistry 18:5294-5299, 1979) and polyA+ RNA by i:he method of Aviv and Leder, Purification of biologically active globin mes<renaer RNA bar chromatoaraohy on oligothymidylic acid-cellulose, Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972. Single stranded cDNA, generated from isolated polyA+ RNA with reverse transcriptase (Maniatis, et al. Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboiratory, Cold Spring Harbor, NY 1982), was used as a template for PCR amplification of the entire coding sequence of lysozyme using oligonucleotide primers based on the published sequence of the rat enzyme by Yeh, et al. Evolutionyf ro~gnt Ivsozymes~ Isolation and sequence of the rat Ivsozy~pe genes, Mol. Physiol. Evol. 2:25-75, 1993. Isolation of the human SP-B cDNA has previously been described (Glasser, S.W., et al. ~IDNA and _g-deduced amino acid sequence of human ~aulmonar~r surfactant associated proteoii~pid SPL IPhe1_, Proc. Natl. Acad. Sci. USA 84:4007-4011, 1987).
The synthesized cDNAs were either generated into a chimeric molecule consisting of the rat lysozyme protein and a carboxyl terminal (C-terminal) propeptide of a human surfactant protein B (SP-B) for evaluation, or recombinant rat lyso;zyme fSEQ ID N0:1 ) alone was generated. Specifically, this chimeric molecule was a fusion protein of residues 1-148 of rat lysozyme SEQ ID N0:1 and residues 270-381 of SEQ ID N0:2, shown in SEQ ID N0:4, forming a lysozyme/surfactant-B fusion protein SEQ ID N0:3.
Pre ra i of Tra eni r in P-B Fusi n Pr in Three lines of transgenic mice were generated that expressed a cDNA construct comprising the entire coding sequence for rat lysozyme SEQ
1D N0:1 and the entire C-terminal propeptide of SP-B along with the preceding ten amino acids from the C-terminal of the mature peptide SEQ ID N0:4. The coding sequence for rat lysozyme SEQ ID N0:1 was cloned in frame with the coding sequence for the C-terminal propeptide and preceding 10 amino acids for human pulmonary surfactant protein B propeptide SEQ ID N0:4. FVB/N
transgenic mice expressing a transgene construct encoding the fusion protein SEQ ID N0:3 under the control of the 3.7 kilobase (kb) human surfactant protein C (SP-C) promoter were generated as described in Lin et al. (J. Biol.
Chem. 1996; 271: 19689-19695) which is expressly incorporated by reference herein in its entirety. -The expression of transgene RNA in these mice was restricted to the distal respiratory epithelium.

Expression of the chimeric protein SEQ ID N0:3 was confirmed by Western blot analysis, as is known to one skilled in the art, using antibody #R96189 generated and characterized as reported by Lin et al., Structural ~.~irements for intu-~~IJlular transport of pulmonary surfactant protein B
SP- (Biochim. Biophys. Acta Mol. Cell. Res. 1312:177-185, 1996), that detects the C-terminal- propeptide of SP-B proprotein. A protein of approximately 29 kDa was detected in transgenic mice using this antibody, as would be predicted by the size of the construct of 15 kDa rat lysozyme and 14 kDa C-terminal prope;ptide and preceding 10 amino acids SEQ ID N0:3. The transgene product was detected in both lung homogenates and in bronchoalveolar lavac~e (BAL) fluids, consistent with secretion of the chimeric protein SEQ ID NO::.. into the alveolar space. Constitutive expression of the chimeric protein SEQ ID N0:3 was not associated with altered lung structure, as assessed by light microscopy evaluation of lung tissue stained with hematoxylin and eosin.
Preparation of Transgenic Mice OvPrexpressing~x~.ozym~
In order to assess the role of lysozyme in pulmonary host defense, transgenic mouse lines were generated in which rat lysozyme SEQ ID N0:1 was targeted to the distal airway epithelium under the direction of the 3.7 kb human surfactant protein C (SP-C) promoter. Seven of twenty-one offspring from fertilized oocyi:e injections were positive for the transgene as assessed by PCR and confirmed by Southern blot analyses of tail DNA (now shown).
Transgenic mice w~:re indistinct from wild type littermates with respect to body weight, lung weight, longevity and reproductive capability. Two transgenic lines (3.5 and 2.6) had increased levels of lysozyme protein in bronchoalveolar lavage fluid.
Efficac~of ~,ysozy,~~,e/SP-B Fusion Protein Five-week old transgenic mice (n = 56) carrying the fusion protein SEQ ID N0:3 (treated) and their wild type litter mates (control) were infected with 106 strain III group B Streptococci (GBS) via intratracheal administration.
Aliquots of GBS were grown in Todd Hewitt broth at 37 ° C
overnight and bacteria were pelleted by centrifugation. The bacterial pellet was suspended in sterile phosphate buffered saline (PBS) at a concentration of 10'/ml. One hundred microliters of bacterial suspension ( 1 O6 bacteria) were drawn into tuberculin syringes fitted with 2'7 gauge needles in preparation for the intratracheal injection. All injections were carried out in a sterile environment.
Five to aix week old mice maintained in clean rooms were used for bacterial clearance studies. Mice were anesthetized with a mixture of nitrous oxide and oxygen. The trachea was exposed through a midline incision and dissection through the thyroid gland. One hundred microliters of bacterial suspension was instilled into the trachea with a 27 gauge needle just below the cricoid cartilage. The two halves of the thyroid muscles were apposed at the midline and the skin incision was closed by approximating the two edges with surgical glue. Animals were housed for either 6 or 24 hours prior to sacrifice.
After either 6 or 24 hours post infection, lungs from treated and control mice were harvested, weighed, homogenized in PBS and plated on BAP
to evaluate formation of GBS colonies. The cultured plates were incubated at .. 12 -37°C for approximatE;ly 16-18 hours (overnight). Colony forming units per gram of lung tissue (CFU/g) were assessed by manual counting of bacterial colonies.
As shown in FIG. 1, transgenic mice harboring the chimeric protein SEQ ID N0:3 had significantly fewer CFU/g of lung tissue at both 6 hours and 24 hours following infection with GBS. The number of CFU in most transgenic mice was even less than the number of bacteria that had been administered. As shown in FIG. 1, transgenic mice had significantly enhanced clearance of GBS from the lungs at both the 6 h and 24 h post infection time points. At 6 h post-infection, BAP inoculated with lung tissue from transgenic (treated) mice had 1.99 ~ 1.4 x 104 CFU/g of tissue, while BAP inoculated with lung tissue from wild type (control) mice had 25.49 ~ 12.43 x 104 CFU/g (p < 0.0061. At 24 h post-infection, BAP inoculated with lung tissue from transgenic (treated) mice had 9.9 ~ 6.43 x 104 CFUIg tissue, while BAP
inoculated with lung tissue from wild type (control) mice had 67.29 ~ 34.2 x 104 CFU/g tissue (p < 0.04).
These results suggested that the expression of lysozyme/surfactant protein-B fusion protein SEQ ID N0:3 in the airway of the transgenic mice facilitated bacterial clearance from the airway. The results show that in mice carrying the lysozyme/surfactant protein-B fusion protein SEQ 1D N0:3, bacterial proliferation was inhibited at 6 hours post infection, while bacterial clearance was enhanced at 24 hours post infection. Transgenic mice which expressed a lysozyme/surfactant protein B fusion protein SEQ 1D
N0:3 in the distal airway were twelve-fold more efficient in clearing bacteria in the airway than their wild type littermates. This lysozyme-produced efficacy is particularly striking, since wild type mice are inherently very efficient in clearing bacteria in the airway.
Two other lines of transgenic mice expressed the lysozyme/surfactant protein-B chimeric protein SEQ ID N0:3 although at lower levels. Bacterial cleairance in these lines was correspondingly lower, although still significantly greater than that in wild type control mice. Lysozyme enzyme activity was increased 40% (relative to wild type littermates) in bronchoalveolar lavade fluid of the transgenic line expressing the highest levels of the lysozyme/SP-B fusion protein SEQ ID N0:3. Since bacterial clearance was enhanced twelve-fold in this line, the antibacterial effect may have been conferred by the SP-B C-terminal propeptide with the preceding amino acids from the mature peptide SEQ ID N0:4 alone, or the result of the combined action of SP-B and ly;sozyme SEQ ID N0:3 components; alternatively the effect may be due to increased lysozyme SEQ ID N0:1 activity.
Analysis of ~yrsozyme Transgene Expression RNA With reference to FIGS. 2A and 2B, expression of the lysozyme transgene was assessed by Northern blot analysis of 2 Ng of total cellular RNA isolated from lung tissue of five-week old transgenic mice (line 3.5). These are shown in FIG. 2A lanes 4 and 5, and in FIG. 2B lanes 3 and 4. Control wild type littermates are shown in FIG. 2A lanes 1-3 and in FIG. 2B
lanes 1 and 2. Both transgenic and control samples were probed with mouse complementary DNA (cDNA) (FIG. 2A) and rat cDNA (FIG. 2B). In FIG. 2A, endogenous mouse lysozyme was the larger of the 2 transcripts. The mouse cDNA probe, because. of cross hybridization between rat lysozyme and mouse cDNA, detected rat lysozyme.
A cDNA , probe specific for rat lysozyme SEQ ID N0:1 detected a " 1 kb transcript, the predicted size of the iysozyme transgene (FIG. 2B).
When the same RNA samples were probed with a mouse lysozyme cDNA
(FIG. 2A), both the larger endogenous mouse lysozyme mRNA and rat lysozyme mRNA were detected. The mouse lysozyme mRNA was used as an internal control for normalization of samples.
Pr in Because rat and mouse lysozyme have very similar molecular weights, it was not possible to resolve the proteins by SDS-PAGE.
Total levels of lysoz:yme (rat and mouse) were estimated by Western blot analysis of equal amounts of protein from lung homogenates or BAL fluid from five-week old transge;nic mice and wild type littermate controls. FIG. 3 shows a Western blot analysis of 1 ,ug of total lung protein from transgenic mouse line 3.5 (lanes 4-6) and control wild type littermates (lanes 1-3). Proteins were fractionated by SD;i-PAGE under non-reducing conditions, blotted onto a nitrocellulose membn~ane and incubated with anti-human lysozyme antibody, which detected both mouse and rat lysozyme (molecular weight of about 15 kD).
As shown in FIG. 3, mice from transgenic line 3.5 (lanes 4-6) had a four-fold increase in the level of lysozyme protein in both lung homogenate and BAl_ fluid over v~riid type littermates (lanes 1-3). Mice from transgenic line 2.6 had a two-fold increase in lysozyme protein compared to control wild type littermates (not shown).

-~ 15 -Lysoz~m~e Enzyme Activity With reference to FIG. 4, lysozyme enzyme activity was assessed in BAL fluid from five-week old transgenic mice from line 3.5 (number of animals (n)=4) and line 2.6 (n=4), and control wild type littermates (n = 7) by a turbidimetric assay. Ten nanograms of BAL fluid protein was incubated with Micrococcus lysodeikticus suspended at an optical density (O.D.) of 1 at 450 nm. Following one hour of incubation at 37°C, the change in O.D. of the suspension was determined. Purified chicken egg white lysozyme was used to generate a standard curve.
As showin in FIG. 4, mice from transgenic line 3.5 had a 17.5-fold increase in lysozyme .activity compared to wild type mice (550 units/ng BAL
protein versus 31 units/ng BAL protein in wild type mice). The data are mean ~ standard error of the mean (SEM), with p = < 0.0001 for wild type versus transgenic line 3.5, p -_ < 0.0001 for wild type versus 2.6, and p = < 0.0008 for transgenic line 3.5 versus transgenic line 2.6. As predicted from Western blot analyses, mice from transgenic line 2.6 had increased lysozyme enzyme activity (205 units/ng BAL protein) relative to wild type controls Ip=0.02), but lower activity relative to transgenic line 3.5.
Spatial Expression of Laisozvme With reference to FIGS. 5A and 5B, cellular localization of lysozyme protein in transgenic and control mice was obtained. Paraffin-embedded lung sections from five-week oid transgenic mice from line 3.5 (FIG 5B) and wild types control littermates (FIG. 5A) were immunostained with hematoxylin/eosin using an anti-human lysozyme antibody which detects both rat and mouse lysozyme and were photographed at a magnification of 80 x.

The overall architecture of lung sections of transgenic mice were indistinct from wild type littermates. There was no evidence of pulmonary edema or vascular congestion, and inflammatory cells were not detected in lung sections from uninfected transgenic mice.
Lysozyme was detected in Type II cells in wild type and transgenic mice, wii:h more intense staining in Type Il cells from transgenic mice. Transgenic mice, in addition, have expression in bronchiolar epithelial cells. In wild type mice, endogenous expression of epithelial lysozyme was restricted to Type II cells, whereas in transgenic mice, lysozyme expression was equally prominent in non-ciliated bronchiolar cells and Type II cells.
Effect of Lysozyme Overexpression on Lung Structure With reference to FIGS. 6A and 6B, gross histologic features of paraffin-embedded, hematoxylin/eosin-stained sections of lungs from uninfected five-week old transgenic mice (FIG. 6A) and control wild type littermates (FIG. 6B) were compared at a magnification of 40 x. Total and differential cell couma (Cytospin° preparations stained with Diff-Quick°) in BAL
fluids were performed.
As shown in FIG. 7, there were no significant differences in either the total cell count or in the distribution of cell types recovered from BAL
fluid in transgenic mice compared to wild type control littermates, with p = 0.98 for total cell counts, p ~= 0.74 for macrophages, and p = 0.95 for lymphocytes.
V
Data are mean ~ SEM.

Effect of Lyso~yrme Overex rp ession on Bacterial Pathogen Clearance from the it Clearance of Grou~~~ B Streptococcus IGBS). To determine if increased lysozyme levels in the airway enhanced clearance of bacteria from the lungs, bacterial counts in quantitative cultures of lung homogenates from transgenic mice and v~rild type littermate controls were compared following an intratracheal injectiorw of 106 colony forming units (CFU) of GBS. All mice survived until sacrifice at six hours post infection.
The results, shown in FIG. 8, are expressed as CFU/g lung tissue ~ SEM, with p =0.0172 in wild type (n = 20) versus transgenic line 3.5 (n =19), and p = 0.1 ~~ in wild type versus transgenic line 2.6 (n =101. Mice from transgenic line 3.5 had a three-fold enhancement of GBS clearance (2.1 ~ 0.1 x 106 CFU/g lung tissue versus 6.8 ~ 0.5 x 106 CFU/g lung tissue in wild type mice, p=0.0172). The incidence of systemic dissemination of infection, as assessed by growth of GBS on plates inoculated with splenic homogenates, was less in transgenic mice (27% versus 60%, p=0.04).
Clearance of GBS from the lungs was enhanced less than two-fold in mice from transgenic line 2..6 (4.2 ~ 0.8 x 1 O6 CFU/g lung tissue versus 7.1 ~ 0.6 x 106 in wild type mice, p =0.19).
~e~,n_c;e of Psseudomonas aeru~rinasa. Transgenic and wild type mice received intratracheal injections with 10' CFU of Pseudomonas aeruginosa. All transgenic mice survived until sacrifice at 24 hours post-infection. In contrast, 20% of infected wild type mice died.
FIG. 9 shows CFU/g lung tissue ~ SEM. Bacterial clearance was enhanced approximately thirty-fold in mice from transgenic fine 3.5 (n =10) ( 1.06 ~ 0.05 x 1 O6 CFU/g lung tissue, compared to 3.24 ~ 0.41 x 10' in wild type littermate controls (n =10, p =0.03). Bacterial clearance was enhanced approximately six-fold in mice from transgenic line 2.6 (n =10) (6.52 ~ 0.71 x 1 O6 CFU/g lung tissue, p = 0.05). Systemic bacterial dissemination was not detected in surviving mice at 24 hours post-infection.
The lysozyme/SP-B fusion protein SEQ ID N0:3 or SEQ ID N0:6 or recombinant lysozyme SEQ ID N0:1 of the invention may be used to treat and/or reduce bacterial colonization of the airway. The latter use would be extremely beneficial in treating individuals with cystic fibrosis, since chronic bacterial colonization of the major airways, particularly by Pseudomonas aeruginosa, with consequent debilitating exacerbations is the major cause of the morbidity and mortality suffered by cystic fibrosis patients.
Cystic fibrosis is a systemic disease in which mucus secretion is altered so that a viscid mucus is produced. Production of altered mucus leads to dilation of the exocrine ducts, destruction of acinar tissue, and replacement of the destroyed tissue by fibrous connective tissue. Involvement of the lungs leads to pneumonia and bronchiectasis. These patients often succumb at a young age to terminal lung infections with Pseudomonas aeruginosa in spite of aggressive antibiotic therapy. Therefore, the bactericidal activity of lysozyme or the lysozyme/surfactant protein-B fusion protein in vivo offers a potential therapeutic strategy for suppressing bacterial colonization of the airways in cystic fibrosis patients without compromising whatever degree of respiratory function the patient exhibits.

It will be appreciated that prophylaxis or therapeutic treatment by the method and composition of the invention may range from total prevention, reduction of the bacterial load, amelioration of the severity of, or elimination of a bacterial infection. The IysozymeiSP-B fusion protein SECT ID N0:3 or SECT ID N0:6 or recombinant lysozyme SECT ID N0:1 may be used to protect against all bacterial ;trains which colonize the respiratory tract such as, for example, Staphylococcus aureus, Streptococcus species, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiellae species, Proteus species, Pseudomonas cepacia, Haemophilus influenzae, Bordetella pertussis, Mycoplasn7a pneumoniae, Legionella pneumophila.
Additionally, the invention may be used to combat gastrointestinal infections. The lysozymeisurfactant protein-B fusion protein SECT ID N0:3 or SECT ID N0:6 may be administered by an enteral route to target the gastrointestinal tract for treating or preventing infections with bacterial strains that colonize the gastrointestinal tract such as, for example, Salmonellae species, Shigellae species, Escherichia coli, and Vibrio species, Yersinia enterocolitica, Camp~lobacter fetus, ssp. jejuni, and Helicobacter pylori. The lysozyme/SP-B may be formulated for oral administered as a solid or liquid in the form of a capsule, tablet, syrup, and so on.
Other variations or embodiments of the invention will also be apparent to one of ordinary skill in the art from the above description. For example, other fusion proteins besides surfactant protein-B, such as members of the structurally related saposin protein family such as saposin A, saposin B, saposin C, saposin D, NK lysin, pore forming peptide of amoebapore A etc., could be generated. ~Jarious modes of administration besides inhalation could be used, such as injection, etc. The fusion protein SEQ ID N0:3 or SEQ ID
N0:6 or recombinant lysozyme SEQ ID N0:1 may be administered either alone or in combination v~rith antibiotic therapy. Thus, the forgoing embodiments are not to be construed ass limiting the scope of this invention.
What is claimed is:

SEQUENCE LISTING
<110> Weaver, Timothy E.
<120> LYSOZYNIE FUSION PROTEINS IN INFECTIONS
<130> CMC-127-232 <160> 6 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 148 <212> PRT
<213> RAT
<400> 1 Met Lys Ala Leu Leu Val Leu Gly Phe Leu Leu Leu Ser Ala Ser Val 1. 5 10 15 Gln Ala Lys Ile Ty:r G1u Arg Cys Gln Phe Ala Arg Thr Leu Lys Arg Asn Gly Met Ser Gly Tyr Tyr Gly Val Ser Leu Ala Asp Trp Val Cys Leu Ala Gln His Gl~a Ser Asn Tyr Asn Thr Gln Ala Arg Asn Tyr Asn Pro Gly Asp Gln Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp Cys Asn Asp G1y Lys Thr Pro Arg Ala Lys Asn Ala Cys Gly I1e Pro Cys Ser Ala Leu Leu Gln Asp Asp Ile Thr Gln Ala IIe Gln Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp Gln Arg His Cys Lys Asn Arg Asp Leu Ser Gly Tyr Ile Arg Asn Cys Gly Val <210> 2 <211> 381 <212> PRT
< 213 > HUMAN
<220>
<221> PROPEP
<222> (0)...(0) <400> 2 Met Ala Glu Ser Hi.s Leu Leu Gln Trp Leu Leu Leu Leu Leu Pro Thr 1 c; 10 15 Leu Cys Gly Pro Gly Thr Ala Ala Trp Thr Thr Ser Ser Leu Ala Cys Ala Gln Gly Pro Glu Phe Trp Cys Gln Ser Leu Glu Gln Ala Leu Gln Cys Arg Ala Leu G7_y His Cys Leu Gln Glu Val Trp Gly His Val Gly Ala Asp Asp Leu Cvs Gln Glu Cys Glu Asp Ile Val His Ile Leu Asn Lys Met Ala Lys G.Lu Ala Ile Phe Gln Asp Thr Met Arg Lys Phe Leu Glu Gln Glu Cys Aan Val Leu Pro Leu Lys Leu Leu Met Pro Gln Cys Asn Gln Val Leu Asp Asp Tyr Phe Pro Leu Val Ile Asp Tyr Phe Gln Asn Gln I:Le Asp Ser Asn Gly Ile Cys Met His Leu Gly Leu Cys Lys Ser Arg Gln Pro Glu Pro Glu Gln Glu Pro Gly Met Ser Asp F?ro Leu Pro Lys Pro Leu Arg Asp Pro Leu Pro Asp Pro Leu Leu Asp Lys Leu Val Leu Pro Val Leu Pro Gly Ala Leu Gln Ala Arg Pro Gly Pro His Thr Gln Asp Leu Ser Glu Gln Gln Phe Pro Ile Pro Leu Pro 'ryr Cys Trp Leu Cys Arg F.la Leu Ile Lys Arg Ile Gln Ala Met Ile Pro Lys Gly Ala Leu Arg Val Ala Val Ala Gln Val Cys Arg Val Val Pro Leu Val Ala Gly Gly Il.e Cys Gln Cys Leu Ala Glu Arg Tyr Ser Val Ile :?45 250 255 Leu Leu Asp Thr heu Leu Gly Arg Met Leu Pro Gln Leu Val Cys Arg Leu Val Leu Arg Cys Ser Met Asp Asp Ser A.La Gly Pro Arg Ser Pro Thr Gly Glu Trp Leu Pro Arg Asp Ser Glu Cys His Leu Cys Met Ser Val Thr Thr Gln :~.:La Gly Asn Ser Ser Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Ala Cys Val Gly Ser Trp Leu Asp Arg Glu Lys Cys Lys Gln Phe Val Glu Gln His Thr Pro Gln Leu Leu Thr Leu Val Pro Arg Gly Trp Asp Ala His Thr Thr Cys Gln Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pro Leu Gln Cys Ile His Ser Pro Asp Leu <210> 3 <211> 260 <212> PRT
< 213 > Ra t an~i Human <220>
<223> Chimer::r, Protein <400> 3 Met Lys Ala Leu Leu Val Leu Gly Phe Leu Leu Leu Ser Ala Ser Val Gln Ala Lys Ile Tyr Glu Arg Cys Gln Phe Ala Arg Thr Leu Lys Arg Asn Gly Met Ser Gly Tyr Tyr Gly 'Jal Ser Leu Ala Asp Trp Val Cys Leu Ala Gln His Glu Ser Asn Tyr Asn Thr Gln Ala Arg Asn Tyr Asn Pro Gly Asp Gln Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp Cys Asn Asp G1y Lys Thr Pro Arg Ala Lys Asn Ala Cys Gly Ile Pro Cys Ser Ala Leu Leu Gln Asp Asp Ile Thr Gln Ala IJ.e Gln Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val A.La Trp Gln Arg His Cys Lys Asn Arg Asp Leu Ser Gly Tyr Ile Arg Asn Cys Gly Val Val Cys Arg Leu Val Leu Arg Cys Ser Met Asp Asp Ser Ala Gly Pro Arg Ser Pro Thr Gly Glu Trp Leu Pro Arg Asp Ser Glu Cys His Leu Cys Met Ser Val Thr Thr Gln Ala Gly Asn Ser Ser Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Ala Cys Val Gly Ser Trp Leu Asp Arg Glu L~~s Cys Lys Gln Phe Val Glu Gln His Thr Pro Gln Leu Leu Thr Leu Val. Pro Arg Gly Trp Asp Ala His Thr Thr Cys Gln Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pro Leu Gln Cys Ile His Ser Pro Asp Leu <210> 4 <211> 112 <212> PRT
<213> Human <220>
<221> PROPEP
<222> (0) . . . (0) <223> C-terminal propeptide + 10 amino acids of mature SP-B
<400> 4 Val Cys Arg Leu Val Leu Arg Cys Ser Met Asp Asp Ser Ala Gly Pro 5 . 10 15 Arg Ser Pro Thr Gly Glu Trp Leu Pro Arg Asp Ser Glu Cys His Leu Cys Met Ser Val Thr Thr Gln Ala Gly Asn Ser Ser Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Ala Cys Val Gly Ser Trp Leu Asp Arg Glu Lys Cys Lys Gln Phe Val Glu Gln His Thr Pro Gln Leu Leu Thr Leu Val Pro Arg Gly 'Crp Asp Ala His Thr Thr Cys Gln Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pro Leu Gln Cys Ile His Ser Pro Asp Leu <210> 5 <211> 148 <212> PRT
<213> Human <220>
<400> 5 Met Lys Ala Leu Ilea Val Leu Gly Leu Ala Leu Leu Ser Val Thr Val 1. 5 10 15 Gln Gly Lys Val Phs= Gly Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly Met Asp Gly Tyr Arg Gly :Lle Ser Leu Ala Asn Trp Met Cys Leu Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly Asp Arg Se:r Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp Cys Asn As;p G1y Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser Cys Ser A1a Leu Leu Gln Asp Asn Ile Ala Asp Ala Ala Ala Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gly Val Arg Ala Trp Ala Ala Trp Arg Asn Arg Cys Gln Asp Arg Asp Val Arg Gln Tyr Val Gln Gly Cys Gly Val <210> 6 <211> 260 <212> PRT
<213> Human and Human <220>
<223> Chimer_Lc Protein <400> 6 Met Lys Ala Leu Ilea Val Leu Gly Leu Ala Leu Leu Ser Val Thr Val Gln Gly Lys Val Phe Gly Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly Met Asp Ghy Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Ala Ala Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gly Val Arg Ala Trp Ala Ala Trp Arg Asn Arg Cys Gln Asp Arg Asp Val Arg Gln Tyr Val Gln Gly Cys Gly Val Va.l Cys Arg Leu Val Leu Arg Cys Ser Met Asp Asp Ser Ala Gly Pro Ax°g Ser Pro Thr Gly Glu Trp Leu Pro Arg Asp Ser 1E;5 170 175 Glu Cys His Leu Cys Met Ser Val Thr Thr Gln Ala Gly Asn Ser Ser Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Ala Cys Val Gly Ser Trp Leu Asp Arg Glu Lvs Cys Lys Gln Phe Val Glu Gln His Thr Pro Gln Leu Leu Thr Leu Va1 Pro Arg Gly Trp Asp Ala His Thr Thr Cys Gln Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pro Leu Gln Cys Ile His Ser Pro Asp Leu

Claims (12)

1. A composition comprising a lysozyme/surfactant protein-B fusion protein selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6.

2. A composition comprising recombinant lysozyme SEQ ID NO:1.

3. The composition of claims 1 or 2 for prophylaxis or therapeutic treatment of a bacterial infection in a mammal.

4. The composition of claims 1 or 2 for prophylaxis or therapeutic treatment of a respiratory bacterial infection.

5. The composition of claim 4 wherein said respiratory infection is in said mammal having cystic fibrosis.

6. The composition of claim 1 for prophylaxis or therapeutic treatment of a gastrointestinal infection in a mammal.

7. A fusion protein selected from the group consisting of SEQ ID
NO:3 and SEQ ID NO:6 having antibacterial activity in a mammal.

8. A recombinant lysozyme SEQ ID NO:1 having antibacterial activity in a mammal.

9. A method of prophylaxis or therapeutic treatment of a bacterial infection in a mammal comprising administering a composition selected from the group consisting of a recombinant lysozyme SEQ ID NO:1 in a pharmaceutically acceptable carrier and a lysozyme/surfactant protein-B fusion protein selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6 in a pharmaceutically acceptable carrier to said mammal at a dose sufficient to prevent or treat the infection.

10. The method of claim 9 wherein the composition is administered by a method selected from the group consisting of inhalation and aerosol installation.

11. The method of claim 9 wherein the mammal is infected with Pseudomonas.

12. The method of claim 9 wherein the mammal has cystic fibrosis.