MXPA94006245A - Antibacterial composition - Google Patents

Abstract

The present invention relates to the use of alpha-lactalbumin in the preparation of preparations to be used in prophylactic therapeutic treatment and / or for use in the diagnosis of infections, preferably of the respiratory tract caused by bacteria, in particular S. pneumoniae and / or H. influenz

Description

"ANTIBACTERIAL COMPOSITION" Inventors and Applicants: HEMANT SABHAR AL, Swedish, domiciled in Bjórn Járnsidas Grándidas 12, S-224 27 Lund, Sweden and CATHARINA SVANBORG, Swedish, domiciled in Arkivgatan 4, S-223 59 Lund, Sweden.

SUMMARY OF THE INVENTION The present invention relates to the use of alpha-lactalbumin in the preparation of preparations to be used in prophylactic therapeutic treatment and / or for use in the diagnosis of infections, preferably of the respiratory tract caused by bacteria, in particular __ > . pneumoniae and / or H. influenzae.

DESCRIPTION TECHNICAL FIELD The present invention relates to a novel antibacterial protein and to compositions in the form of pharmaceutical compositions, human food compositions and animal fodders comprising the protein to be used in the therapeutic and / or prophylactic treatment of infections caused by bacteria, in particular Streptococcus pneumoniae and / or Haemophilus influenzae, as well as a method for diagnosing infections caused by the bacteria. The object of the present invention is to obtain a protein and compositions containing the protein for the prophylactic and / or therapeutic treatment of infections caused by bacteria, in particular Streptococcus pneumoniae and Haemophilus influenzae in the upper airways, ear-nose infections and throat, but also in the lower airways, for example, the lungs preventing adhesion and / or causing a bactericidal effect on these bacteria. Another object is to diagnose infections caused by these bacteria.

BACKGROUND OF THE INVENTION Natural antimicrobial compounds exist in secreted form, as well as in cells of immune and non-immune origin. Human milk has been used as a source for the purification of such compounds. These previously known compounds include antibodies specific for the surface structure of the microorganism, casein, lysozyme and oligosaccharides. The mechanism of action differs between groups of antimicrobial molecules. Antibodies and receptor analogues prevent the adherence of the microorganism to mucosal surfaces. Lysozyme attacks the cell wall, etcetera. The term bacterial adherence means the binding of the bacterium to the surface of the mucosa. This mechanical association is an element for the organism to resist elimination by body fluids to establish a population at the site where important receptors are expressed. In most cases, where the binding mechanism has been identified is a specific process. Bacterial ligands, commonly called adhesins, bind to host receptors. For Gram-negative bacteria, adhesins are commonly associated with villi or fibrils, rigid surface organs that help the bacterium reach the appropriate receptor on the complex cell surface. The fibrils function as lectins, that is, they show specificity for the receptor epitopes provided by the oligosaccharide sequences in glyco-conjugates of the host (13). For Gram-positive bacteria, on the other hand, adhesins are not expressed as an organelle on the surface, but are bound to the components of the cell wall and lipoteichoic acids (21,22). The receptor epitopes for Gram-positive bacteria may consist of oligosaccharide sequences, but may also be provided by the peptides, for example, in connective tissue proteins (10). The functional consequences of adherence depend on the virulence of the bacterial strain, and on the shape of the receptor. The ligand-receptor interaction of the associated cell facilitates colonization and attacks the tissue (8). When the receptor molecule is secreted it will occupy the adhesins and inhibit competitively bound to the receptor bound to the corresponding cell. Human milk is a rich source of such competition-soluble receptor molecules. The ability of specific antibodies to inhibit binding is well established. This was first demonstrated for Vibrio cholera and oral streptococci. Anti-adhesive antibodies can act in either of two ways: 1) antibodies to the binding sites of the adhesin receptor competitively inhibit the interaction with the receptor or 2) antibodies to the bacterial surface molecules, which are not directly involved in the adhesion, can agglutinate the bacteria and therefore reduce the number of organisms available for the link. In any of the above cases, the anti-adhesive activity of the antibody is attributed to the specificity of the co-antigen site. Recently, an alternative mechanism of interaction between secretory IgA and E. coli was identified based on lectin-carbohydrate interactions. Human milk drastically inhibits the binding of Streptococcus pneumoniae and Hemophilus influenzae to nasopharyngeal epithelial cells. It contains antibodies against numerous surface antigens on these organics, for example, phosphoryl choline and polysaccharides of the capsule S. pneumoniae, lipopolysaccharide and proteins of the outer membrane of H. influenzae. Accordingly, some of the anti-adhesive activity in milk resides in the immunoglobulin fraction.

The anti-adhesive activity remaining in the non-immunoglobulin fraction of milk can be explained by two types of molecules: free oligosaccharides and glycoproteins in the casein fraction. Human milk is unique with respect to its content of complex carbohydrates. The free oligosaccharide fraction of milk is dominated by lactoseries and with improved methods of isolation and characterization of carbohydrates, more than 130 oligosaccharides containing up to 20 monosaccharides per molecule have been identified. An anti-adhesive activity against S. pneumoniae in a low molecular weight fraction (<5 kDa) of milk is explained by the free oligosaccharides. On the contrary, there was no such effect against H. influenzae (15). An anti-adhesive activity of high molecular weight components of milk is located in the casein fraction. Human casein drastically reduces the adhesion of S. pneumoniae and H. influenzae (15). This effect was specific species. Alpha-lactalbumin is an etaloprotein, which shows some degree of heterogeneity depending on the saturation of. Ca (II) and / or glycosylation (1). Alpha-lactalbumin acts as a specific protein in the lactose synthase system. During lactation, an alpha-lactalbumin is formed in the mammary gland and alters the substrate specificity of the enzyme galactosiltrans-ferase N-acetyl glucosamine (GlcNAc) to glucose (Glc), allowing the synthesis of lactose: GT UDP-Gal + Glc. Lactose + UDP alpha-LA Multiple forms of alpha-lactalbumin, bovine, pig, sheep and goat have been isolated and well characterized (2, 3). These multiple forms differ in a few amino residues or the number of disulfide bonds (4, 5) but all are active in the lactose synthase system. The physiological importance or functions of these different forms of alpha-lactalbumin is not known. Alpha-lactalbumin has undergone a high rate of evolutionary change and shows homology with lysozyme (1). These two proteins are believed to originate from the same ancestral protein. While lysozyme is known as an anti-bacterial agent, alpha-lactalbumin has not yet been found to have antibacterial functions.

DESCRIPTION OF THE PRESENT INVENTION The present invention describes the identification of a new antibacterial protein or group of milk proteins. The protein consists of a multimeric form of alpha-lactalbumin. In the following, this protein or group of proteins are abbreviated ALLP, Protein Similar to LactoAlbumin Anti-Adhesive. The term "antimicrobial or antibacterial protein" used in the context of the present invention means that in the following a protein, which inhibits the adherence of microorganisms to the tissue- and / or exerts a bactericidal effect on the microorganisms. Other features of the invention will be apparent from the appended claims. The present invention will be described in more detail with reference to the following example.

Experimental part The purification of the bactericidal and active anti-adhesive protein (ALLP). Milk samples from lactating women are selected for anti-adhesive activity against S. pneumoniae and H. influenzae. Approximately 50 1 of breast milk with high anti-adhesive activity is collected from a healthy donor and used for the purification of ALLP.

Approximately 5 1 of the milk is frozen for a time and centrifuged to remove the fat. Casein is prepared from defatted milk by acid precipitation at a pH of 4.6. The ALLP is purified as described in the following: (i) Ion Exchange Interchange of Casein. The casein is divided using an ion exchange column (14 cm x 1.5 cm) packed with DEAE-Tris-acryl M (LKB, Sweden) bound to an FPLC (Pharmacia, S eden) using a gradient of NaCl. 100 mg of the lyophilized casein are dissolved in 10 ml of 0.01 M Tris-HCl, pH 8.5. After centrifugation, the sample is applied directly to the column and runs under the following conditions: buffer A: 0.01 M Tris-HCl, pH 8.5, Shock absorber B: Shock absorber A containing 1M NaCl / 1. Gradient program: from 0-3 mi of 100% of A, of 3-60 mi of 15% of B, of 60-85 mi of 25% of B; 85-87 ml of 100% B, 87-89 ml of 100% B for 2 minutes; of 89-120 ml of 100% A. The gradient was not linear, but was interrupted in the elution of each peak for the best separation. Flow rate: 1 ml / minute, recording 0.2 cm / minute. The dampers are degassed and filtered through a 0.22 μm filter before use. The peaks were verified at 280 nm and the size of the fraction was 3 ml. The fractions are pooled as shown (FIG 1A). Deposits (I-VI) are desalted by dialysis (3.5 kD exclusion membrane) against distilled water for at least 48 hours, lyophilized and tested for anti-adhesive activity. (ii) Gel Chromatography of Deposit VI. 100 mg of the active VI-tank is obtained after repeated FPLC of the casein fractions, dissolved in 7 ml of 0.06 M sodium phosphate buffer, pH 7.0 and applied to a Sephadex® G-50 column (Pharmacia, Sweden ) (93 cm x 2.5 cm). The flow rate of 20 ml / hour, the peaks are verified at 280 nm, fractions of 3 ml are collected and assembled as shown (FIGURE 2A). The deposits are desalted by dialysis, lyophilized, tested for composition and anti-adhesive activity. Commercial alpha-lactalbumin ion exchange chromatography. 20 mg of commercial human or bovine alpha-lactalbumin (Sigma) is dissolved in 2 ml of 0.01 M Tris-HCl, pH 8.5. The ion exchange chromatography of alpha-lactalbumin is subjected to similar conditions as described above for fractionation of casein. The linear NaCl gradient (without interruption), flow rate 1 ml / min. , 3 ml of the fractions were collected and fractionated as shown in FIGURE 1B. The fractions were dialyzed (3.5 kD membrane cut), lyophilized, resuspended to the required concentration and tested for anti-adhesive activity.

Gel Chromatography of the Commercial Alfa-Lactoalbúmina. Approximately 8-10 mg of commercial human or bovine alpha-lactalbumin (Sigma) are dissolved in 3 ml of 0.06 M sodium phosphate buffer, pH 7.0 and fractionated on the Sephadex® G-50 column as described above. The flow rate was 30 ml / h, the peaks are verified at 280 nm, collected and collected fractions of 3 ml as shown (FIGURE 2B). The deposits are desalted by dialysis (3.5 kD exclusion membrane) against distilled water for at least 48 hours, lyophilized, tested for composition and for anti-adhesive activity. 6-8 mg of the material retained and then eluting 1M NaCl during the ion-exchange chromatography of alpha-lactalbumin, dissolved in 5 ml of 0.06 M sodium phosphate buffer, pH 7.0 and subjected to gel chromatography in column C-50 as described in the above. The 3 ml fractions are collected and collected (FIGURE 3). The deposits are desalinated, lyophilized and tested for anti-adhesive activity. Electrophoresis in Polyacrylamide Gradient Gel (PAGGE). The analytical PAGGE is performed using 4-20% of polyacrylamide pre-gelled gels (Bio-Rad, Richmond, CA) in a Mini Protean II Bio-Rad cell. To 10 μl (5-10 mg / ml) of each of the lyophilized fractions, an equal volume of the sample buffer is added (13.1% 0.5 M Tris-HCl, pH 6.8, 10.5% glycerol, 1.2% SDS and 0.05% bromophenol blue). 20 μl of each are loaded into the gel, which is run in Tris-Glycine buffer (pH 8.3) 0.1% SDS at a constant voltage of 200V for approximately 40 minutes. The protein staining is done by immersing the gel in Coomassie Blue solution (0.1% in 40% methanol, 10% acetic acid). For approximately 0.5 h. The fade was by various changes in 40% methanol, 10% acetic acid until a clear background is obtained. Ionic Desorption Mass Spectroscopy. ALLP and commercial alpha-lactalbumin are analyzed by ion desorption mass spectroscopy.

Bacteria S. pneumoniae (CCU3114 and 10175) and H. influenzae (H1998) are used in all experiments. It is known that these strains adhere well to human nasopharyngeal epithelial cells in vitro. These strains were initially isolated from the nasopharynx of children frequent episodes of acute otitis media. Strains are kept lyophilized and transferred to blood agar plates (10175) or agar plates from Levinthal medium (Hi 198). S. pneumoniae is cultured for 9 hours at 37 ° C in liquid medium (17), harvested by centrifugation and suspended in 1 ml of 0.9% NaCl 1% choline. H. influenzae H1998 is cultured for 4 hours in haemophilus medium (18), is harvested by centrifugation and suspended in phosphate buffered saline (PBS).

Inhibition of adhesion Adhesion and adhesion inhibition are tested as previously described (15, 19). In summary, the epithelial cells of the oropharynx of healthy donors (10-7ml) are mixed bacterial suspensions (10 ^ / ml). After incubation of the bacteria and epithelial cells, unbound bacteria are removed by repeated cycles of centrifugation and resuspension in NaCl 1% choline (10175) or PBS (Hi 198). The inhibitory activity of the different fractions is tested by preincubation the bacteria for 30 minutes at 37 ° C before the addition of the epithelial cells. The number of joined epithelial cells is counted the help of an interfering contrast microscope (Ortolux II microscope TE Leitz interference contrast equipment, etzlar). Adherence is given as the average number of bacteria / cell for 40 epithelial cells. Inhibition is given in percent of the value of the control buffer.

Results Properties of the AI_LP ALLP is purified from human milk by fractionation of casein by ion exchange chromatography and fractionation of the tank eluting after 1M NaCl by gel chromatography. The ion exchange fractionation profile of casein is shown in FIGURE 1A. The eluted fractions are pooled as indicated and tested for anti-adhesive activity. Deposit VI retains the anti-adhesive activity of casein; this deposit inhibits the binding of S. pneumoniae and H. influenzae in more than 80% of the control (Table 3). The remaining fractions were inactivated and are no longer analyzed. Fraction VI is fractionated by gel chromatography on the Sephadex® G-50 column. The fractionation profile showed two distinct peaks well separated (FIGURE 2A). The eluted fractions are pooled as shown, desalted and tested for anti-adhesive activity. Fraction K retained 98% of the anti-adhesive activity against S. pneumoniae and 91% of the activity against H. influenzae. Fraction L was inactive (Table 3). The analytical PAGGE of the K fraction showed the presence of bands in the region of 14-15 kD units, one band in the region of 30 kD units and two bands stained in the 100 kD region. The L fraction showed the presence of only one band in the 14-15 kD region (FIGURE 2A, interspersed). Analysis of the N-terminal amino acid sequence showed that the bands of the K-fraction were similar and identical to the N-terminal sequence of human alpha-lactalbumin. The active anti-adhesive protein in the K fraction is designated as Anti-adhesive Lactoalbumin-like Protein (ALLP). ALLP reduces the binding of S. pneumoniae and H. influenzae by approximately 60% at a concentration of 1 mg / ml.

Mass spectroscopy of ALLP The results of the analytical PAGGE suggested that the ALLP must be presented in a multimeric form. By ion laser desorption mass spectroscopy, ALLP showed three different mass fragments (1, 2 and 3) at 14128.7 m / z, 28470.5 m / z and 42787.8 m / z, respectively (FIGURE 4). These fragments are in agreement with the ranges of monomeric mass (14 m / z) dimeric (28 m / z) and trimeric (42 m / z) of the protein.

Comparison of commercial ALLP and alpha-lactalbumin When tested for anti-adhesive activity with commercial alpha-lactalbumin it does not inhibit the adhesion of S. pneumoniae or H. influenzae even at a concentration of 10 mg / ml (Table 4). ALLP showed woven bands in the regions of 14-15 kD, 30 kD and 100 kD, while commercial alpha-lactalbumin has only one band in the 14-15 kD region. The N-terminal amino acid sequence of ALLP showed complete homology with the sequence of human alpha-lactalbumin. The lack of anti-adhesive activity of commercial alpha-lactalbumin, when compared to ALLP, must be seen as a difference in its molecular forms. Therefore, commercial human alpha-lactalbumin is subjected to ion laser desorption mass spectroscopy. The spectrum only shows a mass fragment at 14128.7 m / z corresponding to the monomeric form of alpha-lactalbumin (calculated molecular mass = 14,079 kD). Thus, commercial human alpha-lactalbumin was in the monomeric form and lacked antiadhesive activity, whereas ALLP is found to be multimeric and inhibits the binding of S. pneumoniae and H. influenzae to human oropharyngeal cells in vitro. Ion exchange chromatography of human lactalbumin. To test the effect of ion exchange chromatography on the anti-adhesive effect of commercial human alpha-lactalbumin, 20 mg of the commercial sample is applied on the Tris-acryl column. The ion exchange profile is shown in FIGURE 1B. Approximately 50% of the applied material is retained on the column and eluted after the application of 1M NaCl (arrow, Figure 1B). The different fractions are brought together as shown. After desalting and lyophilizing the fractions are reconstituted to a concentration of about 5-10 mg / ml and tested for anti-adhesion activity.

Anti-adhesive Effect of Human Alpha-lactalbumin After Ion Exchange Chromatography. Prior to ion exchange chromatography, comeroial human alpha-lactalbumin lacked anti-adhesive activity (Table 4). After that, it is subjected to ion exchange chromatography, the deposit or the fraction from which it is retained and eluted with 1M NaCl (fraction LA2, Figure 1B) inhibits the binding of both S. pneumoniae and H. influenzae in more than 95% of the control value (Table 4). The other fraction obtained (LA-.) Was inactive. Human Alpha-lactalbumin Gel Chromatography before and after Ion Exchange Chromatography. Since approximately 50% of the commercial human alpha-lactalbumin had become active after ion exchange chromatography, it was decided to verify the mobility of alpha-lactalbumin and fraction A2 in gel chromatography. The chromatographic profile of the G-50 gel of human alpha-lactalbumin is shown in FIGURE 2B. Alpha-lactalbumin eluted as a single peak, which gave an individual band (14-15 kD) in the PAGGE analysis (intercalated, FIGURE 2B). This LA fraction is found to be inactivated when tested for anti-adhesive activity (Table 4). The gel chromatographic profile of the active LA2 fraction, obtained after the ion-exchange chromatography of alpha-lactalbumin is shown in FIGURE 3. This fraction elutes as two well-separated peaks (1 and 2, FIGURE 3) corresponds to the elution volumes of the K and L peaks of casein (FIGURE 2A). When tested for anti-adhesive activity, fraction 1 retained activity against S. pneumoniae and H. influenzae, while fraction 2 was inactive (Table 4). When the fraction 1 is analyzed by analytical PAGGE, a pattern similar to that of the ALLP is obtained. The bands dyed in the region of 14-15 units 14 kD, the region of 30 units kD and two bands in the region of 100 kD. Fraction 2 gives a single band in the region of 14-15 kD, which correspond to the monomeric alpha-lactalbumin (intercalated, FIGURE 3). Properties of commercial alpha-lactalbumin. Since commercial human alpha-lactalbumin can not be converted to the active multimeric form by ion exchange chromatography, it has been decided to test the activity of bovine alpha-lactalbumin and to test its mobility in gel and ion exchange chromatography. . When tested for anti-adhesive activity, it was found that bovine alpha-lactalbumin is inactive in inhibiting the binding of S. pneumoniae and H. influenzae (Table 5). mg of bovine alpha-lactalbumin are subjected to ion exchange chromatography under similar conditions described above for human alpha-lactalbumin. 50% of the material applied to the column is retained and then eluted with 1M NaCl. The elution pattern is similar to that obtained for human alpha-lactalbumin (FIGURE 1B). the BL fraction? of alpha-lactalbumin, which corresponds to the elution volume of the LA2 fraction of human alpha-lactalbumin (FIGURE 1B) inhibited the binding of __ .. pneumoniae by more than 95% and of f. influenzae in more than 80% of the control value (Table 5). When subjected to gel chromatography on the G-50 column as described above, bovine alpha-lactalbumin eluted as a single peak corresponding to the elution volume of human alpha-lactalbumin (Figure 2 B). In contrast, the material in the BL2 fraction is resolved into two distinctive peaks corresponding to fractions 1 and 2 obtained for human alpha-lactalbumin (Figure 3). The fraction that elutes just after the empty volume of the column (corresponds to fraction 1) retained the anti-adhesive activity, while the other fraction was inactive. The active fraction had a PAGGE pattern similar to that of ALLP, while the inactive fraction stained only one band in the region of 14-15 kD units. In this way, a portion of the commercial bovine alpha-lactalbumin is also converted to the active multimeric form by ion exchange chromatography.

Bactericidal effect The present ALLP is tested with respect to the bactericidal effect in different strains of S. pneumoniae that are known to be resistant to antibiotics and some other strains of streptococcus, E. coli, H. influenzae and M. cath. Therefore, the different bacterial strains are inoculated in growth plates after incubation with the ALLP in different concentrations, the viable counts (CFU) are determined in the inoculation, 0.5 H, 2 h and 4 h (hours), respectively, after the inoculation. Table 1 below shows the viable counts after incubation with a medium containing 10 mg / ml of ALLP compared to the control.

TABLE 1 Viable Accounts (CFU) in Strains of S. pneumoniae after exposure to ALLP Viable Accounts (CFU) Designation of Strain Oh 0.5h 2h 4h 10175 control 2x1 O6 1x106 1x105 1 1 O4 ALLP 2x1 O5 - - - 15006-92 control 1x1 O4 2x1 O4 1x1 O3 - ALLP 2x1 O4 - - - 14060-92 control 2x1 O6 1x105 1x1 O4 - ALLP 2x1 O5 - - - 15256- 92 control 1x1 O6 2x1 O6 2x1 O5 4x1 O4 ALLP 2x1 O6 - - - 14326-92 control 4x1 O5 2x1 O5 2x1 O4 2x1 O3 ALLP 7x1 O4 - - - Prag 1828 control 5x106 2x1 O6 5x1 O5 - ALLP 5x1 O6 - - - 14091 -92 control 3x105 5x1 O5 1x105 - ALLP 7x1 O5 - - - 14117-92 control 2x1 O6 - 2x106 2x1 O6 - ALLP 2x106 - - - 14612-92 control 3x1 O5 1x105 2x1 O4 1x103 ALLP 3x1 O4 - - - Dk 84/87 control 1x1 O7 5x1 O6 2x1 O6 6x1 O4 ALLP 3x105 _ 14007-92 control 1x10D 5x10 4X10-3 ALLP 1x105 14030-92 control 5x106 2x10 2x105 ALLP 5x106 2x10 14423-92 control 6x1 O5 6x10 < 1x106 6x105 ALLP 2x1 O5 3x10 4502-93 control 4x105 ALLP 5x1 O4 SA44165 control 2x105 5x10- ALLP 3x105 1017-92 control 1x106 5x10- 4x10- ALLP 9x105 317-93 control 4x104 1x1 O4 5x103 ALLP 2x1 O3 760-92 control 2x1 O7 2x10C 1x10 1x10 ALLP 8x10ß Hun 859 control 6x105 3x10- 2x10- 2x10- ALLP 3x105 Hun 963 control 1x1 O7 4x10 1x105 ALLP 5x106 BN 241 control 4x1 O6 5x10 2x10 ALLP 2x1 O5 TABLE 2 Variable Accounts (CFU) in Different Bacterial Species Viable Accounts (CFU) Designation of the Cepa Oh 0.5h 2h 4h S. mitis control 1x106 10x1 O6 2x1 O5 1x105 116 ALLP 1x106 - - - S. sanguis control 5x1 O7 3x1 O7 4x1 O7 5x1 O6 197 ALLP 3x1 O7 2x1 O5 2x1 O2 - E. Coli control 6x1 O6 5x1 O6 3x1 O6 3x1 O6 60 ALLP 7x1 O6 5x1 O6 1x107 2x1 O7 4 control 5x1 O6 5x1 O6 5x1 O6 7x1 O6 ALLP 5x1 O6 6x1 O6 1x1 O7 2x1 O7 H. influenzae control 4x1 O7 1x1 O7 4x1 O6 2x1 O5 21594 ALLP 3x1 O7 4x1 O5 < 1x103 < 1x103 21300 control 4x1 O7 2x1 O7 5x1 O6 3x1 O5 ALLP 4x1 O7 2x1 O6 2x1 O4 2x1 O3 M. cath control 4x1 O5 3x1 O5 5x1 O4 2x1 O4 71257 C + ALLP 3x1 O5 2x1 O5 5x1 O3 - 71295 C + control 2x1 O7 1x107 3x1 O6 6x1 O5 ALLP 2x1 O7 5x1 O6 2x1 O6 3x1 O5 C + = That produces beta-lactamase A dose-response curve is formed based on the bactericidal effect in S pneumoniae 10175 at different concentrations of ALLP administration compared to the control (no addition). Therefore ALLP is administered at 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml, respectively. The graph obtained is shown in Figure 5. As is evident from it, neither like 0.1 mg / l of ALLP provides a bactericidal effect in that S. pneumoniae. The viable accounts are further determined using different control proteins, viz, bovine serum albumin (BSA), alfalactoalbumin (bovine origin), lactofe-rhine (bovine origin) at a concentration of 10 mg / ml and control (without protein). As is evident from FIGURE 6, these proteins have no bactericidal effect on S. pneumoniae 10175. A new form of alpha-lactalbumin (ALLP) with anti-adhesive activity and bactericidal effect against respiratory tract pathogens S. pneumoniae and H. influenzae it was isolated and characterized from a human milk sample. The commercial human or bovine alpha-lactalbumin lacked anti-adhesive activity in the assay system. In a portion of the commercial bovine human alpha-lactalbumin was converted to the active form by ion exchange chromatography. The active and non-active forms of alpha-lactalbumin showed different mobilities in gel chromatography and their staining patterns in gel electrophoresis were also different. By analysis of ion desorption mass spectrometry, it was found that ALLP is in the trimeric form, while commercial alpha-lactalbumin was monomeric. Activated forms of commercial human and bovine alpha-lactalbumin showed a gel pattern similar to the trimeric form. A portion of the monomeric form of alpha-lactalbumin was separated from the multimeric form and found to be inactive to inhibit the adhesion of S. pneumoniae and H. influenzae. The three forms of alpha-lactalbumin (mono, di, and tri) existed in some kind of equilibrium after ion exchange chromatography and can not be successfully separated from one another. This proposes that active anti-adhesive alpha-lactalbumin (ALLP) is a multimeric form not previously identified in human milk. The identification of ALLP in a previous casein preparation was a result of its purification that is verified by biological activity (16). It retains all the anti-adhesive activity of casein and thus could be followed during the purification procedures. This form of alpha-lactalbumin present in human milk has not previously been described. Our previous studies showed that the anti-adhesive effect of human milk against S. pneumoniae and H. influenzae was independent of the activity of the specific antibody and was concentrated in a fraction of casein (15). However, it was found that casein has both bactericidal effects and an anti-adhesive effect. A bactericidal effect was present and found to be more pronounced against S. pneumoniae than H. influenzae. The anti-adhesive activity remained intact after the elimination of casein fatty acids. The mechanism of inhibition of ALLP adhesion was found to be independent of its carbohydrate content. The carbohydrate analysis of the ALLP showed the presence of only one unit of monosaccharide associated with the molecule. The elimination of this monosaccharide unit by the glucosidase treatment does not alter the anti-adhesion effect of ALLP. Also, since the commercial forms of human and bovine alpha-lactalbumin could be activated by ion exchange chromatography, it is very unlikely that carbohydrates play any role in the bactericidal or anti-adhesive effect of the ALLP tested by the analysis system. biological. Being predominantly a protein, whey, alpha-lactalbumin are usually purified from the alpha-lactalbumin-rich fractions of the serum. Since the monomeric form and the multimeric forms have different mobilities in gel chromatography, active multimeric forms are lost during purification procedures. It is not surprising that commercial preparations of alpha-lactalbumin lack the anti-adhesive properties in the present system. Genetic variants of alpha-lactalbumin have been isolated from the milk of other mammals including cattle. Most of these forms consist of four sulfide bonds and one form of bovine alpha-lactalbumin with three disulfide bonds that have also been isolated (5). The physiological role of these different forms of alpha-lactalbumin is not known. Current data demonstrate that monomeric alpha-lactalbumin completely lacks biological activity in the present system. Therefore, aggregation and polymerization can be an important event in the anti-adhesion activity of ALLP against S. pneumoniae and H. influenzae. Current data show that multimeric alpha-lactalbumin is active in inhibiting the adhesion of respiratory tract pathogens and thus may play a role in protection against respiratory and gastrointestinal infections. It is also active as a bactericide on at least S. pneumoniae, even those resistant to antibiotics.

COMMENTS S. pneumoniae and H. influenzae are important causes of morbidity and mortality in all age groups. Infections in the respiratory tract, for example meningitis, otitis and sinusitis are caused by bacteria which enter the nasopharyngeal route. Colonization at this site can be an important determinant of the disease (18). The discovery that a specific alpha-lactalbumin derived from human milk as well as bovine milk inhibits the binding of cytoplasm species opens up the possibility of avoiding colonization by specific interference of the junction using these structures. Therefore, the bactericidal effect is also important. The importance of antimicrobial molecules is shown by the protection against infections, which is observed in breastfed infants. Breastfed infants have a reduced frequency of diarrhea, upper respiratory tract infections, and acute otitis media (AOM). The bacterial species discussed in this application are the most frequent bacterial causes of AOM, viz, S. pneumoniae, H. influenzae and Streptococcus pneumoniae. It is evident from the data shown that alpha-lactalbumin obtained from human or bovine milk inhibits the binding of S. pneumoniae and H. influenzae to the epithelial cells of the human respiratory tract in vi tro.

TABLE 3 Bacterial adhesion to oropharyngeal cells after incubation with active human milk, casein and casein fractions obtained after ion exchange chromatography on DEAE-Tris-acryl.

Adhesion S. pneumoniae H. influenzae Sample Average (%) Average (%) Control saline solution 150 (100) 200 (100) Human milk 25 '(17) 70 (35) Casein 4 (3) 10 (5) Fraction VI 14 (9) 22 (11) Fraction K 3 (2) 17 (9) Fraction L 150 (100) 178 (89) TABLE 4 Bacterial adhesion to oropharyngeal cells after incubation with human alpha-lactalbumin and fractions obtained after ion exchange chromatography and gel chromatography Adhesion S. pneumoniae H. influenzae Sample Average (%) Average (%) Control saline solution 138 (100) 130 (100) Human alpha-lactalbumin 124 (90) 110 (85) Fraction LA2 4 (3) 9 (7) Fraction LA 128 (93) 76 (58) TABLE 5 Bacterial adhesion to oropharyngeal cells after incubation with bovine alpha-lactalbumin and fractions obtained after ion exchange chromatography and gel chromatography- Adhesion S. pneumoniae H. influenzae REFERENCES Sample Average (%) Average (%) Control saline solution 138 (100) 130 (100) Bovine alpha-lactalbumin 130 (94) 99 (76) Fraction BL 3 (2) 18 (14) APPLICATIONS The alpha-lactalbumin of the present invention can be administered in the form of a dose unit, in the oral mucosa, an injectable composition or a toxic composition. In any case, the protein is normally administered together with commonly known carriers, fillers / excipients, which are pharmaceutically acceptable. In the case of the protein is administered in the form of a solution for topical use, the solution contains an emulsifying agent for the protein together with a diluent, which can be sprayed into the nasopharynx or can be inhaled in the form of a mist in the upper respiratory tract.

In oral use the protein is normally administered together with a carrier, which can be a solid, semi-solid or liquid diluent or a capsule. These pharmaceutical preparations are another object of the present invention. Typically, the amount of the active compound is between 0.1 to 99% by weight of the preparation, preferably between 0.5 to 20% by weight in the preparations for injection and between 2 and 50% by weight in the preparations for oral administration. In pharmaceutical preparations containing a protein of the present invention in the form of dosage units for oral administration, the compound can be mixed with a solid, powdery carrier such as for example with lactose, sucrose, sorbitol, mannitol, starch, such as potato starch, corn starch, amylo-pectin, cellulose derivatives or gelatin as well as with a antifriction agent such as magnesium stearate, calcium stearate, polyethylene glycol waxes or the like and pressed into tablets. Multi-unit dose granules can also be prepared. The tablets and granules of the above cores can be coated with concentrated sugar solutions, and so on. The cores can also be coated with polymers that change the rate of dissolution in the gastrointestinal tract, such as anionic polymers that have a pka above 5.5. Such polymers are hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate and the polymers sold under the trademark Eudragit S100 and L100. In the preparation of the gelatin capsules, these can be soft or hard. In the first case the active compound is mixed with an oil in the latter case the granules of multiple unit doses are filled in them. Liquid preparations for oral administration may be in the form of syrups or suspension, for example suspensions containing from about 0.2% by weight to about 20% by weight of the active compound described and glycerol and propylene glycol. If desired, such preparations may contain coloring agents, flavoring agents, saccharin and carboxymethylcellulose as a thickening agent. The daily dose of the active compound varies and is dependent on the type of route of administration, but as a general rule it is from 1 to 100 mg / dose of the active compound in the peroral administration and from 2 to 200 mg / dose in the topical administration. The number of applications during 24 hours depends on the route of administration, but it can vary, for example in the case of a topical application in the nose of 3 to 8 times during 24 hours, that is, depending on the flow of phlegm produced by the body treated in therapeutic use. In a prophylactic use, the number may be on the lower side of the given range. The topical form of preference is used in the prophylactic treatment, preferably in connection with an infection caused by a rhinitis virus. The protein can also be used as an additive in foods for young children, particularly for prophylactic reasons, to supply casein in an easy form for the child. Young children usually reject pharmaceutical products for different reasons. The food product may be in the form of a powdery oatmeal base, a cereal base, a milk substitute base or a more complex food product such as the Scotch piece type, which comprises vegetables and pieces of meat frequently in disintegrated form. In the case of the administration of the protein to the animals, they are usually added to the forage, which in addition to the protein contains the commonly used nutrients. According to another aspect of the invention, a process for determining the presence of S. pneumoniae and H. influenzae in the sample taken from the respiratory tract of an animal or human is provided. This process is based on the technique of determining the degree of interaction between the bacteria of the sample and a composition of the present invention. Such an interaction can be determined by the inhibition or induction of the adherence of bacteria to cells or other surfaces.

REFERENCES 1. McKenzie, H.A., White, F.H. Jr adv. Protein Chem. 41: 173, 1991 2. Hopper, K.E. and MaKenzie, H.A. Biochim. Biophys. Acta 295: 532, 1 73 3. Schmidt, D.V. and Ebner, K.E. Biochim. Biophys. Acta 263: 714, 1972 4. Maynard, F. J. Dairy Res. 59: 425, 1992 5. Barman, T.E. Eur. J. Biochim. 37:86, 1973 6. Readhead, K., Hill, T. and Mulloy, B. FEMS Microbiol Lett. 70: 269, 1990 7. Gilin, F., Reinerl, D.S. and Wang, C.S. Science 221: 1290, 1983 8. Fiat, A. -M. , and Jolles, P. Mol. Cell Biochem. 87: 5, 1989 9. Matthews, T.H.J., Nair, C.D.G. , Lawrence, M.K. and Tyrell, D.A.J. Lancent, December, 25: 1387, 1976 10. Andersson, B., Dahmén, J., Fredjd, T., Leffler, H., Magnusson, G., Noori, G., and Svanborg, C., J. Exp. Med., 158: 559, 1983 11. Svanborg, C., Aniansson, G., Mestecky, J., Sabharwal, H., and Wold, A. In Immunology of milk and the neonate, J. Mestecky ed. Plenum Press, New York, 1991 12. Svanborg-Eden, C. and Svennerholm, A. -M. , Infect. Immun. 22: 790, 1978 13. In Microbial lectins and agglutinins, properties and biological activity, Mirelman, D., Wiley, New York, 1986 14. Anderson, B., Porras, 0., Hansson, LA, Lagergard, T. Svanborg-Eden, CJ Infect. Dis. 153: 232, 1986 15. Aniansson, G., Anderson, B., Lindstedt, R., and Svanborg, C., Microbial 'Pathogenesis 8, 365, 1990 16. Sabharwal, H., Hansson, C., Nilsson, AK Saraf, A., Lónnerdahl, B., and Svanborg, C. 1993, sub itted 17. Lacks, S., and Hotchiss, R.D.

Biochim. Biophys. Acta, 38: 508, 1960 18. Branesfors-Helander, P. Acta Pathol. Microbiol, Immunol. Scand. (B), 80: 211, 1972 19. Porras, 0., Svanborg Eden, C., Lagergard, T., and Hansson, L.A. Eur. J. Clin. Microbiol., 4, 310-15, 1985. 20. Vanam, T.C., Brew, K., and Hill, R.L. J. Biol. Chem. 245: 4583, 1970 21. Beachey, E.H. J. Infect. Dis. 142, 325, 1981 22. Anderson, B., Beachey, EH, Tomasz, A., Tuomanen, E., and Svanborg, C., Microbial Pathogenesis, 4, 267, 1988 23. Anderson, B., Eriksson, B ., Falsén, E., et al Infect. Immun. 32, 311-17, 1981 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (15)

1. The use of a multimeric alpha-lactalbumin in the preparation of antibacterial preparations therapeutic and / or prophylactically active against infections, preferably of the respiratory tract, caused by bacteria in particular S. pneumoniae and / or H. influenzae.

2. The use according to claim 1, characterized in that the multimeric alpha-lactalbumin is present in a mixture of monomeric, dimeric and trimeric forms.

3. The use according to claim 2, characterized in that the monomeric, dimeric and trimeric forms are present in a weight ratio of about 15-7: 5-2: 1.

4. The use according to claim 3, characterized in that the monomeric, dimeric and trimeric forms are present in a weight ratio of approximately 10: 3: 1.

5. A pharmaceutical composition characterized in that it comprises a therapeutically active amount of a protein according to any of claims 1-4, for the therapeutic and / or prophylactic treatment of infections, preferably in the respiratory tract, caused by bacteria in particular S. pneumoniae / or H. influenzae.

6. Feed and forage comprising an active amount of a protein according to any of claims 1-4, for the therapeutic and / or prophylactic treatment of infections, preferably in the respiratory tract, caused by bacteria, in particular S. pneumoniae and / or H. influenzae.

7. The method for the prophylactic and / or therapeutic treatment of infections caused by bacteria, in particular S. pneumoniae and / or H. influenzae, characterized in that a therapeutically active amount of a protein according to any of claims 1-4 is administered to mammals, including humans, optionally in combination with therapeutically inert excipients or nutrients.

8. The method according to claim 7, characterized in that the alpha-lactalbumin is administered to prevent adhesion of the virulent bacteria.

9. The method according to claim 7, characterized in that the alpha-lactalbumin is administered to exert a bactericidal effect on the virulent bacteria.

10. The method according to claim 7, characterized in that alpha-lactalbumin is administered to provide a bactericidal effect on a virulent bacterium.

11. The method for diagnosing infections caused by bacteria in particular S. pneumoniae / or H. influenzae, characterized in that a sample of the infected mammals including man, is extracted and determined with respect to the adhesion vis a vis a protein of according to any of claims 1-4.

12. A method for preparing a protein according to any of claims 1-4, characterized in that the monomeric alpha-lactalbumin is subjected to ion exchange chromatography.

13. The method according to claim 12, characterized in that the ion exchange chromatography is carried out in a DEAE-Tris-acryl gel.

14. The method according to claim 13, characterized in that the eluting agent is NaCl having a linear gradient.

15. A method for preparing a therapeutic antibacterial preparation and / or prophylactically active against infections caused, preferably in the respiratory tract by S. pneumoniae and / or H. influenzae, whereby a therapeutically active amount of multimeric alpha-lactalbumin is combined with excipients inert. In testimony of which I sign the present in this City of Mexico, D.F., on August 16, 1994. By: HE ANT SABHARWAL and CATHARINA SVANBORG