EP0989860A1

EP0989860A1 - Methods and compositions for inactivating infectious agents using lactoferrin and related molecules

Info

Publication number: EP0989860A1
Application number: EP99917408A
Authority: EP
Inventors: Andrew G. Plaut; Jiazhou Qiu
Original assignee: New England Medical Center Hospitals Inc
Current assignee: New England Medical Center Hospitals Inc
Priority date: 1998-04-13
Filing date: 1999-04-12
Publication date: 2000-04-05
Also published as: CA2294018A1; WO1999052545A1; EP0989860A4; AU3554099A; JP2002508009A

Abstract

A method for substantially reducing the pathogenicity of an infectious agent, without killing the infectious agent, by removing or degrading a surface protein of the infectious agent, by contacting the infectious agent with substantially pure, non-pasteurized, naturally occurring lactoferrin under conditions sufficient to remove or degrade the protein, is disclosed.

Description

METHODS AND COMPOSITIONS FOR INACTIVATING INFECTIOUS AGENTS USING LACTOFERRIN

AND RELATED MOLECULES

Statement as to Federally Sponsored Research The invention was made with funding from the National Institutes of Health, grants NIDDK DK34928, DE 09677, HD 20859, and Al 19641. The government has certain rights in the invention.

Background of the Invention The invention relates to methods and compositions for inactivating infectious agents, such as bacteria and viruses.

Infections, especially bacterial, fungal, and viral infections, are an increasingly serious health threat. The great variety of microbes and viruses, as well as their ability to develop resistance to the therapeutic agents used to inactivate them, presents a constant challenge in modern medicine. Relatively common infections can cause serious illness, and even death, in immuno- compromised patients. Infections can also affect the long-term health of otherwise-healthy patients; even when the infections themselves are successfully treated, the secondary effects can cause lasting damage to the body.

The vast majority of people experience their first bacterial infection early in life. For example, an especially common early childhood infection is acute otitis media, which is a suppurative infection of the middle ear. By the time they have reached the age of three, 80% of children have suffered from acute otitis media, and 40-50% have experienced at least three episodes. Otitis media accounts for over one-third of all pediatric office visits in the United States and is the most common reason for prescribing oral antibiotics. Following each episode of otitis media, fluid persists in the middle ear for weeks to months, causing hearing impairment that can result in deficiencies in language acquisition, speech development, and cognitive achievement.

Most cases of otitis media are caused by infection with Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis. Infection begins with colonization of the nasopharynx, followed by contiguous spread through the eustachian tube to the middle ear. Colonization is a complex process and involves the interplay of bacterial and host factors. Successful colonization requires that an organism evade local immune responses and overcome clearance by the mucociliary escalator. For example, both S. pneumoniae and H. influenzae secrete an IgAl protease, which specifically cleaves and inactivates human IgAl, the predominant secretory antibody in the upper respiratory tract. In addition, all three of these respiratory pathogens elaborate adhesins, which promote attachment to the host epithelium and prevent the physical removal of bacteria from colonization sites.

Summary of the Invention In one aspect, the invention features a method for substantially reducing the pathogenicity of an infectious agent, without killing the infectious agent, by removing or degrading a surface protein of the infectious agent; the method includes contacting the infectious agent with substantially pure, non- pasteurized, naturally occurring lactoferrin under conditions that are sufficient to remove or degrade the protein. Examples of infectious agents include bacteria, such as H. influenzae, and viruses. Examples of surface proteins include autotransported colonization factors, such as IgAl protease, and -3- adhesins, such as Hap.

In a related aspect, the invention features a method for substantially reducing the pathogenicity of an infectious agent, without killing the infectious agent, by removing or degrading a surface protein of the infectious agent; the method includes contacting the infectious agent with recombinant lactoferrin under conditions sufficient to remove or degrade the protein. In another related aspect, the invention features a method for substantially reducing the pathogenicity of an infectious agent, without killing the infectious agent, by removing or degrading a surface protein of the infectious agent; the method includes contacting the infectious agent with a substantially pure fragment of non-pasteurized, naturally occurring lactoferrin under conditions sufficient to remove or degrade the protein. A preferred fragment is the N-terminal lobe of lactoferrin.

In a second aspect, the invention features a method of inhibiting microbial colonization in a mammal, such as a human, by administering to the mammal a therapeutically effective amount of substantially pure, non- pasteurized, naturally-occurring lactoferrin. In a related aspect, the invention features a method of inhibiting microbial colonization in a mammal by administering to the mammal a therapeutically effective amount of a substantially pure fragment of non-pasteurized, naturally-occurring lactoferrin, such as the N-terminal lobe of lactoferrin.

In a third aspect, the invention features a method for substantially inactivating an infectious agent; the method includes contacting the infectious agent with substantially pure, non-pasteurized, naturally-occurring lactoferrin under inactivating conditions. In a related aspect, the invention features a method for substantially inactivating an infectious agent; the method includes contacting the infectious agent with a substantially pure fragment of lactoferrin -4- under inactivating conditions, where the fragment has at least 100 amino acid residues. In a preferred method, the fragment has at least 200 amino acid residues. For example, a preferred fragment is the N-terminal lobe of lactoferrin. Preferably, the fragment is non-pasteurized and/or is isolated from naturally-occurring lactoferrin.

In a fourth aspect, the invention features an antimicrobial pharmaceutical composition including substantially pure, non-pasteurized, naturally-occurring lactoferrin and a pharmaceutically acceptable carrier. The composition may be formulated, e.g., for administration by the gastrointestinal tract (e.g., by oral administration), by inhalation, by the mucous membranes, or by the eyes. In a related aspect, the invention features an antimicrobial pharmaceutical composition including a substantially pure fragment of non- pasteurized, naturally-occurring lactoferrin and a pharmaceutically acceptable carrier. A preferred fragment is the N-terminal lobe of lactoferrin. In a fifth aspect, the invention features a method for producing an attenuated vaccine including the steps of (a) contacting an infectious agent with lactoferrin under conditions sufficient to substantially inactivate the infectious agent; and (b) formulating the inactivated infectious agent into a vaccine. Preferably, the lactoferrin is non-pasteurized and/or is isolated from a naturally- occurring source. In a related aspect, the invention features a method for producing an attenuated vaccine including the steps of (a) contacting an infectious agent with a substantially pure fragment of lactoferrin under conditions sufficient to substantially inactivate the infectious agent; and (b) formulating the inactivated infectious agent into a vaccine. A preferred fragment is the N-terminal lobe of lactoferrin.

In a sixth aspect, the invention features an attenuated vaccine including a substantially inactivated infectious agent, where the infectious -5- agent is inactivated with lactoferrin. Preferably, the lactoferrin is non- pasteurized and/or is isolated from a naturally-occurring source. In a related aspect, the invention features an attenuated vaccine including a substantially inactivated infectious agent, where the infectious agent is inactivated with a substantially pure fragment of lactoferrin, such as the N-terminal lobe of lactoferrin.

In a seventh aspect, the invention features a substantially pure peptide consisting of the N-terminal lobe of lactoferrin, where the lobe is isolated from non-pasteurized, naturally-occurring lactoferrin. By "non-pasteurized" is meant not subjected to the conditions, such as physical (e.g., heat) or chemical (e.g., acid) conditions, that result in sterilization of a milk product.

By "fragment of lactoferrin" is meant an amino acid sequence having antimicrobial activity, but which is shorter than the full-length lactoferrin protein for any given mammalian species.

By "substantially inactivated" is meant that the infectivity or pathogenicity of an agent is reduced, as measured by any standard assay.

By "substantially modified" is meant that all or a portion of one or more of the factors necessary for infectivity are removed from an infectious agent, or degraded.

By "substantially pure" is meant that a compound, such as lactoferrin or a fragment of lactoferrin, has been separated from components which naturally accompany it, or which are generated during its preparation or extraction. For example, a "substantially pure fragment of lactoferrin" is separated from other fragments of lactoferrin. Preferably the lactoferrin preparation is at least 50%, more preferably at least 80%, and most preferably at least 95%, by weight, free from the other proteins, lipids, and other naturally- occurring molecules with which it is naturally associated. The purity of lactoferrin or fragments of lactoferrin can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. By "inactivating conditions" are meant the conditions, such as time of treatment, temperature, pH, salt composition, and concentration of lactoferrin or lactoferrin fragment, under which infectious agents can be inactivated by the methods and compositions of the invention.

By "infectious agent" is meant an agent, such as a bacterium or a virus, capable of causing disease in animals.

By "antimicrobial" is meant an agent capable of reducing the infectivity or pathogenicity of any microscopic infectious agent, including, without limitation, any bacteria or virus.

By a "surface protein" is meant a protein or protein-like factor found on or near a surface, such as a cell wall or a virus coat, that contributes to the infectivity of an infectious agent.

By "vaccine" is meant an agent effective to confer the necessary degree of immunity against an infectious agent while preferably causing only very low levels of morbidity or mortality in a host organism population. By "pharmaceutically acceptable carrier" is meant any standard pharmaceutical carrier, buffer, or excipient currently used, including, without limitation, phosphate buffered saline solution, water, oil/water emulsions, water/oil emulsions, wetting agents, and adjuvants.

The methods and compositions of the invention are useful for inhibiting microbial, including viral, infections. They can be used to selectively inactivate surface proteins of microbes, such as those necessary for colonization or infectivity. At the same time, they leave other microbial -7- activities unchanged, thus providing useful compositions for the preparation of attenuated vaccines.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings

Figs. 1 A- ID are Western blot analyses showing the effect of lactoferrin on the IgAl protease precursor. Fig. 1 A illustrates removal of the native IgAl protease precursor and the remnant helper domain from wild-type Rd H. influenzae cells by milk whey; Figs. IB and 1C show removal of the IgAl protease preprotein from H. influenzae Rd3-13 cells by human milk whey; and Fig. ID demonstrates removal of the IgAl protease preprotein from H. influenzae cells by recombinant human lactoferrin.

Figs. 2A-2C are Western blot analyses showing that treatment of H. influenzae strain DB1 17 with human milk lactoferrin or A. awamori recombinant human lactoferrin results in degradation of the Hap preprotein and Hapβ. Fig. 2 A shows whole cell lysates of H. influenzae strain DB117 derivatives preincubated with PBS alone, and with PBS and 13 μM human milk whey lactoferrin; Fig. 2B illustrates whole cell lysates of H. influenzae strain DB117 derivatives preincubated with PBS alone, and with PBS and 13 μM A. awamori recombinant human lactoferrin; and Fig. 2C shows culture supernatants of H. influenzae strain DB117 derivatives preincubated with PBS alone, and with PBS and 13 μM A. awamori recombinant human lactoferrin. Figs. 3A-3C demonstrate the effect of human lactoferrin on Hap- mediated H. influenzae adherence to human epithelial cells. Fig. 3A is a graphical representation showing adherence to Chang epithelial cells by

DB117/vector and DB1 17/HapS243A after incubation in PBS, human milk whey lactoferrin, or recombinant lactoferrin; Fig. 3B is a light micrograph showing DB 1 17/HapS243 A adherence to Chang epithelial cell samples after incubation in PBS; and Fig. 3C is a light micrograph showing DB1 17/HapS243A adherence to Chang epithelial cell samples after incubation in recombinant lactoferrin.

Fig. 4 is a Western blot analysis illustrating the effect of the serine protease inhibitor PMSF on lactoferrin-associated proteolysis ofH. influenzae Hap.

Figs. 5A-5D are Western blot analyses showing that the outer membrane proteins P2, P5, and P6 are not removed by exposure to human milk whey.

Description of the Preferred Embodiments The invention is based on the discovery that non-pasteurized lactoferrin isolated from naturally-occurring sources inactivates infectious agents, such as bacteria, without killing the infectious agents. For example, lactoferrin is believed to attenuate the pathogenicity of bacteria by extracting and/or deactivating bacterial cell wall proteins and similar factors that are necessary for colonization and infection, while leaving bacterial viability relatively unaltered. Fragments of lactoferrin can extract and deactivate these proteins as well. In addition, since viruses also have coat proteins that play important roles in their ability to infect host organisms, lactoferrin and its fragments are useful for inactivating viruses as well.

In one particular example, lactoferrin removes the H. influenzae IgAl protease preprotein from bacterial cell walls. Once this protein is separated from the cell wall, it can be inhibited by milk anti-IgAl protease antibodies. Lactoferrin and its fragments also proteolytically degrade, and -9- therefore inactivate, the Hap adhesin on the bacterial surface, thereby diminishing the ability of the bacteria to adhere to epithelial cells.

It is believed that the ability of lactoferrin and its fragments to extract and degrade membrane proteins is dependent, at least in part, on proteolysis; i.e., that lactoferrin is a protease or acquires proteolytic activity through interaction with target proteins. This hypothesis is supported the observation that both extraction and degradation are inhibited by pretreatment of lactoferrin preparations with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. Fragments of lactoferrin can also be used to extract and degrade these membrane proteins. There is no minimum size for the fragments that can be used; the only requirement is that the fragments are large enough to retain proteolytic activity. Lactoferrin fragments may be generated by standard techniques of molecular biology (e.g., by standard deletion procedures) or, particularly for short fragments, by chemical synthetic approaches. Once generated, the fragments are tested for proteolytic activity using any standard assay (including the assays described herein). Glycosylation of these fragments may be accomplished in vivo by production in an appropriate host cell (e.g., a mammalian host cell) or in vitro using purified glycosylation enzymes or cell extracts.

In some applications, however, larger fragments of lactoferrin are preferred. For example, fragments of lactoferrin having at least 100, and more preferably 200, amino acid residues are preferred for some uses.

The active sites responsible for the extraction/degradation capabilities appear to reside on the N-terminal lobe of lactoferrin, which has 334 amino acids. Therefore, fragments of lactoferrin including at least a portion of the N-terminal lobe and having this activity are also useful in the -10- invention. A preferred fragment is the N-terminal lobe itself; this lobe has been shown to be as effective as full-length lactoferrin in extracting the H. influenzae IgAl protease from bacterial cells and in degrading the Hap adhesin.

Lactoferrin from a variety of sources may be used in the methods and compositions described herein; both lactoferrin isolated from natural sources and recombinant lactoferrin have been shown to exhibit proteolytic activity. It is important that the lactoferrin not be pasteurized, as pasteurization destroys the proteolytic activity of lactoferrin. Exemplary sources of lactoferrin include bovine milk and human milk. Lactoferrin and fragments of lactoferrin can also be produced using synthetic or recombinant methods, for example, as described in Stowell et al., Biochem. J. 276: 349-355 (1991). When recombinant lactoferrin or lactoferrin fragments are used, it is important that they are glycosylated in a manner similar to that of naturally-occurring lactoferrin, for example, using the methods described above. Targets for lactoferrin include any number of surface proteins.

Exemplary proteins include IgAl protease and Hap, which belong to the same family of gram-negative bacterial autotransported secretory proteins. IgA proteases are the prototypes of a family of gram-negative bacterial proteins that undergo autosecretion, as described in Jose et al., Mol. Microbiol. 18: 378-380 (1995). The H. influenzae strain Rd protease described herein is synthesized as a 185 kDa protein with four domains, including an N-terminal signal sequence, a central serine protease (IgAp), a highly basic and helical alpha domain (IgAoc), and a carboxy-terminal beta or helper domain (IgAβ) (Poulsen et al., Infect. Immun. 57: 3097-3105 (1989); Pohlner et al., Nature 325: 458-462 (1987)). The signal sequence directs export across the bacterial inner membrane, and is then cleaved. Subsequently, the remainder of the protein (hereafter called the preprotein) inserts into the outer membrane via the beta -11- domain. This domain is predicted to form a β-barrel structure with a hydrophilic channel, thus facilitating translocation of the protease and the alpha domain to the extracellular space. Ultimately, the protease domain gains catalytic activity and cleaves within the alpha domain to release itself from the surface of the organism.

The H. influenzae Hap protein is a nonpilus protein that promotes intimate interaction with human epithelial cells (St. Geme III et al., Mol. Microbiol. 14: 217-233 (1994)). It was originally identified by its ability to confer the capacity for in vitro attachment and invasion when expressed in a nonadherent, noninvasive laboratory strain of H. influenzae. Hap shares significant sequence homology (30-35% identity and 51-55% similarity) with the H. influenzae and Neisseria gonorrhoeae IgAl proteases, and undergoes autosecretion via an analogous pathway. It is produced as a 155 kDa protein with three functional domains, including an N-terminal signal sequence, a surface localized serine protease domain (Hap_s), and a C-terminal outer membrane domain (Hapβ) (Hendrixson et al., Mol. Microbiol. 26: 505-518 (1997)). Ultimately, the Hap_s domain mediates an autoproteolytic cleavage event, releasing itself from Hapβ and from the surface of the organism. It is believed that attachment to host epithelial cells is a function of the preprotein (Hap_s linked to Hapβ), prior to autoproteolytic cleavage. Lactoferrin inactivates this protein by degrading the Hap_s domain.

Thus, these proteins both contain a C-terminal domain that is embedded in the membrane, and an N-terminal protease domain that resides on the surface of the organism until released to the extracellular medium by autoproteolysis of the preprotein (the passenger domain). Proteins sharing these characteristics are also expected to be affected by lactoferrin or its fragments. Examples of such proteins include the polyprotein precursors of -12-

Neisseria gonorrheae, H. mustelae, Bordetella spp., Serratia marcescens, Helicobacter pylori, E. coli, S. flexneri, and B. pertussis.

Because certain other outer membrane proteins of H. influenzae are resistant to the proteolytic effects of lactoferrin, the presence of the N-terminal passenger domain may be important for interaction with lactoferrin and its fragments. For example, P2, P5, and P6 are H. influenzae outer membrane proteins that, like IgAβ and Hapβ, are believed to form β-barrel structures. P2, P5, and P6, however, lack N-terminal passenger domains. These proteins are unaffected by lactoferrin.

Use

Because lactoferrin and its fragments inhibit the colonizing ability of infectious agents, lactoferrin preparations have significant therapeutic potential. Pharmaceutical compositions including lactoferrin, or fragments of lactoferrin, can be formulated for administration by the gastrointestinal tract. Examples of formulations for oral administration include tablets, capsules, and liquid formulations. Oral formulations of lactoferrin can be especially valuable as supplements for infant formulas. When lactoferrin is administered orally, it can enter the bloodstream, e.g., via gastrointestinal absorption, and can thereby affect tissues remote from the site of administration. Lactoferrin and its fragments can also be formulated as eye drops, as nasal sprays, or as any other formulation suitable for inhalation.

Since non-pasteurized lactoferrin and fragments of lactoferrin inactivate proteins necessary for colonization, while leaving bacterial viability relatively unchanged, lactoferrin and its fragments may also be used to produce attenuated vaccines. For example, bacteria may be contacted with lactoferrin under conditions sufficient to extract and/or degrade the proteins in the -13- bacterial cell walls, and the attenuated bacteria may then be formulated into a vaccine. Methods for preparing vaccines are known in the art and can be found, for example, in Vaccines, G. Slorein and E. Martance eds., 2d ed. Saunders, Harcourt-Brace 1994. There now follow particular examples of the inactivation of infectious agents according to the invention. These examples are provided for the purpose of illustrating the invention, and should not be construed as limiting.

Example 1 : Extraction of protease from H. influenzae Rd in Milk Whey

Haemophilus influenzae strain Rd is a nonencapsulated derivative of a serotype Rd strain that secretes type 1 IgAl protease. The Rd strain was grown in brain heart- infusion broth supplemented with hemin (10 μg/ml) and nicotinamide adenine dinucleotide (2 μg/ml) to mid-log phase, and then harvested by centrifugation.

Human milk was obtained 3 to 6 days postpartum from healthy mothers taking no antibiotics, as described in Plaut et al., J. Infect. Dis. 166:43 (1992). All samples were collected in sterile beakers; within 6 hours of collection they were centrifuged at 10,000 x g for 20 min at 4^°C to remove lipids and cells. The resulting whey was stored at -70 C and was prepared for use by thawing slowly, without further modifications. 2 x 10⁹H. influenzae cells were resuspended in 1 ml of the unmodified human milk whey and incubated at 37 C with gentle mixing. Samples were removed at intervals between 2 minutes and 1 hour. Whole cells and supernatants were examined by Western immunoblot using antisera directed against all domains of the preprotein. -14-

As shown in Fig. 1 A, human milk whey removed the native IgAl protease precursor and the remnant helper domain from wild-type Rd H. influenzae cells. Lanes 1 and 2 show broth cultures of Rd cells. The cells in Lane 1 contained preprotein (P), and the remnant helper domain (β) from processed preprotein. The broth supernatant in Lane 2 produced two main bands, both of which were active IgAl proteases released during culture. Lane 3 shows the same Rd cells incubated 1 hour with milk whey, which removed the precursor and beta domains. The precursor (*) was transferred to the milk supernatant in Lane 4; it was unprocessed, since milk contains antibodies that inhibit processing of the precursor in solution. The extracted helper beta domain was unstable in solution, and was not detected. Lactoferrin is indicated by arrow Lf.

The antiserum used was anti-Rd3-13, which reacts with IgAp, IgAα, and IgAβ. Lactoferrins (Lf) were detected by the second antibody, an enzyme- conjugated goat anti-rabbit IgG.

Example 2: Extraction of protease from H. influenzae RD3-13 in

Milk Whey

H. influenzae strain Rd3-13 is an Rd derivative that expresses enzymatically inactive IgAl protease which cannot autoprocess, leading to the accumulation of preprotein in the bacterial outer membrane. Mid-log phase Rd3-13 bacteria were incubated in milk whey, and aliquots were removed at the times shown. Just before extraction, the cell-associated preprotein ran at a higher than expected position on an electrophoretic gel.

Bacterial pellets (Fig. IB) and their corresponding whey supernatants (Fig. IC) were examined using unadsorbed rabbit anti-Rd3-13 preprotein.

After incubation for 10 minutes in milk, only a small amount of IgAl protease -15- preprotein remained cell-associated; by 60 minutes, extraction was complete. Following transfer to the supernatant, the protein was very slowly degraded to lower-molecular-weight species. Solid arrowheads show the preprotein, and the brackets designate degradation products of the preprotein in whey. The controls, in lanes C, were Rd3-13 cells incubated for 60 minutes in buffer alone. Preprotein in the controls remained associated with the bacterial cells.

Quantitation of colony forming units of Rd or Rd3-13 after incubation in milk for two hours, when nearly all of the preprotein had been extracted, showed no effect on viability.

Example 3: Determination of Active Constituents of Milk Whey

Milk whey proteins were fractionated by precipitating the proteins with acetone, then subjecting them to anion exchange (DE 52, Whatman, England), followed by molecular sieve chromatography (Biogel P 200, Pharmacia, Richmond, CA). All steps were performed in neutral buffers, at room temperature or 4^°C in neutral buffers. The resulting fractions were tested for activity. In experiments with both Rd and Rd3-13, only fractions containing lactoferrin reproduced the findings with unmodified milk whey.

Highly purified recombinant forms of the full-length protein from two sources, baby hamster kidney (BHK) cells, and Aspergillus awamori recombinant lactoferrin (provided by Agennix Corporation, and described in Stowell et al., Biochem. J. 276, 349-355 (1991)), as well as the wild-type N- lobe of human lactoferrin produced in BHK cells, were tested.

Recombinant proteins were used at a concentration of 1 mg/ml (13 μM), approximating levels of lactoferrin in human milk (Masson et al., Clin. Chim. Acta 14, 735 (1966)). The results are shown in Fig. ID. Lanes Al-4 are unmodified human milk whey; Lanes Bl-4 are baby hamster kidney -16- recombinant human lactoferrin. Lanes Al and Bl show Rd3-13 cells (the arrow P shows the preprotein). Lanes A2 and B2 show cells after incubation in whey (A) or 13 μM recombinant lactoferrin (B); Lanes A3 and B3 show the corresponding supernatants. Lanes A4 and B4 show milk and lactoferrin controls containing no bacteria. The antiserum used was anti-Rd3-13, which reacts with IgAp, IgAa, and IgAb. Lactoferrins (Lf) were detected by the second antibody, an enzyme-conjugated goat anti-rabbit IgG.

As shown in Fig. ID, lactoferrin purified from BHK cells removed the IgAl protease preprotein (*) from strain Rd3-13, and then slowly degraded the extracted protein (Lanes Bl-4, brackets). The N-lobe of human lactoferrin had an identical effect. In addition, both sources of lactoferrin caused an upward shift of the preprotein. Lactoferrin iron content, which was varied according to the protocol of Mazurier and Spik (Biochim. Biophys. Acta 629:399-408 (1980)), had no influence on either extraction or degradation. To ensure that no other proteins were present in the recombinant lactoferrin preparations, molecular mass measurements of these proteins were carried out by mass spectroscopy using a Maldi-Tof linear instrument. The intact, glycosylated BHK lactoferrin was 79,338 daltons, and glycosylated N- lobe was 36,890 daltons. Both of these values were very close to the predicted values for these species.

Example 4: Effect of Human Milk Lactoferrin on Hap Adhesin The effect of 13 μM human milk lactoferrin on the Hap adhesin, which is structurally similar to IgAl protease, was also examined. Fig. 2A shows the analysis of whole cell lysates of H. influenzae strain DB117 derivatives preincubated with PBS alone (left), and with PBS and 13 μM human milk whey lactoferrin (right). Fig. 2B illustrates the analysis of whole -17- cell lysates of H. influenzae strain DBl 17 derivatives preincubated with PBS alone (left), and with PBS and 13 μM A. awamori recombinant human lactoferrin (right). Fig. 2C shows the analysis of culture supematants of H. influenzae strain DBl 17 derivatives preincubated with PBS alone (left), and with PBS and 13 μM A. awamori recombinant human lactoferrin (right). Western analysis was performed with antiserum Rab730, which reacts with the Hap preprotein, Hap_s, and Hapβ. The gels in all panels were loaded as follows: Lane 1, DBl 17/vector with PBS; Lane 2, DBl 17/wild type Hap with PBS; Lane 3, DBl 17/HapS243A with PBS; Lane 4, DBl 17/Hapβ with PBS; lane 5, DBl 17/vector with lactoferrin; lane 6, DBl 17/wild type Hap with lactoferrin; Lane 7, DBl 17/HapS243A with lactoferrin; and Lane 8, DBl 17/Hapβ with lactoferrin. Arrowheads indicate the Hap preprotein and Hapβ, arrows indicate Hap degradation products, and asterisks indicate Hap_s. As shown in Figs. 2A-2C, lactoferrin treatment of strain DBl 17 expressing wild-type Hap resulted in proteolysis, rather than extraction of Hap. The preprotein and Hapβ were lost, and a C-terminal fragment slightly smaller than Hapβ (39 kDa vs. 45 kDa) appeared.

To determine whether proteolysis depended on Hap serine protease activity, the effect of lactoferrin on DB 117 expressing Hap with a mutated active site serine (HapS243A) was examined. This protein lacks autoproteolytic activity and remains in the outer membrane in preprotein form. Western analysis of whole cells revealed loss of the Hap preprotein and generation of a Hap C-terminal fragment (Fig. 2A, Lanes 3 and 7). Treatment of DB 1 17 expressing a Hap derivative containing the Hap signal sequence fused to Hapβ also resulted in generation of the cell-associated 39 kDa C- terminal fragment (Fig. 2 A, Lanes 4 and 8), indicating that proteolysis of the exposed segment of Hapβ by lactoferrin could take place in the absence of the entire Hap_s domain.

Example 5: Effect of Recombinant Human Lactoferrin on Hap As shown in Fig. 2B, 13 μM recombinant human lactoferrin prepared from A. awamori generated two products, one being the same 39 kDa C-terminal fragment observed with milk-derived lactoferrin, and the other being a slightly smaller C-terminal fragment. Further analysis revealed that Hap_s or a related fragment of the Hap preprotein, was liberated into the supernatant (Fig. 2C).

Experiments comparing the proteolysis of Hap by 87 nM, 217 nM, 430 nM naturally-occurring human lactoferrin with 13 μM recombinant lactoferrin established a dose-response relationship, with proteolysis detectable but incomplete after treatment of cells for 1 hour with the lowest concentration.

Additional studies with BHK recombinant human lactoferrin yielded results that paralleled those obtained with A. awamori recombinant protein. As seen with IgAl protease, the recombinant N-lobe behaved exactly like the full- length protein.

Example 6: Inhibition of Hap-Mediated Attachment Strain DBl 17 expressing HapS243A was incubated for 1 hour in PBS alone, and in PBS with 13 μM lactoferrin. It was washed twice, and then inoculated onto a monolayer of Chang epithelial cells. Following incubation for 30 minutes, adherence was quantitated as described by St. Geme III et al., Proc. Natl. Acad. Sci. USA 90: 2875 (1993). Adherence is reported relative to DBl 17/HapS243A after incubation in PBS, which was normalized to 100%. Fig. 3A illustrates adherence to Chang epithelial cells by -19-

DB1 17/vector and DBl 17/HapS243A after incubation in PBS, PBS with 13 μM human milk whey lactoferrin, or PBS with 13 μM A. awamori recombinant lactoferrin. Figs. 3B and 3C show light micrographs of DBl 17/HapS243A associated with Chang epithelial cells samples after staining with Giemsa stain. The sample in Fig. 3B was incubated in PBS, and the sample in Fig. 3C was incubated with 13 μM A. awamori recombinant lactoferrin.

DBl 17 expressing HapS243A demonstrated augmented in vitro adherence compared with DBl 17 expressing wild-type Hap, reflecting the fact that attachment is mediated by the preprotein form of Hap, which remains intact and cell-associated when the active site serine is mutated. As shown in Fig. 3 A, treatment of DBl 17/HapS243A with either milk-derived or recombinant lactoferrin resulted in an 85-97% decrease in Hap-mediated adherence. DBl 17/vector served as a negative control and was nonadherent, regardless of lactoferrin treatment.

Example 7: Effect of Serine Protease Inhibitor PMSF on Lactoferrin-

Associated Proteolysis of H. influenzae Hap To determine whether lactoferrin was functioning as a serine protease, the ability ofphenylmethylsulfonyl fluoride (PMSF), a broad inhibitor of serine proteases, to inhibit degradation of Hap was examined. The results are shown in Fig. 4. DBl 17/HapS243A was incubated in PBS (Lane 1), PBS with 430 nM A. awamori recombinant lactoferrin (Lane 2), PBS with 430 nM recombinant lactoferrin and 7.5% isopropanol (Lane 3), or PBS with recombinant lactoferrin and 7.5 mM PMSF in isopropanol (Lane 4). Whole cell lysates were prepared and examined by Western blot analysis with antiserum Rab 730, which reacts with the Hap preprotein, Hap_s, and Hapβ. The arrowhead indicates the Hap preprotein, and the arrows indicate Hap -20- degradation products. As shown in Fig. 4, the partial proteolysis of Hap produced by 430 nM recombinant lactoferrin was significantly inhibited by 7.5 mM PMSF.

Lactoferrin extraction of the IgAl protease preprotein was also inhibited in the presence of 10 mM PMSF or 10 mM diisopropylfluorophosphate (DFP, a second serine protease inhibitor).

Example 8: Determination of the Specificity of the Interaction of

Lactoferrin and H. influenzae Proteins

The H. influenzae major outer membrane proteins P2, P5, and P6 are predicted to form β-barrel structures that include a series of transmembrane antiparallel amphipathic β sheets (Vachon et al., Biochim. Biophys. Acta 861 : 74-82 (1986); Nelson et al., Infect. Immun. 56: 128-134 (1988); Deich et al., J. Bacteriol. 170: 489-498 (1988); Munson et al., Infect. Immun. 61 : 4017-4020 (1993)), as do IgAβ and Hapβ. However, P2, P5, and P6 lack the characteristic large extracellular domains that link IgAβ and Hapβ to their N- terminal passenger domains in the autotransported proteins.

Logarithmic phase cells of H. influenzae were incubated with saline (sal) or whey; the results are shown in Figs. 5A-5D. Cells were centrifuged, and the pellets (Lanes P) and corresponding supernatants (Lanes S) were examined by immunoblot assay. The panel marked IgA protease (Fig. 5A) was probed with rabbit serum #331, an antiserum that recognizes IgAp, IgAα, and IgAβ. The other panels (Figs. 5B-5D) were probed with monoclonal antibodies specific for the proteins noted: OMP P2: antibody 6G3; OMP P5: antibody 2C7; OMP P6: antibody 7F3. Proteins were detected with protein A peroxidase and horseradish peroxidase color developer. Cells in the OMP P2 panel (Fig. 5B) were strain 1479 for which antibody 6G3 is specific. Cells in all other -21- panels were Rd3-13. Molecular mass markers (as kDa) are on the right.

As shown in Figs. 5A-5D, the IgA protease precursor was translocated to supernatant from cells by milk whey, while the P2, P5, and P6 outer membrane proteins were unaffected. All three proteins remained cell associated.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

What is claimed is:

Claims

-22-Claims

1. A method for substantially reducing the pathogenicity of an infectious agent, without killing said infectious agent, by removing or degrading a surface protein of said infectious agent, said method comprising contacting said infectious agent with substantially pure, non-pasteurized, naturally occurring lactoferrin under conditions sufficient to remove or degrade said protein.

2. The method of claim 1, wherein said infectious agent is a bacterium or a virus.

3. The method of claim 2, wherein said infectious agent is H. influenzae.

4. The method of claim 1, wherein said protein is an autotransported colonization factor, an IgAl protease, an adhesion, or Hap.

5. A method for substantially reducing the pathogenicity of an infectious agent, without killing said infectious agent, by removing or degrading a surface protein of said infectious agent, said method comprising contacting said infectious agent with recombinant lactoferrin under conditions sufficient to remove or degrade said protein.

6. A method for substantially reducing the pathogenicity of an infectious agent, without killing said infectious agent, by removing or degrading a surface protein of said infectious agent, said method comprising contacting said infectious agent with a substantially pure fragment of non- -23- pasteurized, naturally occurring lactoferrin under conditions sufficient to remove or degrade said protein.

7. A method of inhibiting microbial colonization in a mammal comprising administering to said mammal a therapeutically effective amount of substantially pure, non-pasteurized, naturally-occurring lactoferrin.

8. The method of claim 7, wherein said mammal is a human.

9. A method of inhibiting microbial colonization in a mammal comprising administering to said mammal a therapeutically effective amount of a substantially pure fragment of non-pasteurized, naturally-occurring lactoferrin.

10. The method of claim 6 or 9, wherein said fragment is the N- terminal lobe of lactoferrin.

1 1. A method for substantially inactivating an infectious agent comprising contacting said infectious agent with substantially pure, non- pasteurized, naturally-occurring lactoferrin under inactivating conditions.

12. A method for substantially inactivating an infectious agent comprising contacting said infectious agent with a substantially pure fragment of lactoferrin under inactivating conditions, wherein said fragment has at least 100 amino acid residues.

13. The method of claim 12, wherein said fragment has at least 200 -24- amino acid residues, is the N-terminal lobe of Icatoferrin, is non-pasteurized, or is isolated from naturally-occurring lactoferrin.

14. An antimicrobial pharmaceutical composition comprising substantially pure, non-pasteurized, naturally-occurring lactoferrin and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein said composition is formulated for administration by the gastrointestinal tract, by inhalation, by the mucous membranes by the eyes, or orally.

16. An antimicrobial pharmaceutical composition comprising a substantially pure fragment of non-pasteurized, naturally-occurring lactoferrin and a pharmaceutically acceptable carrier.

17. The composition of claim 16, wherein said fragment is the N- terminal lobe of lactoferrin.

18. A method for producing an attenuated vaccine comprising the steps of

(a) contacting an infectious agent with lactoferrin under conditions sufficient to substantially inactivate said infectious agent; and

(b) formulating said inactivated infectious agent into a vaccine.

19. The method of claim 18, wherein said lactoferrin is non- pasteurized, or is isolated from a naturally-occurring source. -25-

20. A method for producing an attenuated vaccine comprising the steps of

(a) contacting an infectious agent with a substantially pure fragment of lactoferrin under conditions sufficient to substantially inactivate said infectious agent; and

(b) formulating said inactivated infectious agent into a vaccine.

21. The method of claim 20, wherein said fragment is the N-terminal lobe of lactoferrin.

22. An attenuated vaccine comprising a substantially inactivated infectious agent, wherein said infectious agent is inactivated with lactoferrin.

23. The vaccine of claim 22, wherein said lactoferrin is non- pasteurized, or is isolated from a naturally-occurring source.

24. An attenuated vaccine comprising a substantially inactivated infectious agent, wherein said infectious agent is inactivated with a substantially pure fragment of lactoferrin.

25. The vaccine of claim 24, wherein said fragment is the N- terminal lobe of lactoferrin.

26. A substantially pure peptide consisting of the N-terminal lobe of lactoferrin, wherein said lobe is isolated from non-pasteurized, naturally- occurring lactoferrin.