Abstract:
A method and composition for prophylaxis and/or treatment of bacterial infections, particularly bacterial respiratory 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 is administered in a pharmaceutically acceptable medium to an individual. The fusion protein 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:
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 award by the National Institutes of Health. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to prophylactic and therapeutic uses of 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 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. 
     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. 
     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. 
     Human lysozyme is an 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 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. 
     Surfactant protein-B (SP-B) is one of the protein components of pulmonary surfactant. Pulmonary surfactant, a complex mixture of phospholipids and proteins, is synthesized and secreted by alveolar type II epithelial cells, a specialized exocrine cell. Normal respiratory function requires pulmonary surfactant for maintenance of alveolar patency. 
     A method and composition for the prophylaxis and/or 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 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). The composition prevents or treats a respiratory infection such as occurs frequently in individuals with cystic fibrosis, or a gastrointestinal infection. 
     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 rat 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 Iysozyme/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. 
    
    
     These and other methods and compositions will be apparent in light of the following FIGURE and detailed description. 
     BRIEF DESCRIPTION OF THE FIGURE 
     FIG. 1 is a graph showing bacterial clearance from lungs of transgenic (lysozyme/surfactant protein-B fusion protein) and control mice. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Preparation of Fusion Protein 
     Rat lysozyme is a hydrophobic peptide of 148 amino acids SEQ ID NO:1. Human lysozyme 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 of 79 amino acids that avidly associates with surfactant phospholipids in the alveolar airspace. Human SP-B is synthesized by alveolar type II epithelial cell 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 177 amino acids, and a carboxyl 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. 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 improved method for isolating Type II cells in high yield and purity, Am. Rev. Respir. Dis. 134:140-145, 1986). 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 18:5294-5299, 1979) and polyA  +   RNA by the method of Aviv and Leder, Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose, Proc. Nati. Acad. Sci. USA 69:1408-1412, 1972. Single stranded cDNA, generated from isolated polyA30 RNA with reverse transcriptase (Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 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. Evolution of rodent lysozymes: Isolation and sequence of the rat lysozyme genes, Mol. Physiol. Evol. 2:25-75, 1993. 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 84:4007-4011, 1987). 
     The synthesized cDNAs were 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). Specifically, this chimeric molecule was a fusion protein of residues 1-148 of rat lysozyme SEQ ID NO:1 and residues 270-381 of SEQ ID NO:2, shown in SEQ ID NO:4, forming a Iysozyme/surfactant-B fusion protein SEQ ID NO:3. 
     Preparation of Transgenic Mice 
     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 10 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. 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 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. 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 by using this antibody, as would be predicted by the size of the construct of 15 kDa rat lysozyme and 14 kDa C-terminal propeptide and preceding 10 amino acids SEQ ID NO:3. The transgene product was detected in both lung homogenates and in bronchoalveolar lavage 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. 
     Efficacy of 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 10 6  strain IlIl 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 7  /ml. One hundred microliters of bacterial suspension (10 6  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. Lungs and spleen tissues were harvested, weighed and homogenized in PBS. Dilutions of homogenized lung tissues and spleen were then plated on BAP. Plates were incubated overnight at 37° C. 
     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 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 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±1.4×10 4  CFU/g of tissue, while BAP inoculated with lung tissue from wild type (control) mice had 25.49±12.43×10 4  CFU/g (p&lt;0.006). At 24 h post-infection, BAP inoculated with lung tissue from transgenic (treated) mice had 9.9±6.43×10 4  CFU/g tissue, while BAP inoculated with lung tissue from wild type (control) mice had 67.29±34.2×10 4  CFU/g tissue (p&lt;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. 
     The lysozyme/SP-B fusion protein SEQ ID NO:3 or SEQ ID NO:6 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 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 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 Iysozyme/SP-B fusion protein SEQ ID NO:3 or SEQ ID NO:6 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, Campylobacter fetus, ssp. jejune, and Helicobacter pylon. 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 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. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- &lt;160&gt; NUMBER OF SEQ ID NOS: 6- &lt;210&gt; SEQ ID NO 1&lt;211&gt; LENGTH: 148&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: RAT- &lt;400&gt; SEQUENCE: 1- Met Lys Ala Leu Leu Val Leu Gly Phe Leu Le - #u Leu Ser Ala Ser Val#                15- Gln Ala Lys Ile Tyr Glu Arg Cys Gln Phe Al - #a Arg Thr Leu Lys Arg#            30- Asn Gly Met Ser Gly Tyr Tyr Gly Val Ser Le - #u Ala Asp Trp Val Cys#        45- Leu Ala Gln His Glu Ser Asn Tyr Asn Thr Gl - #n Ala Arg Asn Tyr Asn#    60- Pro Gly Asp Gln Ser Thr Asp Tyr Gly Ile Ph - #e Gln Ile Asn Ser Arg#80- Tyr Trp Cys Asn Asp Gly Lys Thr Pro Arg Al - #a Lys Asn Ala Cys Gly#                95- Ile Pro Cys Ser Ala Leu Leu Gln Asp Asp Il - #e Thr Gln Ala Ile Gln#           110- Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gl - #y Ile Arg Ala Trp Val#       125- Ala Trp Gln Arg His Cys Lys Asn Arg Asp Le - #u Ser Gly Tyr Ile Arg#   140- Asn Cys Gly Val145- &lt;210&gt; SEQ ID NO 2&lt;211&gt; LENGTH: 381&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: HUMAN&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: PROPEP&lt;222&gt; LOCATION: (0)...(0)- &lt;400&gt; SEQUENCE: 2- Met Ala Glu Ser His Leu Leu Gln Trp Leu Le - #u Leu Leu Leu Pro Thr#                15- Leu Cys Gly Pro Gly Thr Ala Ala Trp Thr Th - #r Ser Ser Leu Ala Cys#            30- Ala Gln Gly Pro Glu Phe Trp Cys Gln Ser Le - #u Glu Gln Ala Leu Gln#        45- Cys Arg Ala Leu Gly His Cys Leu Gln Glu Va - #l Trp Gly His Val Gly#    60- Ala Asp Asp Leu Cys Gln Glu Cys Glu Asp Il - #e Val His Ile Leu Asn#80- Lys Met Ala Lys Glu Ala Ile Phe Gln Asp Th - #r Met Arg Lys Phe Leu#                95- Glu Gln Glu Cys Asn Val Leu Pro Leu Lys Le - #u Leu Met Pro Gln Cys#           110- Asn Gln Val Leu Asp Asp Tyr Phe Pro Leu Va - #l Ile Asp Tyr Phe Gln#       125- Asn Gln Ile Asp Ser Asn Gly Ile Cys Met Hi - #s Leu Gly Leu Cys Lys#   140- Ser Arg Gln Pro Glu Pro Glu Gln Glu Pro Gl - #y Met Ser Asp Pro Leu145                 1 - #50                 1 - #55                 1 -#60- Pro Lys Pro Leu Arg Asp Pro Leu Pro Asp Pr - #o Leu Leu Asp Lys Leu#               175- Val Leu Pro Val Leu Pro Gly Ala Leu Gln Al - #a Arg Pro Gly Pro His#           190- Thr Gln Asp Leu Ser Glu Gln Gln Phe Pro Il - #e Pro Leu Pro Tyr Cys#       205- Trp Leu Cys Arg Ala Leu Ile Lys Arg Ile Gl - #n Ala Met Ile Pro Lys#   220- Gly Ala Leu Arg Val Ala Val Ala Gln Val Cy - #s Arg Val Val Pro Leu225                 2 - #30                 2 - #35                 2 -#40- Val Ala Gly Gly Ile Cys Gln Cys Leu Ala Gl - #u Arg Tyr Ser Val Ile#               255- Leu Leu Asp Thr Leu Leu Gly Arg Met Leu Pr - #o Gln Leu Val Cys Arg#           270- Leu Val Leu Arg Cys Ser Met Asp Asp Ser Al - #a Gly Pro Arg Ser Pro#       285- Thr Gly Glu Trp Leu Pro Arg Asp Ser Glu Cy - #s His Leu Cys Met Ser#   300- Val Thr Thr Gln Ala Gly Asn Ser Ser Glu Gl - #n Ala Ile Pro Gln Ala305                 3 - #10                 3 - #15                 3 -#20- Met Leu Gln Ala Cys Val Gly Ser Trp Leu As - #p Arg Glu Lys Cys Lys#               335- Gln Phe Val Glu Gln His Thr Pro Gln Leu Le - #u Thr Leu Val Pro Arg#           350- Gly Trp Asp Ala His Thr Thr Cys Gln Ala Le - #u Gly Val Cys Gly Thr#       365- Met Ser Ser Pro Leu Gln Cys Ile His Ser Pr - #o Asp Leu#   380- &lt;210&gt; SEQ ID NO 3&lt;211&gt; LENGTH: 260&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Rat and Human&lt;220&gt; FEATURE:&lt;223&gt; OTHER INFORMATION: Chimeric Protein- &lt;400&gt; SEQUENCE: 3- Met Lys Ala Leu Leu Val Leu Gly Phe Leu Le - #u Leu Ser Ala Ser Val#                15- Gln Ala Lys Ile Tyr Glu Arg Cys Gln Phe Al - #a Arg Thr Leu Lys Arg#            30- Asn Gly Met Ser Gly Tyr Tyr Gly Val Ser Le - #u Ala Asp Trp Val Cys#        45- Leu Ala Gln His Glu Ser Asn Tyr Asn Thr Gl - #n Ala Arg Asn Tyr Asn#    60- Pro Gly Asp Gln Ser Thr Asp Tyr Gly Ile Ph - #e Gln Ile Asn Ser Arg#80- Tyr Trp Cys Asn Asp Gly Lys Thr Pro Arg Al - #a Lys Asn Ala Cys Gly#                95- Ile Pro Cys Ser Ala Leu Leu Gln Asp Asp Il - #e Thr Gln Ala Ile Gln#           110- Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gl - #y Ile Arg Ala Trp Val#       125- Ala Trp Gln Arg His Cys Lys Asn Arg Asp Le - #u Ser Gly Tyr Ile Arg#   140- Asn Cys Gly Val Val Cys Arg Leu Val Leu Ar - #g Cys Ser Met Asp Asp145                 1 - #50                 1 - #55                 1 -#60- Ser Ala Gly Pro Arg Ser Pro Thr Gly Glu Tr - #p Leu Pro Arg Asp Ser#               175- Glu Cys His Leu Cys Met Ser Val Thr Thr Gl - #n Ala Gly Asn Ser Ser#           190- Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Al - #a Cys Val Gly Ser Trp#       205- Leu Asp Arg Glu Lys Cys Lys Gln Phe Val Gl - #u Gln His Thr Pro Gln#   220- Leu Leu Thr Leu Val Pro Arg Gly Trp Asp Al - #a His Thr Thr Cys Gln225                 2 - #30                 2 - #35                 2 -#40- Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pr - #o Leu Gln Cys Ile His#               255- Ser Pro Asp Leu       260- &lt;210&gt; SEQ ID NO 4&lt;211&gt; LENGTH: 112&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Human&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: PROPEP&lt;222&gt; LOCATION: (0)...(0)#10 amino acids ofATION: C-terminal propeptide + mature SP-B- &lt;400&gt; SEQUENCE: 4- Val Cys Arg Leu Val Leu Arg Cys Ser Met As - #p Asp Ser Ala Gly Pro#                15- Arg Ser Pro Thr Gly Glu Trp Leu Pro Arg As - #p Ser Glu Cys His Leu#            30- Cys Met Ser Val Thr Thr Gln Ala Gly Asn Se - #r Ser Glu Gln Ala Ile#        45- Pro Gln Ala Met Leu Gln Ala Cys Val Gly Se - #r Trp Leu Asp Arg Glu#    60- Lys Cys Lys Gln Phe Val Glu Gln His Thr Pr - #o Gln Leu Leu Thr Leu#80- Val Pro Arg Gly Trp Asp Ala His Thr Thr Cy - #s Gln Ala Leu Gly Val#                95- Cys Gly Thr Met Ser Ser Pro Leu Gln Cys Il - #e His Ser Pro Asp Leu#           110- &lt;210&gt; SEQ ID NO 5&lt;211&gt; LENGTH: 148&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Human and Human&lt;220&gt; FEATURE:- &lt;400&gt; SEQUENCE: 5- Met Lys Ala Leu Ile Val Leu Gly Leu Ala Le - #u Leu Ser Val Thr Val#                15- Gln Gly Lys Val Phe Gly Arg Cys Glu Leu Al - #a Arg Thr Leu Lys Arg#            30- Leu Gly Met Asp Gly Tyr Arg Gly Ile Ser Le - #u Ala Asn Trp Met Cys#        45- Leu Ala Lys Trp Glu Ser Gly Tyr Asn Thr Ar - #g Ala Thr Asn Tyr Asn#    60- Ala Gly Asp Arg Ser Thr Asp Tyr Gly Ile Ph - #e Gln Ile Asn Ser Arg#80- Tyr Trp Cys Asn Asp Gly Lys Thr Pro Gly Al - #a Val Asn Ala Cys His#                95- Leu Ser Cys Ser Ala Leu Leu Gln Asp Asn Il - #e Ala Asp Ala Ala Ala#           110- Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gl - #y Val Arg Ala Trp Ala#       125- Ala Trp Arg Asn Arg Cys Gln Asp Arg Asp Va - #l Arg Gln Tyr Val Gln#   140- Gly Cys Gly Val145- &lt;210&gt; SEQ ID NO 6&lt;211&gt; LENGTH: 260&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Human and Human&lt;220&gt; FEATURE:&lt;223&gt; OTHER INFORMATION: Chimeric Protein- &lt;400&gt; SEQUENCE: 6- Met Lys Ala Leu Ile Val Leu Gly Leu Ala Le - #u Leu Ser Val Thr Val#                15- Gln Gly Lys Val Phe Gly Arg Cys Glu Leu Al - #a Arg Thr Leu Lys Arg#            30- Leu Gly Met Asp Gly Tyr Arg Gly Ile Ser Le - #u Ala Asn Trp Met Cys#        45- Leu Ala Lys Trp Glu Ser Gly Tyr Asn Thr Ar - #g Ala Thr Asn Tyr Asn#    60- Ala Gly Asp Arg Ser Thr Asp Tyr Gly Ile Ph - #e Gln Ile Asn Ser Arg#80- Tyr Trp Cys Asn Asp Gly Lys Thr Pro Gly Al - #a Val Asn Ala Cys His#                95- Leu Ser Cys Ser Ala Leu Leu Gln Asp Asn Il - #e Ala Asp Ala Ala Ala#           110- Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gl - #y Val Arg Ala Trp Ala#       125- Ala Trp Arg Asn Arg Cys Gln Asp Arg Asp Va - #l Arg Gln Tyr Val Gln#   140- Gly Cys Gly Val Val Cys Arg Leu Val Leu Ar - #g Cys Ser Met Asp Asp145                 1 - #50                 1 - #55                 1 -#60- Ser Ala Gly Pro Arg Ser Pro Thr Gly Glu Tr - #p Leu Pro Arg Asp Ser#               175- Glu Cys His Leu Cys Met Ser Val Thr Thr Gl - #n Ala Gly Asn Ser Ser#           190- Glu Gln Ala Ile Pro Gln Ala Met Leu Gln Al - #a Cys Val Gly Ser Trp#       205- Leu Asp Arg Glu Lys Cys Lys Gln Phe Val Gl - #u Gln His Thr Pro Gln#   220- Leu Leu Thr Leu Val Pro Arg Gly Trp Asp Al - #a His Thr Thr Cys Gln225                 2 - #30                 2 - #35                 2 -#40- Ala Leu Gly Val Cys Gly Thr Met Ser Ser Pr - #o Leu Gln Cys Ile His#               255- Ser Pro Asp Leu       260__________________________________________________________________________