WO2000029588A1

WO2000029588A1 - Lysozyme fusion proteins in infections

Info

Publication number: WO2000029588A1
Application number: PCT/US1999/027403
Authority: WO
Inventors: Timothy Edward Weaver; Henry Toyin Akinbi
Original assignee: Children's Hospital Medical Center
Priority date: 1998-11-18
Filing date: 1999-11-18
Publication date: 2000-05-25
Also published as: BR9915218A; CA2349837A1; EP1129201A1; JP2002530083A; AU2345900A; AU759743B2

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

LYSOZYME FUSION PROTEINS IIM INFECTIONS

Related Applications

This application is a Continuation-ln-Part of U.S. Application Serial

No. 09/1 93,877 filed November 1 8, 1 998, now pending.

The U.S. Government has a paid-up license in this invention and

the right in iimited 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 awarded by the National Institutes of Health.

Field of the Invention

The invention relates to prophylactic and therapeutic uses of

recombinant lysozyme and a lysozyme/surfactant protein-B fusion protein in

bacterial infections.

Background of the Invention

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 that have become resistant to classic antibiotic treatment regimens. In

individuals infected with resistant strains, antibiotic administration results in

incomplete and ineffective 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 lack 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 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, lysozyme 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 ( 1 5

kilodaltons), 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 lysozyme to the bronchial serous

submucosal glands, alveolar macrophages and lamellar bodies in Type II

alveolar epithelial cells. Approximately 80% of lysozyme secreted into the

airway comes from the mucosal layer of the upper airways.

In vitro, lysozyme has been demonstrated to act independently to

cause bacterial death. It is known that one way lysozyme kills bacteria is by

hydrolyzing the glycosidic bond between C-1 of N-acetylmuramic acid and C-4

of N-acetylglucosamine 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 neutrophils, 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 surfactant 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.

Summary of the Invention

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 lysozyme/surfactant protein-B (SP-B) fusion protein SEQ ID NO:3

(rat lysozyme SEQ ID NO: 1 fused with the carboxyl terminal SP-B propeptide

and the preceding ten amino acids of the mature SP-B peptide SEQ ID NO:4),

or a lysozyme/SP-B fusion protein SEQ ID NO:6 (human lysozyme SEQ ID

NO:5 fused with the carboxyl terminal SP-B propeptide and the preceding ten

amino acids of the mature SP-B peptide SEQ ID NO:4), or recombinant

lysozyme alone SEQ ID NO: 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 NO:3 or SEQ ID NO: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 invention is still further directed to a fusion protein SEQ ID

NO:3, comprising a rat lysozyme SEQ ID NO: 1 and a carboxyl terminal SP-B

propeptide with the ten terminal amino acids of the mature peptide SEQ ID

NO:4, having antibacterial activity in a mammal.

The invention is still further directed to a fusion protein SEQ ID

NO:6, comprising a human lysozyme SEQ ID NO:5 and a carboxyl terminal SP-

B propeptide with the ten terminal amino acids of the mature peptide SEQ ID

NO: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 NO:3 or SEQ ID NO: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 NO: 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 NO: 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 is 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 NO: 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 NO: 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. FIG. 5A is 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.

Detailed Description of the Preferred Embodiment

Preparation of Recombinant Lysozyme and SP-B and Fusion Protein

Rat lysozyme is a hydrophobic peptide of 1 48 amino acids SEQ

ID NO: 1 . Human lysozyme SEQ ID NO:5 also has 1 48 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 NO:2. Mature SP-B is generated by

sequential cleavage of a 23 amino acid signal peptide, an amino terminal (N-

terminal) propeptide of 1 77 amino acids, and a carboxy terminal (C-terminal)

propeptide of 102 amino acids. The C-terminal propeptide has been shown to 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 synthesized. The protocol used for synthesis was that

described in Akinbi et al., J. Biol. Chem. 1 997; 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 improved

method for isolating Type II cells in high yield and purity. Am. Rev. Respir. Dis.

1 34: 1 40-145, 1 986). Total RNA was isolated from Type II cells by the

method of Chirgwin, et al., The isolation of biologically active ribonucleic acid

from sources enriched in ribonuclease. Biochemistry 1 8:5294-5299, 1 979)

and polyA⁺ RNA by the method of Aviv and Leder, Purification of biologically

active globin messenger RNA by chromatography on oligothvmidylic

acid-cellulose. Proc. Natl. Acad. Sci. USA 69: 1 408-141 2, 1 972. Single

stranded cDNA, generated from isolated polyA⁺ RNA with reverse

transcriptase (Maniatis, et al. Molecular Cloning: A Laboratory Manual. Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY 1 982), 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. Evolution of rodent lysozymes: Isolation and sequence of the rat

Ivsozyme genes. Mol. Physiol. Evol. 2:25-75, 1 993. Isolation of the human

SP-B cDNA has previously been described (Glasser, S.W., et al. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated

proteolipid SPL (Phe).. Proc. Natl. Acad. Sci. USA 84:4007-401 1 , 1 987).

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 lysozyme (SEQ ID NO: 1 ) alone was generated. Specifically,

this chimeric molecule was a fusion protein of residues 1 -1 48 of rat lysozyme

SEQ ID NO: 1 and residues 270-381 of SEQ ID NO:2, shown in SEQ ID NO:4,

forming a lysozyme/surfactant-B fusion protein SEQ ID NO:3.

Preparation of Transgenic Mice Overexpressing Lysozyme/SP-B Fusion Protein

Three lines of transgenic mice were generated that expressed a

cDNA construct comprising the entire coding sequence for rat lysozyme SEQ

ID NO: 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 NO:4. The

coding sequence for rat lysozyme SEQ ID NO: 1 was cloned in frame with the

coding sequence for the C-terminal propeptide and preceding 1 0 amino acids

for human pulmonary surfactant protein B propeptide SEQ ID NO:4. FVB/N

transgenic mice expressing a transgene construct encoding the fusion protein

SEQ ID NO: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. 1 996; 271 : 1 9689-1 9695) 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 NO:3 was confirmed

by Western blot analysis, as is known to one skilled in the art, using antibody

#R961 89 generated and characterized as reported by Lin et al., Structural

requirements for intracellular transport of pulmonary surfactant protein B

(SP-B) (Biochim. Biophys. Acta Mol. Cell. Res. 1 31 2: 1 77-1 85, 1 996), 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 1 5 kDa rat lysozyme and 14

kDa C-terminal propeptide and preceding 1 0 amino acids SEQ ID NO:3. The

transgene product was detected in both lung homogenates and in

bronchoalveolar lavage (BAL) fluids, consistent with secretion of the chimeric

protein SEQ ID NO:3 into the alveolar space. Constitutive expression of the

chimeric protein SEQ ID NO: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 Overexpressing Lysozyme

In order to assess the role of lysozyme in pulmonary host defense,

transgenic mouse lines were generated in which rat lysozyme SEQ ID NO: 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 oocyte injections were positive for the transgene as assessed

by PCR and confirmed by Southern blot analyses of tail DNA (now shown).

Transgenic mice were 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.

Efficacy of Lysozvme/SP-B Fusion Protein

Five-week old transgenic mice (n = 56) carrying the fusion protein

SEQ ID NO: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 1 0⁷/ml. One

hundred microliters of bacterial suspension ( 1 0⁶ bacteria) were drawn into

tuberculin syringes fitted with 27 gauge needles in preparation for the

intratracheal injection. All injections were carried out in a sterile environment.

Five to six 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 37 °C for approximately 1 6-1 8 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 NO: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 ± 1 2.43 x 1 0⁴ CFU/g

(p < 0.006). At 24 h post-infection, BAP inoculated with lung tissue from

transgenic (treated) mice had 9.9 ± 6.43 x 10⁴ CFU/g tissue, while BAP

inoculated with lung tissue from wild type (control) mice had

67.29 ± 34.2 x 1 0⁴ CFU/g tissue (p < 0.04).

These results suggested that the expression of

lysozyme/surfactant protein-B fusion protein SEQ ID NO: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 ID NO: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 ID

NO: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 NO:3 although at lower

levels. Bacterial clearance 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 lavage fluid of the transgenic line expressing the highest levels

of the lysozyme/SP-B fusion protein SEQ ID NO: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 NO:4 alone, or the result of the combined

action of SP-B and lysozyme SEQ ID NO:3 components; alternatively the effect

may be due to increased lysozyme SEQ ID NO: 1 activity.

Analysis of Lysozyme Transgene Expression

RNA With reference to FIGS. 2A and 2B, expression of the

lysozyme transgene was assessed by Northern blot analysis of 2 μg 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 NO: 1 detected

a ^" 1 kb transcript, the predicted size of the lysozyme 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.

Protein Because rat and mouse lysozyme have very similar

molecular weights, it was not possible to resolve the proteins by SDS-PAGE.

Total levels of lysozyme (rat and mouse) were estimated by Western blot

analysis of equal amounts of protein from lung homogenates or BAL fluid from

five-week old transgenic mice and wild type littermate controls. FIG. 3 shows

a Western blot analysis of 1 μg 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 SDS-PAGE under non-reducing conditions, blotted onto a

nitrocellulose membrane and incubated with anti-human lysozyme antibody,

which detected both mouse and rat lysozyme (molecular weight of about

1 5 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 wild 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). Lysozyme 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 shown in FIG. 4, mice from transgenic line 3.5 had a 1 7.5-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 (p = 0.02), but

lower activity relative to transgenic line 3.5.

Spatial Expression of Lysozyme

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 old transgenic mice from line 3.5

(FIG 5B) and wild type 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, with more intense staining in Type II 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 counts (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.

Data are mean ± SEM. Effect of Lysozyme Overexpression on Bacterial Pathogen Clearance from the Airway

Clearance of Group B Streptococcus (GBS) . 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 wild type littermate controls were compared following an

intratracheal injection of 1 0⁶ 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 = 0.01 72 in wild type (n = 20) versus transgenic line 3.5

(n = 1 9), and p = 0.1 9 in wild type versus transgenic line 2.6 (n = 1 0). Mice

from transgenic line 3.5 had a three-fold enhancement of GBS clearance (2.1

± 0.1 x 1 0⁶ CFU/g lung tissue versus 6.8 ± 0.5 x 1 0⁶ CFU/g lung tissue in

wild type mice, p = 0.01 72). 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 10⁶ CFU/g lung tissue versus 7.1 ± 0.6

x 10⁶ in wild type mice, p = 0.1 9).

Clearance of Pseudomonas aeruginosa. Transgenic and wild type

mice received intratracheal injections with 1 0⁷ 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 iine 3.5 (n = 10) ( 1 .06 ± 0.05 x 1 0⁶ CFU/g lung tissue, compared to 3.24 ± 0.41 x 1 0⁷ in

wild type littermate controls (n = 1 0, p = 0.03). Bacterial clearance was

enhanced approximately six-fold in mice from transgenic line 2.6 (n = 1 0) (6.52

± 0.71 x 1 0⁶ 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 NO:3 or SEQ ID NO:6

or recombinant lysozyme SEQ ID NO: 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 lysozyme/SP-B fusion protein SEQ ID NO:3 or

SEQ ID NO:6 or recombinant lysozyme SEQ ID NO: 1 may be used to protect

against all bacterial strains 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, Mycoplasma pneumoniae, Legionella pneumophila.

Additionally, the invention may be used to combat gastrointestinal

infections. The lysozyme/surfactant protein-B fusion protein SEQ ID NO:3 or

SEQ ID NO: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, Campy/obacter fetus, ssp. jej^'uni, 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. Various modes of administration besides inhalation could

be used, such as injection, etc. The fusion protein SEQ ID NO:3 or SEQ ID

NO:6 or recombinant lysozyme SEQ ID NO: 1 may be administered either alone

or in combination with antibiotic therapy. Thus, the forgoing embodiments are

not to be construed as limiting the scope of this invention.

What is claimed is:

Claims

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.

1 0. The method of claim 9 wherein the composition is administered

by a method selected from the group consisting of inhalation and aerosol

installation.

1 1 . The method of claim 9 wherein the mammal is infected with

Pseudomonas.

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