JP2002530083A - Lysozyme fusion protein in infection - Google Patents

Abstract

  (57) [Summary] Methods and compositions for the prophylactic and / or therapeutic treatment of bacterial infections, especially respiratory bacterial infections. A fusion protein of the carboxy-terminal propeptide of lysozyme and surfactant protein-B (SP-B) with the preceding 10 amino acids of the mature SP-B peptide, or recombinant lysozyme alone, is contained in a pharmaceutically acceptable medium. And administered to an individual. The fusion protein or recombinant lysozyme can be selected to be delivered to a target site of infection, such as the lung or gastrointestinal tract. The methods and compositions solve problems associated with conventional antibiotic treatments, such as ineffectiveness and the growth of antibiotic-resistant strains.

Description

DETAILED DESCRIPTION OF THE INVENTION

Related Application This application is a continuation-in-part of US Patent Application Serial No. 09 / 193,877, filed November 18, 1998, and currently pending.

[0002] The United States government has a pay-as-you-go license for the present invention, and the terms and conditions of grant numbers R01-HL56285 and HL56285S awarded by the National Institutes of Health. Possesses, on a limited basis, the right to seek the patent owner to license others on reasonable terms, similar to that provided by US Pat.

[0003] The present invention relates to the prophylactic and therapeutic uses of recombinant lysozyme and lysozyme / surfactant protein-B fusion proteins in bacterial infections.

BACKGROUND OF THE INVENTION [0004] Bacterial infections remain the leading drivers of global morbidity and mortality. Antibiotics administered against bacterial infections are often safe and effective, but there is a widespread problem for all bacterial species that have become resistant to classical antibiotic treatment . Thus, in individuals infected by resistant species, administration of antibiotics is incomplete and ineffective, requiring additional treatment with the spread of resistant species. Therefore, prophylactic measures and alternative treatments that do not rely on antibiotics are desired.

[0005] Immunely compromised, suboptimal, deficient in the fully functioning immune system, such as newborn or elderly patients, or diseases involving the respiratory tract, such as cystic fibrosis. Alternatively, in some individuals, such as those with ulcerative colitis or diseases that affect the gastrointestinal tract, such as sprue, bacterial infections can have serious consequences that can lead to serious illness or even death. As an example, the production of altered mucus in cystic fibrosis patients leads to dilatation of the exocrine tract, destruction of acinar tissue, and replacement of the disrupted tissue by fibrous connective tissue. With the lungs, it leads to pneumonia and bronchitis. In these patients, even though substantial bacterial colonization in the lungs is present, less systemic dissemination of the infection indicates that systemic immunity is essentially intact, There is still a risk of lung infection. These patients often die in their teens or early twenties, ultimately due to lung infection, despite aggressive antibiotic treatment.

[0006] The respiratory and gastrointestinal organs are sites of frequent bacterial infections in healthy individuals. Normal respiratory organs have natural clearance mechanisms that help prevent bacterial colonization. These mechanisms include mucus gels that act as barriers to bacterial invasion, the driving force of the fibrils on the epithelial lining of the airways, and antimicrobial humoral factors such as the secretory immunoglobulins IgA and IgM, the proteins lactoferrin, beta-lysine and fibronectin , The secretion of complement components and the enzyme lysozyme. Among these antimicrobial factors, lysozyme is the best established antimicrobial substance for airway secretion.

[0007] Human lysozyme is a naturally occurring enzyme, known to have a bactericidal effect in vitro, and is therefore regarded as a promising means for the prevention and / or treatment of bacterial infections. Lysozyme is a small (15 kilodalton) basic protein produced in most tissues. It is secreted and present from many bodily secretions, such as mucus. In the lung, lysozyme was located in the lamellar bodies of type II alveolar epithelial cells, alveolar macrophages and bronchial serum submucosal tissue glands by immunohistological methods. About 80% of the lysozyme secreted into the respiratory tract is from the mucosal layer of the upper respiratory tract.

In vitro, lysozyme has been shown to act independently, resulting in bacterial killing. One method by which lysozyme kills bacteria is the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine in bacterial polysaccharide cell walls.
It is known that this is due to hydrolysis of a glycosidic bond between Lysozyme also kills bacteria by acting synergistically with other proteins, such as complement or antibodies, that lyse bacterial cells. Lysozyme produced by polymorphonuclear leukocytes such as neutrophils inhibits the chemotaxis of polymorphonuclear leukocytes and limits the production of oxygen free radicals after infection. This reduces the degree of inflammation, but at the same time,
Increase the phagocytosis of these cells. Lysozyme is also considered to be involved in the response to airway tissue damage.

[0009] While the in vitro antibacterial effects of lysozyme are well published, there was no method in the prior art to predict the use of these effects of lysozyme in vivo. Previous reports also suggest that sustained lysozyme administration can be harmful.

[0010] Lung surfactant, a complex mixture of phospholipids and proteins, is alveolar type II
It is synthesized and secreted by special exocrine cells, epithelial cells. Surfactant protein-B (SP-B) is one of the protein components of lung surfactant. Normal respiratory function requires pulmonary surfactant to maintain alveolar patency.

[0011] Therefore, there is a need for methods and compositions for preventing and / or treating bacterial infections, especially respiratory bacterial infections that are frequent in patients with cystic fibrosis.

SUMMARY OF THE INVENTION The present invention relates to compositions for the prophylactic or therapeutic treatment of bacterial infections in mammals. This composition comprises a lysozyme / surfactant protein-B (SP-B) fusion protein of SEQ ID NO: 3 (the carboxyl terminal SP-B propeptide and the preceding of the mature SP-B peptide of SEQ ID NO: 4). Or the lysozyme / SP-B fusion protein of SEQ ID NO: 6 (carboxyl-terminal SP-B propeptide and the mature SP-B peptide of SEQ ID NO: 4) fused to 10 amino acids Of human lysozyme of SEQ ID NO: 5 fused to the preceding 10 amino acids), or recombinant lysozyme of SEQ ID NO: 1 alone. The composition prevents and treats respiratory or gastrointestinal tract infections that are frequent in individuals suffering from cystic fibrosis, or may occur in individuals with other types of respiratory illness. . The use of recombinant lysozyme according to the present invention reduces mortality and also is a bacterium that is a major respiratory tract pathogen in patients with cystic fibrosis, such as Pseudomonas aeruginos
promotes in vivo elimination of a). Furthermore, the increased lysozyme activity in bronchoalveolar lavage fluid resulting from the administration of lysozyme is not associated with altered lung function or structure, or chronic inflammatory disease.

[0013] The present invention also relates to SEQ ID NO: 3 or SEQ ID NO: in a pharmaceutically acceptable composition.
A method for the prophylactic or therapeutic treatment of bacterial infections in mammals by administering the 6 lysozyme / SP-B fusion proteins in a dosage regimen sufficient to prevent or treat the infection. The route of administration can be parenteral (eg, by inhalation) or oral.

The present invention also includes a rat lysozyme of SEQ ID NO: 1 and a carboxyl-terminal SP-B propeptide having the ten terminal amino acids of the mature peptide of SEQ ID NO: 4, which have antibacterial activity in mammals. , SEQ ID NO: 3.

[0015] The invention further encompasses a human lysozyme of SEQ ID NO: 5 and a carboxyl-terminal SP-B propeptide having the ten terminal amino acids of the mature peptide of SEQ ID NO: 4, which have antibacterial activity in mammals. The fusion protein of SEQ ID NO: 6.

The present invention further provides an individual with the lysozyme / S of SEQ ID NO: 3 or SEQ ID NO: 6.
A method for treating a respiratory bacterial infection in an individual suffering from cystic fibrosis by administering a P-B fusion protein. The fusion protein can be administered by aerosol device and / or inhalation.

The present invention also relates to a method for preventing or treating bacterial infection in a mammal,
This method comprises combining recombinant lysozyme of SEQ ID NO: 1 in a pharmaceutically acceptable composition with a dose sufficient to prevent or treat the infection, eg, for the treatment of a bacterial infection in a patient suffering from cystic fibrosis. It is performed by administering a sufficient dose.

The present invention still further relates to a composition comprising the recombinant lysozyme of SEQ ID NO: 1 having antibacterial activity.

[0019] These and other methods and compositions are considered to be apparent in light of the following figures and detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preparation of Recombinant Lysozyme and SP-B and Fusion Protein Rat lysozyme is a 148 amino acid hydrophobic peptide of SEQ ID NO: 1. Human lysozyme of SEQ ID NO: 5 also has 148 amino acids and has 69% homology with rat lysozyme.

Surfactant protein-B (SP-B) is a hydrophobic peptide with 79 amino acids that is affinity-associated with the surfactant phospholipid in the alveolar air space. Human SP-B is synthesized by alveolar type II epithelial cells as a prepropeptide having 381 amino acids of SEQ ID NO: 2. Mature SP-B is produced by sequential cleavage of a signal peptide having 23 amino acids, an amino-terminal (N-terminal) propeptide having 177 amino acids, and a carboxyl-terminal (C-terminal) propeptide having 102 amino acids. Generated. This C-terminal propeptide may serve to maintain the size of the lamellar bodies that store SP-B and to determine the size of the intracellular surfactant pool. It is shown.

[0022] Complementary DNA (cDNA) corresponding to rat lysozyme and human SP-B was synthesized. The scheme used for this synthesis is described by Akinbi et al. In J. Biol. Chem., 1997, 27.
2: 9640-9467, which is expressly incorporated herein by reference in its entirety. Complementary DNA to rat lysozyme was generated as follows. An improved method for isolating alveolar type II epithelial cells from Dobbs et al.
g Type II cells in high yield and purity) (Am. Rev. Respir. Dis., 134: 140-14)
5,1986) from adult rat lungs. Total RNA
Is described by Chirgwin et al. (“Isolation of biologically active ribonucleic acid from ribonuclease-rich sources”).
c acid from sources enriched in ribonuclease), Biochemistry, 18: 5294-5299
PolyA ⁺ RNA was isolated from type II cells by the method of Aviv and Leder (“Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose”). of b
iologically active globin messenger RNA by chromatography on oligothymid
Natl. Acad. Sci., USA, 69: 1408-1412,1972). Single-stranded cDNA generated from isolated polyA ⁺ RNA using reverse transcriptase (Maniatis et al., “Molecular Cloning: Experimental Guidelines” (Molecular
Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, 1982), using the published sequence of the rat enzyme by Yeh et al. ("Evolution of rodent lysozyme: isolation and sequence of the rat lysozyme gene" (Evolution of rodent l).
ysozymes: Isolation and sequence of the rat lysozyme genes), Mol.Physiol.E
vol., 2: 25-75, 1993) as a template for PCR amplification of the complete coding sequence of lysozyme using oligonucleotide primers. Human SP-BcD
The isolation of NA has already been disclosed ("cDNA and deduced amino acid sequence of human pulmonary surfactant-as cDNA of human lung surfactant-associated proteolipid SPL (Phe)" by Glasser, SW et al.).
sociated proteolipid SPL (Phe)), Proc. Natl. Acad. Sci., USA, 84: 4007-4011,198
7).

The synthesized cDNA is generated for evaluation into a chimeric molecule consisting of rat lysozyme protein and the carboxyl-terminal (C-terminal) propeptide of human surfactant protein-B (SP-B), or recombinant rat. Lysozyme (SEQ ID NO: 1) alone was generated. In particular, this chimeric molecule is shown in SEQ ID NO: 4, residues 1-148 of rat lysozyme of SEQ ID NO: 1 and SEQ ID NO: 4.
2 is a fusion protein with two residues 270-381, forming the lysozyme / surfactant-B fusion protein of SEQ ID NO: 3.

Preparation of Transgenic Mice Overexpressing Lysozyme / SP-B Fusion Protein The complete coding sequence of rat lysozyme of SEQ ID NO: 1 and the preceding 10 amino acids from the C-terminus of the mature peptide of SEQ ID NO: 4 SP-B complete with C-
Three lines of transgenic mice expressing the cDNA constructs, including the terminal propeptide, were created. The coding sequence for rat lysozyme of SEQ ID NO: 1 is replaced by the preceding one of the human lung surfactant protein-B
Cloned in frame with 0 amino acids and the coding sequence for the C-terminal propeptide. FVB / N transgenic mice expressing a transgenic construct encoding the fusion protein of SEQ ID NO: 3 were engineered with Lin et al. Under the control of a 3.7 kilobase (kb) human surfactant protein-C (SP-C) promoter. (J. Biol. Chem., 1996, 271).
: 19689-19695) (cited from this publication and incorporated herein in its entirety).
. Expression of transgenic RNA in these mice was restricted to distal respiratory epithelium.

The expression of the chimeric protein of SEQ ID NO: 3 was generated by Western blot analysis known to those skilled in the art as reported by Lin et al. Antibody # R96 for detecting peptides
189 (“Structural requiremants for intracellular transport of pu”
lmonary surfactant protein B (SP-B)), Biochim.Biophys.Acta Mol.Cell.Res., 1
312: 177-185, 1996). A protein of about 29 kDa was detected in transgenic mice using this antibody, which was
Predicted by the size of the construct consisting of 5 kDa rat lysozyme and 14 kDa C-terminal propeptide and the preceding 10 amino acids. This transgenic product was detected in both lung homogenate and bronchoalveolar lavage (BAL) fluid, consistent with secretion of the chimeric protein of SEQ ID NO: 3 into the alveolar space. Constitutive expression of this SEQ ID NO: 3 chimeric protein was not associated with alteration of lung structure. This is assessed by light microscopic evaluation of lung tissue stained with hematoxylin and eosin.

Preparation of Transgenic Mice Overexpressing Lysozyme To assess the role of lysozyme in the defense of lung hosts, a transgenic mouse strain, rat lysozyme of SEQ ID NO: 1, was replaced with a 3.7 kb human surfactant protein. The distal airway epithelium was targeted in the direction of the -C (SP-C) promoter. Seven of the 21 offspring from the fertilized oocyte injection were positive for the transgene as assessed by PCR, which was confirmed by Southern blot analysis of tail DNA (shown herein). Transgenic mice were indistinguishable from litter wild-type mice in terms of body weight, lung weight, longevity and reproductive potential. Two transgenic lines (3.5 and 2.6) contained increased levels of lysozyme protein in bronchoalveolar lavage fluid.

Efficacy of lysozyme / SP-B fusion protein Five-week-old transgenic mice carrying the fusion protein of SEQ ID NO: 3 (n = 56) (treatment group) and their wild-type littermates (control) were infected 10 ⁶ strains III group B Streptococci cock Shi (Streptococci) (GBS) by intratracheal administration. Aliquots of GBS were grown overnight in Todd Hewitt broth at 37 ° C., and the bacteria were pelleted by centrifugation. The bacterial pellet was suspended in sterile phosphate buffered saline (PBS) at a concentration of 10 ⁷ / ml. 100 microliters of this bacterial suspension (10 ⁶ bacteria) were collected into a tuberculin syringe equipped with a 27 gauge needle in preparation for intratracheal instillation. All injections were performed in a sterile environment.

Mice aged 5-6 weeks, kept in a clean room, were used for bacterial exclusion tests.
Mice were anesthetized with a mixture of nitric oxide and oxygen. The trachea was exposed through a central incision and an incision through the thyroid. 100 microliters of the bacterial suspension
Using a 7 gauge needle, it was introduced into the trachea just below the cricoid cartilage. The bisected thyroid muscles were juxtaposed in the middle and the skin incision was closed with surgical adhesive by bringing the two edges close together. Animals were housed for 6 or 24 hours and then sacrificed.

At 6 or 24 hours post-infection, lungs were collected from treated and control mice, weighed, homogenized in PBS, and then plated on BAP to form GBS colonies. Was evaluated. These culture plates are
Incubate at 37 ° C. for approximately 16-18 hours (overnight). Colony forming units per gram of lung tissue (CFU / g) were evaluated by manual counting of bacterial colonies.

As shown in FIG. 1, transgenic mice carrying the chimeric protein of SEQ ID NO: 3 have significantly less CFU / lung tissue at both 6 and 24 hours after GBS infection. g. The number of CFUs in most transgenic mice could even be lower than the number of bacteria administered. As shown in FIG. 1, transgenic mice were 6 hours and 24 hours after infection.
At both time points, there was considerable accelerated GBS clearance from the lungs. At 6 hours post-infection, BAP inoculated with lung tissue from transgenic (treated) mice had 1.99 ± 1.4 × 10 ⁴ CFU / g tissue, whereas BAP inoculated with lung tissue from wild-type (control) mice was 25.49.
Had ± 12.43 × 10 ⁴ CFU / g tissue (p <0.006). At 24 hours post-infection, BAP inoculated with lung tissue from transgenic (treated) mice had 9.9 ± 6.43 × 10 ⁴ CFU / g of tissue, whereas BAP inoculated with lung tissue from wild type (control) mice was 67.29.
Had ± 34.2 × 10 ⁴ CFU / g tissue (p <0.04).

[0031] These results suggest that expression of the lysozyme / surfactant protein-B fusion protein of SEQ ID NO: 3 in the airways of transgenic mice promotes bacterial clearance from the airways. This result indicates that in mice harboring the lysozyme / surfactant protein-B fusion protein of SEQ ID NO: 3, bacterial growth was suppressed at 6 hours post-infection and bacterial elimination was enhanced at 24 hours post-infection. It indicates that it was done. Transgenic mice expressing the lysozyme / surfactant protein-B fusion protein of SEQ ID NO: 3 in the distal airways were more than 12-fold more efficient at eliminating bacteria in the airways compared to their wild-type littermates. The efficacy produced by this lysozyme is particularly pronounced in wild-type mice, since bacterial elimination in the respiratory tract is originally very efficient.

The transformed mice of the two other strains expressed the lysozyme / surfactant protein-B chimeric protein of SEQ ID NO: 3, albeit at low levels. Bacterial elimination in these lines was considerably reduced, but was still significantly higher when compared to wild-type control mice. Lysozyme enzyme activity was increased by 40% in bronchoalveolar lavage fluid of transgenic lines expressing the highest level of lysozyme / SP-B fusion protein of SEQ ID NO: 3 (compared to wild-type littermates). In this strain, the antibacterial effect was conferred by the SP-B C-terminal propeptide with the preceding amino acid from the mature peptide of SEQ ID NO: 4, since bacterial elimination was increased 12-fold, or It can be said that this was the result of the effect of the combination of the lysozyme component of SEQ ID NO: 3 and SP-B; otherwise, this effect was due to the increased lysozyme activity of SEQ ID NO: 1. it can.

Analysis of Lysozyme Transgene Expression RNA : Referring to FIGS. 2A and 2B, the expression of the lysozyme transgene was determined by total cell RN isolated from lung tissue of 5 week old transgenic mice (line 3.5).
A2 μg was evaluated by Northern blotting analysis. These are shown in lanes 4 and 5 of FIG. 2A and lanes 3 and 4 of FIG. 2B. Control wild-type littermates were from lanes 1-3 in FIG. 2A and lanes 1 and 2 in FIG. 2B.
Is shown in 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 larger than the two transcripts. Since rat lysozyme was cross-hybridized with mouse cDNA, rat lysozyme was detected by the mouse cDNA probe.

The rat lysozyme-specific cDNA probe of SEQ ID NO: 1 detected a １1 kb copy, the expected size of the lysozyme transgene (FIG. 2B).
). When the same RNA sample was probed using mouse lysozyme cDNA (
FIG. 2A), larger endogenous mouse lysozyme mRNA and rat lysozyme m
Both RNAs were detected. This mouse lysozyme mRNA was used as an internal control for sample normalization.

Proteins : Rat lysozyme and mouse lysozyme could not be resolved by SDS-PAGE because of their very similar molecular weights. Total levels of lysozyme (rats and mice) were estimated by Western blot analysis of proteins from equivalent lung homogenates or BAL fluid from 5 week old transgenic mice and wild type littermates. 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). The protein was converted to SDS under non-reducing conditions.
-Fractionated by PAGE, blotted on nitrocellulose membrane and then incubated with anti-human lysozyme antibody. This detected both mouse and rat lysozyme (molecular weight about 15 kD).

As shown in FIG. 3, mice from transgenic line 3.5 (lanes 4-6) were treated with wild-type littermate mice (lanes 4-6) in both lung homogenate and BAL fluid.
It had a 4-fold increase in lysozyme protein levels compared to lanes 1-3). Mice from transgenic line 2.6 had 2-fold increased lysozyme protein levels compared to control wild-type littermates (not shown).

Lysozyme enzyme activity : Referring to FIG. 4, the lysozyme enzyme activity was measured using strain 3.5.
Turbidity analysis on BAL fluid from 5 weeks old transgenic mice from (animal number (n) = 4) and strain 2.6 (n = 4) and from control wild-type littermates (n = 7) It was evaluated by the method. Ten nonagrams of BAL fluid protein were suspended at an optical density (OD) of 1 at 450 nm (Micrococcus lysodicus).
ccus lysodeikticus). After incubation at 37 ° C. for 1 hour, the O.D. D. Was measured. A standard curve was prepared using purified chicken egg white lysozyme.

As shown in FIG. 4, mice from transgenic line 3.5
It had 17.5-fold increased lysozyme activity compared to wild-type mice (550 units / ngBAL versus 31 units / ngBAL protein in wild-type mice).
protein). The data are mean ± standard error of the mean (SEM), p = <0.0001 for wild-type versus transgenic line 3.5, and for wild-type versus transgenic line 2.6. , P = <0.0001, and for the transgenic line 3.5 vs. the transformed line 2.6, p = <0.000
8 As expected from Western blot analysis, mice from transgenic line 2.6 had increased lysozyme enzyme activity (205 units / ng BAL protein) compared to wild type control (p = 0.02
), The activity was lower than that of the transgenic line 3.5.

Spatial Expression of Lysozyme With reference to FIGS. 5A and 5B, the subcellular localization of the lysozyme protein in transgenic and control mice was obtained. 5 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 that detects lysozyme in both rats and mice and photographed at 80x magnification.

The overall structure of the lung sections of the transgenic mice did not differ from the wild-type littermates. There was no evidence of pulmonary edema or vascular congestion, and no inflammatory cells were detected in lung sections from uninfected transgenic mice.

Lysozyme was detected in type II cells in wild-type and transgenic mice and stained more strongly in type II cells from transgenic mice. In addition, transgenic mice expressed in bronchial 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 bronchial cells and type II cells.

Effect of Lysozyme Overexpression on Lung Structure Referring to FIGS. 6A and 6B, uninfected 5-week-old transgenic mice (
Overall histological characteristics of paraffin-embedded, hematoxylin / eosin-stained sections of lungs from FIG. 6A) and control wild-type littermates (FIG. 6B) were compared at 40 × magnification. Total and differential cell counts in BAL fluid (Cytospin® specimen stained with Diff-Quick®) were performed.

As shown in FIG. 7, there was no particular difference in the total cell number or distribution of cell types collected from BAL fluid when comparing wild-type littermates and transgenic mice. In this case, p = 0.98 for the total cell number, p = 0.74 for macrophages, and p = 0.95 for lymphocytes. Data are means ± SEM.

Effect of Lysozyme Overexpression on Bacterial Pathogen Elimination from the Airways To determine whether increased lysozyme levels in the excluded airways of Group B Streptococcus (GBS) increased bacterial clearance from the lungs,
The number of bacteria in quantitative cultures of lung homogenates from transgenic mice and nontransgenic littermates controls were compared after intratracheal injection of GBS 10 ⁶ colony forming units (CFU). All animals survived to sacrifice at 6 hours post-infection.

The results shown in FIG. 8 are expressed as CFU / g lung tissue ± SEM, and for wild type (n = 20) vs. transgenic line 3.5 (n = 19), p = 0
. 0172, and p = 0.19 for the wild-type versus transgenic line 2.6 (n = 10). Mice from transgenic line 3.5
Had a 3-fold increase in GBS rejection [2.1 ± 0.1 × 10 ⁶ CFU / g
Lung tissue vs. 6.8 ± 0.5 × 10 ⁶ CFU / g lung tissue lung tissue, p = 0.0
172]. Systemic dissemination of infection was less in transgenic mice as assessed by GBS growth on plates inoculated with spleen homogenate (2
7% vs. 60%, p = 0.04). GBS elimination from the lung was enhanced to less than 2-fold in mice from transgenic line 2.6 (4.2 ± 0.8).
× 10 ⁶ CFU / g lung tissue vs. 7.1 ± 0.6 × 10 ⁶ CFU / g lung tissue lung tissue, p = 0.19).

Elimination of Pseudomonas aeruginosa : Transgenic and wild-type mice were subjected to intratracheal injection with 10 ⁷ CFU of Pseudomonas aeruginosa. All transgenic mice survived to sacrifice at 24 hours post-infection. In contrast, 20% of infected wild-type mice died.

FIG. 9 shows CFU / g lung tissue ± SEM. Bacterial elimination was enhanced approximately 30-fold in mice from the transgenic line 3.5 (n = 10) (3.24 ± 0.41 × 10 ⁷ CFU / g lung tissue in wild-type littermate controls (n = 10 , P = 0.05
1.06 ± 0.05 × 10 ⁶ CFU / g lung tissue). Bacterial exclusion
Mice from transgenic line 2.6 (n = 10) were stimulated approximately 6-fold (6
. 52 ± 0.71 × 10 ⁶ CFU / g lung tissue, p = 0.05). No systemic bacterial dissemination was found in the surviving mice at 24 hours post-infection.

The lysozyme / SP-B fusion protein of SEQ ID NO: 3 or SEQ ID NO: 6, or the recombinant lysozyme of SEQ ID NO: 1 according to the present invention is used for the treatment and / or reduction of bacterial colonization in the respiratory tract can do. The use of the latter is particularly beneficial for treating individuals suffering from cystic fibrosis. This is due to chronic bacterial colonization of the main airways, especially Pseudomonas aerugin
Chronic bacterial colonization by osa) is a major contributor to the morbidity and mortality experienced by cystic fibrosis patients, along with the resulting debilitation.

[0049] Cystic fibrosis is a systemic disease in which mucus secretion is altered so that sticky mucus is produced. The production of altered mucus leads to dilatation of the exocrine tract, destruction of acinar tissue, and replacement of the disrupted tissue by fibrous connective tissue. The involvement of the lungs leads to pneumonia and bronchitis. These patients are often young, despite aggressive antibiotic treatment, with Pseudomonas aeruginos
succumb to the final lung infection due to a). Thus, the in vivo bacterial killing activity of lysozyme or lysozyme / surfactant protein-B fusion protein suppresses bacterial colonization of the respiratory tract of patients with cystic fibrosis, regardless of what respiratory function they exhibit. Provide a powerful therapeutic strategy for:

[0050] Prophylactic and therapeutic treatment with the methods and compositions according to the present invention comprises comprehensive prevention,
Obviously, it can cover a range of reduced bacterial load, reduced or eliminated severity of bacterial infection. Lysozyme / SP-B of SEQ ID NO: 3 or SEQ ID NO: 6
The fusion protein, or the recombinant lysozyme of SEQ ID NO: 1, can be used for protection against all bacterial species that colonize the respiratory tract, such as the following bacterial species: Staphylococcus aureus, Streptococcus species, Streptococcus pneumoniae
(Streptococcus pneumoniae), Neisseria meningi
tidis), Neisseria gonorrhoeae, Klebsiella species
(Klebsiellae species), Proteus species, Pseudomonas
Cepacia (Pseudomonas cepacia), Haemophyl influenzae (Haemophyl
us influenzae), Bordetella pertussis, Mycoplasma pneumoniae, Legionella pneumoniae
(Legionella pneumophia).

Furthermore, the present invention can be used in the fight against gastrointestinal infections. The lysozyme / surfactant protein-B fusion protein of SEQ ID NO: 3 or SEQ ID NO: 6 is a bacterial species that colonizes the gastrointestinal tract, such as Salmonella sp.
almonellae species), Sigellae species, Escherichia coli (E
scherichia coli and Vibrio species, Yersinia enterocolitica, Campylobacter phytas ssp.Jejuni, and Helicobacter pylori.
For the treatment or prevention of infections such as P. bacterium pylori), it can be administered by targeted intestinal routes to the gastrointestinal tract. Lysozyme / SP-B can be formulated for oral administration in solid or liquid form, such as capsules, tablets, syrups, and the like.

[0052] Other variations or embodiments of the invention will also be deemed apparent to those skilled in the art from the above description. As an example, besides surfactant protein-B, saposin (S
aposin) -A, saposin-B, saposin-C, saposin-D, NK lysine (NKly
Other fusion proteins can be produced, such as structurally related members of the saposin protein family, such as sin), a pore-forming peptide of amoebapore A. Various administration methods other than inhalation, such as injection, can also be used. The fusion protein of SEQ ID NO: 3 or SEQ ID NO: 6, or the recombinant lysozyme of SEQ ID NO: 1, can be administered alone or in combination with antibiotic therapy. Therefore, the above embodiments are not intended to limit the scope of the present invention.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Histogram showing bacterial elimination from lungs of transgenic mice (lysozyme / surfactant protein-B fusion protein) and control mice.

FIG. 2A is a photograph showing the expression of recombinant lysozyme of SEQ ID NO: 1 in a transgenic mouse strain using mouse lysozyme-complementary DNA (cDNA) as a probe, and FIG. 1 is a photograph showing the expression of recombinant lysozyme of SEQ ID NO: 1 in the same transgenic mouse strain.

FIG. 3 is a photograph showing analysis of lysozyme protein expression.

FIG. 4. Histogram of lysozyme activity in bronchoalveolar lavage (BAL) fluid.

FIG. 5A is a photograph showing the localization of lysozyme cells in wild-type mice.
B is a photograph showing lysozyme cell localization in transgenic mice.

FIG. 6A is a photograph showing the lung structure of a transgenic mouse that overproduces lysozyme, and FIG. 6B is a photograph showing the lung structure of a wild-type mouse.

FIG. 7 is a histogram showing the cell composition of BAL fluid.

FIG. 8. Group B Streptococcus (Strep) from lung at 6 hours post-infection.
tococcus) (GBS) exclusion histogram.

FIG. 9. Pseudomonas aeruginosa (P from lung at 24 hours post-infection)
Histogram showing the exclusion of S. aeruginosa).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 C12N 9/42 C07K 19/00 15/00 ZNAA C12N 9/42 A61K 37/54 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, A, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZWF terms (reference) 4B024 AA01 BA07 BA80 CA01 GA11 HA01 4B050 CC03 CC05 DD11 GG06 LL01 4C076 AA24 AA93 BB27 CC31 4C084 AA01 AA02 AA03 BA41 BA44 CA53 DA41 DC02 MA13 MA56 NA14 ZA59 ZA66 ZB35 4H045 AA10 AA30 BA41 CA40 DA89 EA29 FA74

Claims (12)

[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 the recombinant lysozyme of SEQ ID NO: 1. 3. A composition according to claim 1 for the prophylactic or therapeutic treatment of a bacterial infection in a mammal. 4. A composition according to claim 1 for prophylactic or therapeutic treatment of a respiratory bacterial infection. 5. The composition according to claim 4, wherein said respiratory infection is an infection in a mammal suffering from cystic fibrosis. 6. The composition according to claim 1, for the prophylactic or therapeutic treatment of a gastrointestinal tract infection in a mammal. 7. A fusion protein having antibacterial activity in a mammal, selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 6. 8. Recombinant lysozyme of SEQ ID NO: 1 having antibacterial activity in mammals. 9. A method for the prophylactic or therapeutic treatment of a bacterial infection in a mammal, comprising the recombinant lysozyme of SEQ ID NO: 1 in a pharmaceutically acceptable carrier and the sequence in a pharmaceutically acceptable carrier. Administering a composition selected from the group consisting of a lysozyme / surfactant protein-B fusion protein selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 6 to the mammal at a dose sufficient to prevent or treat the infection. The above method, comprising: 10. The method according to claim 9, wherein the composition is administered by a method selected from the group consisting of inhalation and aerosol devices. 11. The method according to claim 9, wherein the mammal is infected with Pseudomonas. 12. The method of claim 9, wherein the mammal has cystic fibrosis.