WO1997005884A1 - Aliment lacte pour nourrissons et additifs correspondants - Google Patents

Aliment lacte pour nourrissons et additifs correspondants Download PDF

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Publication number
WO1997005884A1
WO1997005884A1 PCT/US1996/012447 US9612447W WO9705884A1 WO 1997005884 A1 WO1997005884 A1 WO 1997005884A1 US 9612447 W US9612447 W US 9612447W WO 9705884 A1 WO9705884 A1 WO 9705884A1
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Prior art keywords
iga protease
lactoferrin
iga
precursor
protease
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PCT/US1996/012447
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English (en)
Inventor
Andrew Plaut
Jiazhou Qiu
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New England Medical Center Hospitals, Inc.
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Application filed by New England Medical Center Hospitals, Inc. filed Critical New England Medical Center Hospitals, Inc.
Priority to AU66418/96A priority Critical patent/AU6641896A/en
Publication of WO1997005884A1 publication Critical patent/WO1997005884A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39508—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from milk, i.e. lactoglobulins

Definitions

  • This invention relates to infant formulas and infant formula additives that help protect against infection.
  • the maternal/infant collaboration consists of initial "passive" antibodies from the mother's milk during the period in which the child's secretory immune system matures, and then the child's own “active" antibodies.
  • Bacterial colonization involves the attachment and proliferation of bacteria to mucosal surfaces. Under certain circumstances these microorganisms cause disease. Certain bacteria that colonize and infect infants continuously secrete IgA proteases that can cleave both maternal and infant IgA. For example, all strains of Haemophilus influenzae (HI) , Neisseria meningitidis, and Streptococcus pneumoniae that have been studied produce these IgA proteases and cause serious infections in children. Conditions caused by these bacteria include otitis media, pneumonia, bronchitis, sinusitis, septicemia and meningitis. These bacterial infections are a major problem in all countries and even in major U.S. cities.
  • HI Haemophilus influenzae
  • Neisseria meningitidis Neisseria meningitidis
  • Streptococcus pneumoniae that have been studied produce these IgA proteases and cause serious infections in children.
  • Conditions caused by these bacteria include otitis media
  • HI is the predominant pathogen that causes otitis media.
  • IgA proteases are encoded as a single large polypeptide chain by iga genes in the bacterial chromosome. The proteases are secreted from bacterial cells by an autocatalytic processing mechanism. Bacterial IgA proteases cleave and inactivate human IgA on mucosal surfaces and, in this way, allow bacterial pathogens to evade the mucosal immune mechanisms. IgA cleavage impairs function by dissociating the Fab and Fc regions of IgA. In addition, free Fab fragments may also bind key microbial antigens, blocking access by other protective antibodies.
  • Certain infant formulas have been designed to protect the infants that drink the formula against bacterial and rotavirus infections. Modification of infant formula typically involves immunizing a pregnant cow with certain bacteria or their toxins, and then extracting the desired antibody from the colostrum (the first milk after calving) for addition to formula products. Such antibodies are known to protect children against infection by several intestinal pathogens including enterotoxigenic E. coli, Vibrio cholerae , and rotaviruses. Summary of the Invention Applicants have discovered that, in the presence of lactoferrin, IgA protease precursor undergoes an alternative form of processing, which renders the entire IgA protease precursor susceptible to neutralizing antibodies which specifically bind the precursor.
  • the invention features, in one aspect, an infant formula which includes pasteurized milk (e.g. , colostrum) , active lactoferrin (e.g. , non- pasteurized lactoferrin) , and an antibody which specifically binds (i.e., inhibits the proteolytic activity of) either an IgA protease or an IgA protease precursor, or both an IgA protease and an IgA protease precursor.
  • the invention features a method for preparing a pasteurized infant formula.
  • the method involves immunizing a non-human mammal with all or a portion of an IgA protease or an IgA protease precursor; collecting from the mammal milk (e.g., colostrum) including antibodies which specifically bind (i.e., inhibit the proteolytic activity of) an IgA protease or an IgA protease precursor, or both; pasteurizing the milk; and preparing the infant formula using the pasteurized milk and active (e.g., non- pasteurized) lactoferrin.
  • mammal milk e.g., colostrum
  • active e.g., non- pasteurized
  • the invention also features a method for producing an infant formula additive, involving immunizing a pregnant non-human mammal with all or a portion of an IgA protease or an IgA protease precursor; collecting from the mammal milk (e.g. , colostrum) including antibodies which specifically bind (i.e., inhibit the proteolytic activity of) an IgA protease, an IgA protease precursor, or both an IgA protease and an IgA protease precursor; pasteurizing the milk; and mixing pasteurized milk with active (e.g., non-pasteurized) lactoferrin to produce the infant formula additive.
  • colostrum e.g., colostrum
  • active e.g., non-pasteurized
  • the invention further features a method for inhibiting processing of an IgA protease precursor on a cell.
  • This method involves contacting the cell with a formulation comprising a pasteurized excipient, and active (e.g., pasteurized) lactoferrin, thereby producing an extracted IgA protease precursor; and contacting the extracted IgA protease precursor protein with an antibody which specifically binds (i.e., inhibits the proteolytic activity of) the IgA protease precursor.
  • the invention features a method for inhibiting proteolysis of an IgAl antibody by an IgA protease.
  • This method involves contacting a cell which includes a precursor of the IgA protease with a formulation that includes a pasteurized excipient, and active (non-pasteurized) lactoferrin, thereby producing an extracted IgA protease precursor.
  • the method also involves contacting the extracted IgA protease precursor with an antibody which specifically binds (i.e., inhibits) the proteolytic activity of the IgA protease.
  • a pharmaceutical formulation which includes a pasteurized excipient; active (e.g., pasteurized) lactoferrin; and an antibody that binds (i.e., inhibits) the proteolytic activity of an IgA protease, an IgA protease precursor, or both an IgA protease and an IgA protease precursor.
  • the invention includes a pharmaceutical formulation comprising active (e.g., pasteurized) lactoferrin and an antibody that specifically binds (i.e., inhibits) an IgA protease-like protein, an IgA protease precursor-like protein, or both an IgA protease- like protein and an IgA protease precursor-like protein.
  • active e.g., pasteurized
  • lactoferrin an antibody that specifically binds (i.e., inhibits) an IgA protease-like protein, an IgA protease precursor-like protein, or both an IgA protease- like protein and an IgA protease precursor-like protein.
  • an antibody that specifically binds the helper section of an IgA protease precursor is included within the invention.
  • the infant formula includes non-human milk or colostrum, such as cow's colostrum.
  • milk encompasses colostrum and the subsequent lacteal secretions.
  • the formula includes colostrum (i.e., the antibody-rich first secretion at the termination of pregnancy) .
  • the immunization is performed during the last month of gestation, and the antibodies are collected by collecting colostrum.
  • the animal e.g., cow
  • the colostrum or milk is collected after the termination of the second pregnancy since colostrum or milk collected at that point has higher levels of the desired antibodies.
  • the antibody which specifically binds an IgA protease precursor-like protein binds a vacuolating cytotoxin
  • Tsh protein e.g., chicken E. coli Tsh protein
  • HAP protein hemagglutinating adherence protein
  • lactoferrin which is employed in any of the various aspects of the invention can be derived from any mammal; preferably, the lactoferrin is human or bovine in origin. Lactoferrin from other ruminants (e.g., sheep and goats) is also preferred. Such lactoferrin can be isolated from a secretion (e.g. , milk) of the mammal, or the lactoferrin can be produced with recombinant DNA techniques using standard techniques. Recombinant lactoferrin used in the invention can be produced in vitro or in vivo . For example, lactoferrin produced in cultured mammalian tissue is suitable in the invention.
  • Lactoferrin produced in a prokaryotic cell can also be used in the invention since there is no apparent requirement for the carbohydrates found in naturally occurring lactoferrin.
  • conventional gene expression methods can be used to overexpress lactoferrin (e.g., in yeast, baculovirus, or in a mammal (e.g., a transgenic cow) , and lactoferrin obtained from such a mammal can be used in the invention.
  • colostrum or milk used in the invention can be processed before preparing the infant formulas of the invention.
  • fat and casein may be removed.
  • processing steps which include heating or acidification should not be performed after addition of lactoferrin to a formula.
  • processing can include the step of reducing the volume of the colostrum by filtering through a 100,000 MW filter.
  • antibodies e.g., IgG
  • the term "infant formula” includes colostral or other milk preparations that include the inhibitors or antibodies of the invention and, if desired, purified inhibitors that are fed to an infant orally in combination with any other suitable beverage.
  • antibody includes not only complete antibodies but immunologically-active fragments thereof, which include any antibody fragments that are effective to inhibit the proteolytic activity of IgA proteases, IgA protease-like proteins, IgA protease precursors, or IgA protease precursor-like proteins.
  • Antibodies raised against the entire protease precursor-like protein, the helper section of the precursor-like protein, or the mature protease-like protein all are included. Also included are monoclonal antibodies that specifically bind any portion of the IgA protease-like protein or precursor-like protein, and which inhibit the proteolytic activity of the protein.
  • protease includes not only the complete protease but immunologically-active fragments thereof, which include any peptide fragments that give rise to antibodies, e.g., against the complete IgA protease, when used as an immunogen.
  • lactoferrin which retains the ability to extract an IgA protease precursor or protease precursor-like protein from the membrane of a cell.
  • lactoferrin is active if it is purified or produced without substantial heating (substantial heating is considered incubation at 56°C for 30 minutes or 90°C for 5 minutes) or acidification (incubation at pH 4.0 or below).
  • conventional pasteurization techniques e.g., heating milk for 30 minutes at 68°C
  • lactoferrin is inactive in the presence of 15 mM EGTA or EDTA.
  • active lactoferrin includes derivatives of, and those portions of lactoferrin that retain the above-described extraction activity.
  • the lactoferricin domain of lactoferrin is not in and of itself considered active lactoferrin because it is insufficient to extract the protease precursor.
  • Active lactoferrin can be produced with recombinant DNA techniques (e.g., purification from a eukaryotic cell engineered to express lactoferrin in vitro) .
  • the exogenous lactoferrin can be purified from a natural source of lactoferrin.
  • purified active bovine lactoferrin can be admixed with pasteurized bovine colostrum or milk containing appropriate antibodies in preparing an infant formula of the invention.
  • the active lactoferrin may be derived from a species (e.g., a cow) which is different from the species (e.g., a goat) from which milk or colostrum of the formula is derived.
  • the invention offers several advantages.
  • lactoferrin to extract the IgA protease precursor-like protein from the cell membrane, and by using antibodies directed against the protease precursor-like protein or mature protease-like protein, the invention provides a means for inhibiting every IgA protease or protease precursor-like protein in a sample.
  • every IgAl antibody of every specificity in the person e.g., infant
  • This type of protection against the bacterial IgA protease does not require killing the bacterium.
  • immune complexes formed by antibodies that bind the IgA protease or its precursor can be delivered to M cells for generation of an immune response more readily than is the IgA protease or protease precursor in the absence of such antibodies.
  • IgA protease precursors from gram negative cells and proteases from gram positive cells are extracted by lactoferrin and thus are both susceptible to neutralizing antibodies. This effect of lactoferrin is unaffected by iron-saturation of the lactoferrin, thus expanding the applicability of the method.
  • Figs. IA and IB are photographs of gels showing that the IgA protease precursor is extracted when the cells are grown in whey.
  • Fig. 2 is a photograph of a gel showing that the IgA protease precursor is extracted when the cells are grown in substantially pure human lactoferrin.
  • Fig. 3 is a photograph of a gel showing that the IgA protease precursor is extracted when the cells are grown in pure, recombinant lactoferrin.
  • Figs. 4A to 4C are photographs of gels showing that processing of the extracted IgA protease precursor is inhibited in the presence of secretory IgAl (slgAl) .
  • Fig. 5 is a chart showing the titers obtained after immunization of 18 cows with IgA proteases.
  • Fig. 6 is a chart showing the titers obtained after a second immunization and calving.
  • Fig. 7 is a chart providing a second example of titers obtained after immunization of cows with IgA protease.
  • Figs. 8A and 8B are graphs showing the difference in inhibiting titre against various IgA proteases having the same or different serogroups as the immunizing enzyme.
  • Fig. 9 is a chart providing the protease inhibition titers of cows after a second set of immunizations.
  • Fig. 10A is a reproduction of an autoradiogram of an electrophoresis gel showing the activity of IgA protease when added to infant formulas.
  • Fig. 10B is a reproduction of an autoradiogram of an electrophoresis gel showing the inactivity of IgA protease when added to infant formulas supplemented with the bovine antibodies of the invention.
  • IgA protease precursor-like proteins are proteolytically processed into the mature enzymes in reactions that may be autocatalytic in nature.
  • the IgA protease precursor of HI exists as a 184 kD transmembrane protein that undergoes autocatalytic cleavage to produce a 109 kD secreted enzyme.
  • the 70-80 kD helper section i.e., beta domain
  • the IgA protease precursor migrates as a species of 210 kD
  • the secreted enzyme migrates as a species of 116 kD
  • the helper section migrates as a species of 120 kD.
  • IgA protease precursor-like protein undergoes an alternative form of processing. For example, when an HI cell containing an IgA protease precursor is treated with lactoferrin, the entire protease precursor is extracted from the cell. Extraction of the entire protease precursor occurs at only low levels (10-20%) in normal growth media or buffer lacking lactoferrin. In the absence of inhibitory antibodies, the extracted protease precursor can be further processed to release the protease.
  • IgA Protease Precursor in Whey This experiment demonstrates that the IgA protease precursor of HI undergoes alternative processing when a normal milk whey (i.e., de-fatted milk) is used as the culture medium.
  • This experiment employed a strain of Haemophilus influenza (HI) that has a mutation in the igra gene encoding the IgA protease precursor.
  • the mutant IgA protease precursor is unable to cleave the mature protease off of the protease precursor.
  • the mutant HI cells were grown in a BHI medium until they reached mid-log phase (approximately 5 hours).
  • Several 0.4 ml aliquots of cells were taken from the culture, and the cells were washed once with saline.
  • the 0.4 ml aliquots of cells then were grown in 0.2 ml whey for various lengths of time (2, 5, 10, 20, 40, or 80 minutes).
  • As a control an aliquot of cells was grown in saline for 80 minutes.
  • the cells were harvested by centrifugation, and 25% of the cell pellet and 100 ⁇ l of the milk supernatant from each culture were analyzed by western blot analysis using antibodies raised against gel-purified the IgA protease.
  • Growth of HI cells in milk results in extraction of the IgA protease precursor from the cell surface into the culture supernatant.
  • the IgA protease precursor becomes undetectable in the cell pellet, and it appears in the culture supernatant (Figs. IA and IB; compare lanes 2-7) .
  • the ability of whey to extract the IgA protease precursor from the cell is proportional to the concentration of whey in the sample. Dilution of the whey led to a diminished ability to extract the IgA protease precursor from the cell. In contrast to the behavior of the IgA protease in milk, the protease was not extracted when cells were grown for 80 minutes in saline (Figs. IA and IB, lane 8) . These data thus indicate that a component of milk promotes extraction of the entire IgA protease precursor from the cell.
  • these same antibodies specifically bound the IgA protease, the IgA protease precursor, and a recombinant helper section of the IgA protease precursor.
  • these antibodies crossreact with the 210 kD unprocessed IgA protease precursor of an HI strain which has a mutation in the iga gene.
  • the enzyme portion of the IgA protease is no longer attached to the membrane; however, the helper section remains in the membrane.
  • Treatment of cells having a membrane-bound helper section with whey leads to extraction of the helper section of the IgA protease precursor.
  • Treatment of cells having an intact membrane-bound protease precursor with whey leads to extraction of the unprocessed protease precursor.
  • the extracted helper section exhibits a decrease in mobility by SDS-PAGE.
  • the whey was fractionated on a DE-52 anion exchange column, with the desired activity appearing in the non- binding fraction. Proteins from the DE-52 column flowthrough then were separated by size on a BioGel P-200 gel filtration column. From the final pooled column fractions containing the desired activity, western blot analysis was used to identify lactoferrin as the agent which causes extraction of the IgA protease precursor. Confirmation that Lactoferrin is the Extracting Agent: To confirm that lactoferrin is the extracting agent, lactoferrin from several sources was assayed for its ability to extract the IgA protease precursor.
  • substantially purified, but active, human and bovine lactoferrin each were able to extract the IgA protease precursor from the outer membrane of HI.
  • the ability of human lactoferrin (Sigma Cat. No. L-0520) to extract the IgA protease precursor is illustrated in Fig. 2.
  • iga mutant HI cells were grown to mid-log phase in BHI medium, then incubated in human lactoferrin at a concentration of 4 mg/ml or 2 mg/ml.
  • substantially purified human lactoferrin was able to extract the protease precursor from the cell membrane.
  • incubation of the cells in TBS buffer did not result in release of the protease precursor from the cell membrane.
  • Recombinant lactoferrin was used to confirm that lactoferrin extracts the IgA protease precursor from cells. This recombinant lactoferrin was produced in eukaryotic cells, and is iron-saturated. In this experiment, HI iga mutant cells were grown to mid-log phase in BHI, then incubated for 1 hour in 0.5 mg/ml recombinant lactoferrin. As illustrated in Fig. 3, recombinant lactoferrin also extracted the entire IgA protease precursor from the outer membranes of wild type (HI Rd " ) and two mutant strains (HI 3-13 and HI 2-25DK) of HI.
  • Serum transferrin Human plasma containing serum transferrin and purified serum transferrin were also tested for their ability to extract the IgA protease precursor from cell membranes. Serum transferrin is approximately 20% as effective as lactoferrin in extracting the IgA protease precursor from the membrane, suggesting that the iron- transporting property of these proteins is not the dominant factor responsible for extraction of the protease precursor.
  • lactoferrin to extract the IgA protease precursor from a cell membrane is sensitive to heat, pH, and the presence of chelators such as EGTA.
  • bovine milk that has been pasteurized or acidified lacks this desired activity of lactoferrin.
  • iron resaturation of acidified lactoferrin does not restore this activity of lactoferrin.
  • fresh bovine milk is able to extract the IgA protease precursor from the outer membrane.
  • Figs. 4A-C Processing of the Extracted Protease Precursor: Processing of the extracted protease precursor into the mature protease is inhibited by secretory IgAl (slgAl) . This result is illustrated in Figs. 4A-C.
  • HI cells were grown to mid-log phase in BHI, and 0.6 ml aliquots were incubated at 37°C for 45 minutes in milk, slgAl-depleted milk, or slgAl-depleted milk supplemented with slgAl. Following the incubation in milk, the cultures were centrifuged, and the cell-free milk supernatants were further incubated at 37°C for various lengths of time (0.75 to 24 hours) (Figs.
  • the IgA protease precursor subsequently was detected by western blot analysis using antibodies directed against the IgA protease. These results indicate that the IgA protease precursor remains substantially unprocessed in milk containing slgAl. In contrast, the protease precursor is processed over time when it is extracted into milk that lacks slgAl (i.e., jacalin-treated milk) (Fig. 4B) . The addition of slgAl to slgAl depleted milk blocks processing of the protease precursor.
  • HI cells were grown to mid-log phase, washed with saline, then aliquotted into 0.3 ml cultures. The cultures were incubated for 1 hour in 0.2 ml of milk, IgA depleted milk, or IgA depleted milk supplemented with IgA. The cells were pelleted and washed with saline.
  • the IgA protease activity in 30 ⁇ l aliquots of the supernatants was detected by assaying the supernatants for cleavage of radioactively labeled IgAl.
  • the combination of lactoferrin and IgA antibodies blocks the IgA protease from cleaving IgAl.
  • Lactoferrin to Extract IgA Protease From a Gram Positive Bacterium: To determine whether lactoferrin also is able to extract the membrane-bound IgA protease from gram positive bacteria, we measured the ability of lactoferrin to extract the IgA protease of Streptococcus sangui ⁇ .
  • the S . sanguis IgA protease lacks substantial homology to HI IgA protease, and differs from that of HI in that it is a metalloprotease rather than a serine protease. After 6-8 hours of growth, 50% of the IgA protease of this bacterium stays on the cell, and 50% is released into the culture supernatant.
  • the membrane- bound protease is thought to play a role in initiation of attachment and colonization of other pathogenic bacteria on the surface of saliva-coated teeth.
  • lactoferrin to a culture of S . sanguis causes approximately 70% of the IgA protease to be released in 1 hour; by contrast, only 10% of the protease is released when cells are grown for 1 hour in the absence of lactoferrin.
  • lactoferrin effect is not limited to gram negative bacteria, as lactoferrin is able to remove the protease from S . sangui ⁇ .
  • the activity of the protease can be inhibited with antibodies (e.g., antibodies raised against the extracted and gel- purified protease) according to the invention.
  • IgA Precursor-like Proteins Proteins from several other bacteria also are functionally or structurally related to the HI IgA protease precursor, and lactoferrin is expected to extract these IgA protease precursor-like proteins from cell membranes.
  • the precursor-like protein of vacuolating cytotoxin of Helicobacter pylori has a helper section, and the protein is substantially similar to IgA protease. Extraction and neutralization of this toxin can protect against conditions, such as chronic gastritis, which are attributed to H. pylori .
  • the Tsh protein of chicken E . coli shares over 60% sequence identity with HI IgA protease precursor.
  • the Tsh protein includes a helper section. Tsh serves as an agglutinin, facilitating attachment of chicken E. coli to epithelial cells.
  • extraction of this protein with lactoferrin alone, or in combination with antibodies directed against Tsh is a means for inhibiting septicemia caused by this pathogen in animals.
  • HAP protein of HI is associated with the ability of the organism to bind and enter host epithelial cells.
  • the protein shares sequence identity with the catalytic domain of IgA protease and with Tsh protein.
  • the HAP outer membrane protein is processed in a manner similar to that of IgA protease. Extraction of any of these proteins, combined with binding of the protein with antibodies (e.g., antibodies raised against the extracted and gel-purified protein) , is expected to inhibit the pathogenic effects of these bacteria (e.g., by precipitating the HAP protein) .
  • lactoferrin specifically extracts IgA protease precursor-like proteins from the outer membranes of cells, or if other cell surface proteins also are extracted.
  • proteins on HI cells were labeled with 35 S-methionine, and the cells were treated with lactoferrin.
  • the IgA protease precursor constituted approximately 80% of the labeled protein that was extracted from HI.
  • lactoferrin is specific for IgA protease precursor-like proteins.
  • the invention provides infant formulas which inhibit bacterial IgA proteases from cleaving human IgAl, the only known substrate for this class of proteases.
  • Formulations that include lactoferrin and antibodies that inhibit IgA proteases or IgA protease precursors or related proteins also can be used to inhibit the pathogenic effects of bacteria expressing these proteins in adults.
  • lactoferrin extracts IgA proteases or protease precursor-like proteins from the outer membranes of pathogenic bacteria. Once extracted, the proteases or protease precursor-like proteins are targeted by antibodies provided in the formula or formulation. Useful antibodies are those which specifically bind the protease precursor or protease precursor-like protein or a processed protease or protease-like protein. Antibodies that bind the precursor-like protein prevent processing of the precursor-like protein, while antibodies that bind the protease-like protein directly inhibit the activity of the protease.
  • the antibodies which are used in the invention can be raised against all or a portion of the mature IgA protease or protease-like protein, or all or a portion of the IgA protease precursor or protease precursor-like protein. Many, if not all, antibodies raised against such proteins will be able to inhibit the ability of the protease precursor to process itself, or the ability of the protease to cleave its target molecule (e.g., IgAl). Methods for preparing appropriate antibodies are described below.
  • a non-human mammal e.g., a cow
  • a non-human mammal e.g., a cow
  • a bacterial IgA protease precursor or protease precursor-like protein or a peptide fragment thereof e.g., the mature IgA protease
  • bovine immunization to obtain milk, or preferably colostrum, containing antibodies is preferably performed as follows. During the last month of gestation, the cow transfers a very large amount of circulating plasma IgG into its udder.
  • a parenteral (subcutaneous, intramuscular, etc.) injection of an IgA protease (or the preferred antigen) is preferably made during this period to stimulate high levels of circulating (plasma) antibody that eventually are transferred to the colostrum and milk.
  • This colostrum is then processed by conventional methods to produce the infant formula, formula additive, or pharmaceutical formulation. Because the desired activity of lactoferrin is sensitive to heat and acidification, processing steps which involve heat or acidification should not be carried out after lactoferrin is added.
  • a person e.g., an infant
  • the formula protects the mucosa in all of the areas where ingested milk normally contacts the mucosa, i.e., the oral cavity, nasopharynx, bronchial passages, and intestine. Because simple ingestion of maternal milk normally is sufficient to deliver antibodies to these mucosal surfaces, ingestion of the formula of the invention is expected to provide an adequate means for delivery of the formula to the preferred mucosa.
  • pregnant cows received two injections of IgA proteases mixed with 1 mg of the adjuvant Quil A.
  • Each immunization employed 3 ml saline and 1 mg IgA protease (or 6 mg IgA protease for a "cocktail") .
  • Injections were given approximately 28 and 14 days before expected calving. Inoculation was in four sites: 1 ml injected subcutaneously into each shoulder, and 0.5 ml injected intramuscularly into the gluteal region on each side) .
  • the maternal colostrum (approximately four liters) then was collected by venipuncture (bovine colostral whey proteins typically consist of 40% immunoglobulins, 35% beta-lactoglobulin, 15% lactalbumin and small amounts of albumin and other proteins) .
  • the colostrum was centrifuged for 30 minutes at 12,000 x g at 4°C to remove the fat (which floats to the top of the sample) .
  • the casein then was precipitated by incubating the colostrum for 1 hour with 15 units of rennin.
  • the curd subsequently was tightened by heating the sample to 56°C for 10 minutes, and the casein was removed by centrifugation at 4°C for 25 minutes at 12,000 x g. If desired, the colostrum may be frozen at this point.
  • active lactoferrin is added to the processed colostrum or milk to a concentration of 0.01-10 mg/ml, preferably 0.5-1.0 mg/ml.
  • the pH of the formula should not be permitted to drop below pH 4.0, preferably pH 5.0, and the formula should not be heated significantly (e.g., 90°C for 5 minutes, or 56°C for 30 minutes) .
  • lactoferrin is active at a temperature 18-37°C.
  • the colostrum or milk may be tested by culture for sterility, and it can be frozen until used.
  • Monoclonal IgA Protease Antibodies Monoclonal antibodies directed against bacterial IgA protease precursor-like proteins also can be used according to the invention. These antibodies or antibody fragments can be added to infant formulas in the same way as the bovine antibodies or bovine antibody containing colostrum described above.
  • Monoclonal antibodies useful in the invention can be made by standard techniques by immunizing mice with all or a portion of an IgA protease precursor-like protein, fusing the murine splenocytes with appropriate myeloma cells, and screening the antibodies produced by the resultant hybridoma lines for the requisite ability to bind the IgA protease precursor-like protein (e.g., by means of an ELISA assay) . A subsequent screening may be necessary to select binding antibodies that also inhibit or remove the protease or protease precursor. Monoclonal antibody production and screening can be performed according to Uchiyama et al. (J. Immunol . 126:1393,
  • useful antibodies may be isolated from a combinatorial library produced by the method of Huse et al. (Science 246:1275, 1989).
  • the invention can employ not only intact polyclonal or monoclonal antibodies, . but also an immunologically-active antibody fragment, for example, a Fab or (Fab) 2 fragment; an antibody heavy chain, an antibody light chain; a genetically engineered single- chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or a chimeric antibody, for example, an antibody that contains the binding specificity of a murine or bovine antibody, but in which the remaining portions are of human origin.
  • an immunologically-active antibody fragment for example, a Fab or (Fab) 2 fragment
  • an antibody heavy chain an antibody light chain
  • a genetically engineered single- chain Fv molecule Ladner et al., U.S. Patent No. 4,946,778
  • a chimeric antibody for example, an antibody that contains the binding specificity of a murine or bovine antibody, but in which the remaining portions are of human origin.
  • IgA Protease Inhibition Assav A suitable enzyme assay uses human myeloma IgAl immunoglobulin trace labelled with 125 I as a substrate. Fragmentation products of the IgAl heavy chain are measured by first separating these fragments on polyacrylamide gels. A suitable antibody inhibits protease activity by binding to the protease precursor or the protease. In this example, the enzyme activity is assayed. Generally, the assay involves quantitating the amount of IgAl Fab produced over time. The Fab produced can be measured by measuring release of the Fd fragment, which is the heavy chain of the Fab region.
  • a typical reaction mixture has a volume of 75 ⁇ l, consisting of 25 ⁇ l stock substrate solution (2 mg/ml purified IgAl with 2% labeled IgAl) , 25 ⁇ l of a colostrum dilution containing the antibody (or a Tris/HCl/BSA buffer control) , and 25 ⁇ l active IgA protease (Haemophilus, Neisseria or
  • IgA protease any IgA protease can be used. After the enzyme and antibody were incubated for 30 minutes at 37°C to allow them to interact, the IgAl substrate was added, and incubation continued at the same temperature. Aliquots of 10 ⁇ l were removed at 10-minute intervals over a 40 minute period and hydrolysis of IgAl measured as follows.
  • sample buffer contains 12.5% glycerol, 1.2% sodium dodecyl sulfate, 1.2% mercaptoethanol, and 0.001% bromphenol blue dye.
  • Aliquots of 100 ⁇ l were electrophoresed on 9% PAGE gels after which the gels were stained with 0.05% Coomassie Brilliant Blue, dried, and autoradiographed at -70°C using Kodak XAR5 film with an intensifying screen.
  • the developed autoradiograph was used as a template for cutting the stained and dried gel into segments consisting of 1) any uncleaved IgA heavy chain and 2) the 22 kD Fd alpha fragment representing that portion of the heavy chain within the Fab fragment digestion product. Radioactivity in the uncleaved heavy chain and in its Fd product were counted on a Beckman Biogamma Counter, background counts from control digests were subtracted from each value, and quantitation of IgA protease activity was expressed as a percentage of the heavy chain that has been cleaved using the formula:
  • FIG. 5 An inhibition assay from the colostrum of 18 cows is shown in Fig. 5.
  • the titers for 18 cows immunized with individual IgA proteases for the first time during a single calving are provided.
  • the preferred antigens are IgA proteases of Neisseria and Haemophilus .
  • Inhibiting antibodies were detected in milk obtained after a second immunization and second calving (Fig. 6) .
  • the levels of inhibition obtained with colostrum are higher than those obtained with milk, and antibodies from the second calving are more potent than those obtained with the first calving
  • the samples were normalized for IgG level. Even when IgG levels are accounted for, colostral antibodies were approximately 6 to 10-fold more inhibitory than were milk antibodies.
  • Fig. 7 provides a second set of examples of IgAl protease inhibition activities from serum, colostrum, and milk after a first lactation.
  • inhibiting antibodies were produced when the immunizing antigen was Hie, HIb, N. meningitidis IC or 2R.
  • antibody titers were greatest in colostrum.
  • Inhibiting antibodies were also obtained in serum at 14 or 21 days after calving (Fig. 7) .
  • the bovine colostral antibodies of the invention differ in inhibiting titre against various IgA proteases.
  • IgA proteases from HI of other serotypes (groups b (HiB) , e (HiE) , f (HiF) , and C (HiC) ) were inhibited less than 100 at the same level of activity.
  • Fig. 8A shows that enzymes having the same cleavage type as that of the immunogen are better inhibited.
  • it is preferable to use several enzymes to immunize each cow such that various, e.g. , Haemophilus , proteases are blocked.
  • antibodies from cows immunized with each protease can be combined for addition to infant formula.
  • Fig. 8B shows the inhibitory curves of cow #236, which was immunized with type 2 IgA protease of HI serotype e.
  • the reciprocal inhibitory titer was 3180.
  • the three proteases having type 2 specificity HiC 3 (2), HiE 27 (2), and HiE (2)
  • These curves in Fig. 8B show that in addition to serogroup specificity as shown in Fig. 8A, certain of the bovine antibodies of the invention also show more specificity for enzymes of the correct cleavage type.
  • this cow had a colostrum titer of 1280 and a milk titer of 20, which were the highest levels of all the cows, even though no inhibition was detectable after the first calving.
  • Bovine IgA Protease Antibody Using an anti-bovine IgG antiserum we have shown that the IgA protease antibody in bovine colostrum is IgG. This was done by exposing enzymes on western blots to the colostral preparations as a first antibody, and examining the isotype of the binding antibody by various antibodies against cow serum. Furthermore, chromatography of the inhibiting colostrum on Biogel P200 columns shows that the ability to inhibit co-elutes precisely with the IgG peak.
  • the inhibiting property of bovine colostral IgG is localized in the Fab fragment (as anticipated for an antibody) .
  • Papain enzyme/protein ratio of 1/100 w/w
  • proteolysis was stopped with Ca ++ added in excess, and the material examined on western blot analysis; this showed about 80% hydrolysis.
  • the Fab fragments were isolated by column chromatography on Biogel P100, and identified by their reduced molecular weight (changed elution position) and light chain content as determined by appropriate antisera.
  • This Fab fragment material was tested as an IgA protease inhibitor, and found to be 2-3 fold less effective than the starting material before proteolysis. Thus, the entire IgG antibody molecule is not required for IgA protease inhibition, although some inhibiting power could be lost by isolation of the monovalent Fab region of the antibody.
  • Lactoferrin and non-human antibodies to bacterial IgA protease-like proteins or protease precursor-like proteins can be added to bovine milk- based infant formulas. Like human milk, such formulas will inhibit IgA protease-like proteins and protease precursor-like proteins in the fluids bathing the nasopharynx and upper respiratory passages of the infant. The advantage of such inhibition is that, in general, the child's own IgAl will be protected from cleavage by the IgA proteases. Thus, all IgAl antibodies will benefit, not only those directed against the IgAl protease producing bacteria themselves.
  • the processed colostrum containing the antibodies can be diluted or concentrated and used in combination with lactoferrin as an infant formula additive.
  • Standard infant formulas include the SIMILAC® line of formulas (Ross Laboratories, Columbus, Ohio) and the SMA® line of formulas (Wyeth Laboratories, Philadelphia,
  • the colostral antibodies plus lactoferrin also can be used undiluted as an infant formula, with or without added nutrients.
  • the bovine antibodies to bacterial IgA proteases can also be purified from the colostrum and added along with lactoferrin to infant formula directly.
  • the antibodies and lactoferrin may be dried into a powder form to be added to formulas, or even maternal milk.
  • 10A and 10B are autoradiographs of polyacrylamide electrophoresis gels of 125 I-IgAl substrate subjected to IgA protease. These autoradiographs show uncleaved (IgA heavy chain) substrate, and fragments arising from proteolysis (Fab) . In all cases the protease assay was carried out for 30 minutes.
  • Fig. 10A shows that IgA protease is active when added to five commercial infant formulas
  • Fig. 10B shows that the IgA protease is inactive when added to formula supplemented with the inhibiting bovine antibodies of the invention.
  • Fig. 10A shows the gel resulting from Haemophilus influenzae type 1 IgA protease added to a number of commercial infant formulas, along with trace amounts of
  • the top two lanes are controls: the top lane is substrate without protease, which resulted in no cleavage products; the second lane is substrate and protease in buffer (not formula) to show the basic activity of the protease added to the formula products.
  • the subsequent five lanes show that unmodified formulas cannot affect activity of IgA protease; the Fab fragment is the cleavage product.
  • Fig. 10B shows that the Haemophilus influenzae IgA protease used above in the gel of Fig. 10A is inactive in infant formula (here NUTRAMIGEN®) supplemented by an anti-IgA protease from immune cow colostrum.
  • infant formula here NUTRAMIGEN®
  • the bottom two lanes are substrate and enzyme controls.
  • the top four lanes are NUTRAMIGEN® formula containing varying amounts of cow #1 immune colostrum: from top to bottom, the ratio of colostrum:formula (vol:vol) is 1:640, 1:1280, 1:2560, and 1:5120.
  • the enzyme added to all these dilutions is inactive, as shown by the inability to fragment any 125 I-IgA substrate during 30 minutes.
  • the bovine antibodies of the invention should be fed to an infant in combination with lactoferrin, preferably as a formula supplement, at a dosage of about 1.0 to 2.0 grams of bovine colostral antibody protein/kg/day and 0.01 mg/ml to 10 mg/ml of lactoferrin/kg/day, preferably 0.5-1.0 mg/ml, equally distributed over all feedings.
  • This dosage must be adjusted dependent on the titer of the antibodies in the particular colostral preparation used as the infant formula additive, because different colostrum preparations have different percentages of the IgA protease antibodies compared to total antibodies in the preparation.
  • the invention includes formulations that are made without milk, but which include lactoferrin and antibodies that inhibit the proteolytic activities of IgA proteases, protease precursors, protease-like proteins, and protease precursor-like proteins.
  • Non-milk based formulations that include antibodies and lactoferrin can be used as infant formulas.
  • Milk or non- milk formulations of the invention can be used to inhibit infections in adults.
  • S . sanguis is a major initiator of plaque formation which causes dental caries in adults. Lactoferrin and inhibiting antibodies can thus be formulated into a mouthwash or beverage, for example, for use in adults.
  • the formulations can also be used in treating Pneumococcus infections in adults and children.
  • the invention can be formulated for intrabronchial or intranasal administration.
  • Haemophilus influenza also infects adults, and the invention thus can be used to inhibit HI IgA proteases in adults as well.

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Abstract

La présente invention concerne des aliments lactés pour nourrissons, des additifs pour aliments lactés pour nourrissons et des formulations pharmaceutiques incluant une lactoferrine active combinée avec des anticorps qui se lient de façon spécifique à des protéines semblables à l'Iga protéase ou des protéines semblables au précurseur de l'Iga protéase. Ces aliments lactés pour nourrissons protègent les personnes qui boivent ces aliments lactés pour nourrissons ou ces formulations contre les effets pathogènes imputables aux bactéries ou à d'autres agents infectieux, les virus par exemple, qui infectent les voies respiratoires supérieures.
PCT/US1996/012447 1995-08-07 1996-07-29 Aliment lacte pour nourrissons et additifs correspondants WO1997005884A1 (fr)

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Cited By (10)

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WO1998006425A1 (fr) * 1996-08-12 1998-02-19 A+ Science Invest Ab Traitement et prevention d'infections, d'inflammations et/ou de tumeurs a l'aide de lactoferrine et/ou de lactoferricine
WO1999065329A2 (fr) * 1998-06-16 1999-12-23 Regen Biotech Limited Complement dietetique
EP0989860A1 (fr) * 1998-04-13 2000-04-05 New England Medical Center Hospitals, Inc. Methodes et compositions permettant d'inactiver des agents infectieux au moyen de lactoferrine et de molecules associees
EP1017286A1 (fr) * 1997-09-22 2000-07-12 Sepragen Corporation Separation sequentielle de proteines de lactoserum et leurs preparations
US6258383B1 (en) * 1998-08-14 2001-07-10 Lactoferrin Products Company Dietary supplement combining colostrum and lactoferrin in a mucosal delivery format
WO2003043517A2 (fr) * 2001-04-03 2003-05-30 Sweetfrice U.S.A., Inc. Compositions dentifrices
WO2004041004A1 (fr) * 2002-11-06 2004-05-21 Coloplus Ab Composition d'aliment pour animaux ou de produit alimentaire
US6852700B1 (en) 1996-10-03 2005-02-08 Ludwig Hirzfeld Institute Of Immunology And Experimental Therapy, Polish Academy Of Sciences Colostrinin, and uses thereof
WO2011130799A1 (fr) * 2010-04-23 2011-10-27 Probiotec Limited Compositions pharmaceutiques
EP2560679A1 (fr) * 2010-04-23 2013-02-27 Probiotec Limited Traitement de l'eczéma

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WO1993010818A1 (fr) * 1991-12-04 1993-06-10 New England Medical Center Hospitals, Inc. Preparation lactee pour nouveau-nes et additif pour cette preparation

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WO1993010818A1 (fr) * 1991-12-04 1993-06-10 New England Medical Center Hospitals, Inc. Preparation lactee pour nouveau-nes et additif pour cette preparation

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INFECTION AND IMMUNITY, 01 November 1995, Vol. 63, No. 11, LOMHOLT et al., "Distinct Antigenic and Genetic Properties of the Immunoglobulin A1 Protease Produced by Haemophilus Influenzae Biogroup Aegyptius Associated with Brazilian Purpuric Fever in Brazil", pages 4389-4394. *
J. OF DENTAL RESEARCH, 10 March 1993, Vol. 72, WITTLER et al., "Neutralization of Streptococcus-Sanguis IgA1 Protease by Lactoferrin and Transferrin", page 327. *
MOLECULAR MICROBIOLOGY, 01 January 1994, Vol. 11, No. 1, MORELLI et al., "Immunogenicity and Evolutionary Variability of Epitopes within IgA1 Protease from Serogroup A Neisseria Meningitidis", pages 175-187. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006425A1 (fr) * 1996-08-12 1998-02-19 A+ Science Invest Ab Traitement et prevention d'infections, d'inflammations et/ou de tumeurs a l'aide de lactoferrine et/ou de lactoferricine
US6852700B1 (en) 1996-10-03 2005-02-08 Ludwig Hirzfeld Institute Of Immunology And Experimental Therapy, Polish Academy Of Sciences Colostrinin, and uses thereof
EP1017286A4 (fr) * 1997-09-22 2004-06-09 Sepragen Corp Separation sequentielle de proteines de lactoserum et leurs preparations
EP1017286A1 (fr) * 1997-09-22 2000-07-12 Sepragen Corporation Separation sequentielle de proteines de lactoserum et leurs preparations
EP0989860A4 (fr) * 1998-04-13 2000-07-19 New England Medical Center Inc Methodes et compositions permettant d'inactiver des agents infectieux au moyen de lactoferrine et de molecules associees
EP0989860A1 (fr) * 1998-04-13 2000-04-05 New England Medical Center Hospitals, Inc. Methodes et compositions permettant d'inactiver des agents infectieux au moyen de lactoferrine et de molecules associees
WO1999065329A2 (fr) * 1998-06-16 1999-12-23 Regen Biotech Limited Complement dietetique
GB2354153A (en) * 1998-06-16 2001-03-21 Regen Biotech Ltd Dietary supplement
GB2354153B (en) * 1998-06-16 2002-10-16 Regen Biotech Ltd Dietary supplement
WO1999065329A3 (fr) * 1998-06-16 2000-07-13 Regen Biotech Limited Complement dietetique
US6258383B1 (en) * 1998-08-14 2001-07-10 Lactoferrin Products Company Dietary supplement combining colostrum and lactoferrin in a mucosal delivery format
US6410058B2 (en) * 1998-08-14 2002-06-25 Marcus B. Gohlke Methods of use for dietary compositions comprising lactoferrin and colostrum
US6475511B2 (en) 1998-08-14 2002-11-05 Lactoferrin Products Company Dietary supplement combining colostrum and lactoferrin in a mucosal delivery format
WO2003043517A2 (fr) * 2001-04-03 2003-05-30 Sweetfrice U.S.A., Inc. Compositions dentifrices
WO2003043517A3 (fr) * 2001-04-03 2003-09-18 Frederic Dana Compositions dentifrices
WO2004041004A1 (fr) * 2002-11-06 2004-05-21 Coloplus Ab Composition d'aliment pour animaux ou de produit alimentaire
AU2003274883B2 (en) * 2002-11-06 2009-08-27 Coloplus Ab A feed or food product composition
WO2011130799A1 (fr) * 2010-04-23 2011-10-27 Probiotec Limited Compositions pharmaceutiques
EP2560679A1 (fr) * 2010-04-23 2013-02-27 Probiotec Limited Traitement de l'eczéma
EP3202416A1 (fr) * 2010-04-23 2017-08-09 Probiotec Limited Composition comprenant lactoferrin et immunoglobuline
EP3210618A1 (fr) * 2010-04-23 2017-08-30 Probiotec Limited Composition comprenant lactoferrin et immunoglobuline pour le traitement de l'eczéma

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