The present invention provides an immunogenic conjugate comprising biologically deacylated gram-negative bacterial moieties linked to D. discoideum proteinase 1, as well as novel subunits thereof, and methods of making and using the conjugates in vaccines to treat sepsis and other infectious complications.

DETAILED DESCRIPTION OF THE INVENTION

The LPS Polysaccharide Antigen

Traditional methods of detoxifying LPS for antigen usage employ non-specific acid- or base-catalyzed hydrolytic processes to remove fatty acids from LPS polysaccharide antigens, and these processes cause undesired modifications of polysaccharide epitopes, as discussed hereinabove. In contrast, the present biological detoxification process relies on enzymes, produced by D. discoideum cells, to hydrolyze amide and ester bonds that link fatty acids to LPS. Because these enzymatic modifications are highly specific, this biological processing selectively removes toxic components, while preserving non-toxic epitopes needed for eliciting protective antibodies. Accordingly, the biological method of LPS detoxification, unlike chemical detoxification processes, completely deacylates LPS without hydrolyzing covalent bonds that link either diglucosamine, non-reducing terminal KDOs, or ethanolamine pyrophosphate groups to the polysaccharide of LPS.

The present method employs E. coli J5 as a source of PS antigen to exemplify the present method. As discussed hereinabove, native LPS from this organism can elicit antibodies cross-reactive with LPS from several other kinds of gram-negative bacteria. Biological processing of E. coli J5 by D. discoideum cells was employed as a means for detoxifying E. coli J5 LPS because previous studies indicated that D. discoideum cells naturally produced deacylated LPS derivatives as end-products of bacterial catabolism (D. Malchow et al., Eur. J. Biochem., 2, 469 (1967); 7, 239 (1969)). These studies also suggested that D. discoideum cells metabolically removed ester-linked and amide-linked fatty acids from the lipid A portion of LPS, but did not hydrolyze glycosidic bonds in the polysaccharide portion of LPS. In addition, these studies indicated that antibodies elicited against native LPS recognized some LPS catabolites produced by D. discoideum. However, it was not known that D. discoideum could metabolize the J5 strain of E. coli. Also, before the present invention, it was not known whether the forms of deacylated LPS generated by D. discoideum, whatever their structure, would have activity as immunogenic epitopes that would elicit antibodies that in turn, could recognize native forms of LPS. Further, prior art did not provide a method for isolating deacylated, E. coli J5 LPS from D. discoideum cultures.

Thus, the present invention represents the first reported use of a cellular slime mold, such as D. discoideum, to biologically extract and detoxify bacterial LPS in a form that it is useful as a vaccine antigen. The embodied biological method for producing detoxified LPS antigens is more economical and more efficacious than chemical processes used previously to prepare LPS vaccine antigens. Unlike previous isolation methods, the new biological method does not require toxic solvents to extract LPS. Further, the new process does not require that LPS be chemically fractionated before it is detoxified. Instead, detoxified LPS antigens are obtained directly as water-soluble end-products that are produced by cultures of D. discoideum cells grown on bacteria as a food source. The antigens are readily purified from D. discoideum culture media by selective filtration processes and by fractional precipitation of their barium salts in ethanol-water mixtures.

According to the present detoxification method, bacteria are cultured in liquid media, collected, and washed with a salt solution containing potassium chloride and magnesium chloride. When E. coli J5 was added to media containing both magnesium chloride and potassium chloride, the bacterial cells formed into aggregates that were readily phagocytosed by D. discoideum. The embodied method for culturing D. discoideum with bacteria uses phosphate-free media containing 5 mM to 50 mM potassium chloride and 1 mM to 10 mM magnesium chloride. The optimal concentrations of these salts may be different when different strains of bacteria are used in the embodied methods. For example, for E coli J5, the preferred concentrations for potassium chloride and magnesium chloride are 15 mM and 5 mM, respectively.

Washed bacteria are suspended in the same salt solution and seeded with D. discoideum spores or D. discoideum amoebae. The resulting suspension is incubated with stirring and aeration at a constant temperature between 15° C. and 25° C. Growth and aggregation of D. discoideum cells is tracked by periodic, microscopic examination of culture samples.

Incubation is continued until D. discoideum cells cease growing and collect into multi-celled aggregates. When these conditions are met, stirring and aeration of the cultures are discontinued, and the aggregated D. discoideum cells are permitted to sediment from the culture media. The culture media is then separated from the sediment and filtered to remove residual cells. Next, the media filtrate is mixed with 0.2 to 0.5 volumes of ethanol and the mixture is supplemented with a water-soluble barium salt. The addition of barium ions causes the formation of barium-antigen complexes that precipitate and sediment from the ethanol-media mixture.

The method for precipitating LPS antigens from D. discoideum culture media is novel. In a previous study (D. Malchow et al., cited above), LPS derivatives in D. discoideum culture media were concentrated by a multi-step method involving centrifugation, evaporation, and dialysis processes. These methods are undesirable for purifying LPS derivatives intended for use as vaccine antigens—first, because the centrifugation and evaporation processes are costly to perform at large scale; and second, because deacylated LPS antigens from E. coli J5 readily permeate conventional dialysis membranes.

In the present method for concentrating LPS antigens from filtered culture media, the media is adjusted to contain between 10 and 50% ethanol, and between 1 to 10 mM barium ions. A common, water soluble salt of barium, such as barium acetate or barium chloride, is used a source of barium ions. It is within the scope of these methods to substitute an alternative divalent cation for barium. For example, calcium ions may be more suitable than barium ions for precipitating some kinds of LPS antigens produced in D. discoideum cultures.

After incubation for at least 10 hours at a temperature of 0° C.-10° C., the sediment is collected, suspended in water, and treated with acid in order to remove barium ions from the PS antigen. Following this treatment, the PS antigen solution is neutralized by addition of an appropriate amount of a base such as potassium hydroxide, and the solubilized antigen is further purified by selective filtration and by fractional precipitation from solutions containing various concentrations of ethanol and various buffers.

Purified PS antigen, obtained by the present methods, has a sugar composition similar to that determined previously for the polysaccharide portion of native LPS from E. coli J5 (S. Muller-Loennies et al., Eur. J. Biochem., 260, 235 (1999)). The ratio of KDO:heptose:glucosamine:glucose:N-acetylglucosamine in purified antigen preparations was about 2:3:2:1:1, respectively. Phosphorous-31 NMR indicated that phosphate occurred in purified antigen molecules as diphosphodiester, and phosphomonoester forms. The structure of the PS antigen is depicted in FIG. 2.

In a final purification step, antigen is treated with a phosphomonoesterase to remove the 1′-phosphate from the diglucosamine group in each antigen molecule. This treatment generates one aldehyde or acetal functional group in each antigen molecule, that can be further modified, e.g., for direct attachment to the carrier protein, or reacted with a variety of (bis)functional linking molecules. This hydrolysis reaction is depicted in FIG. 3, step (1).

Linker Molecules

Following introduction of an aldehyde or ketal into the molecule, these groups can be reacted with a bis-functional linker such as adipic dihydrazide (ADH), followed by reduction of the Schiff base, to incorporate a linker group that can be used for subsequent conjugation of PS antigen molecules to carrier protein. This reaction is depicted in FIG. 3, step 2. In this derivatization reaction, antigen is incubated at about 20-40° C. for about 20 hours in a solution of formamide containing about 10% v/v sodium acetate at pH 5, or in an aqueous buffer between pH 4-6, containing an excess of sodium cyanoborohydride. Sodium borohydride may be subsequently added to derivatization reactions to enhance reduction of hydrazone bonds formed between antigen and ADH. These conditions support reactions that form antigen-hydrazide molecules that contain covalent hydrazide bonds linking aldehyde groups in antigen to α-hydrazide groups in ADH. The aldehyde group participating in this reaction represents the anomeric carbon in the reducing-end glucosamine of each antigen molecule.

Other linkers are available and can be used to link two aldehyde moieties, two carboxylic acid moieties, or mixtures thereof. Such linkers include (C₁-C₆) alkylene dihydrazides, (C₁-C₆)alkylene or arylene diamines, ω-aminoalkanoic acids, alkylene diols or oxyalkene diols or dithiols, cyclic amides and anhydrides and the like. For example, see U.S. Pat. No. 5,739,313.

Carrier Protein and Modifications Thereof

To prepare the present carrier molecule for the PS antigens, Proteinase 1, a lysosomal cysteine proteinase, was purified from D. discoideum cells by a novel method. Previously, Proteinase 1 was purified by methods that employed two or more chromatographic steps (G. L. Gustafson et al., J. Biol. Chem., 254, 12471 (1979); D. P. Mehta et al., J. Biol. Chem., 271, 10897 (1996); T. Ord et al., Arch. Biochem. Biophys., 339, 64 (1997)). These earlier methods were unsuitable for use in the present method because they resulted in poor recovery of purified enzyme, and the chromatographic steps were not desirable for large-scale production of the enzyme. The novel steps in the present method of Proteinase 1 purification include steps wherein the enzyme is precipitated from aqueous ethanol in the presence of barium acetate, and a step wherein the enzyme is precipitated in the presence of high concentrations of ammonium sulfate. By substituting these novel steps for chromatographic fractionation, it is possible to manufacture purified enzyme in much higher yield and at a much greater scale than achieved previously.

To convert purified Proteinase 1 to a form suitable for use as a carrier protein, the proteinase is reacted with sodium periodate in an aqueous, buffer adjusted to a pH between pH 5 and pH 6. The preferred concentration of periodate in this reaction mixture is between 50 mM and 150 mM, and the preferred reaction temperature is between -20° C. and 20° C., preferably about 0° C., and the desired reaction is the oxidative conversion of diol groups in the N-acetylglucosamine-1-phosphate (GlcNAcP) residues to dialdehyde groups.

It is believed that other proteins containing GlcNAcP-serine moieties, such as analogous lysosomal cysteine proteinases, can be obtained from D. discoideum or from other slime molds, including other species of Dictyostelium or species of Polysphondylium.

The practice of the present invention can be enhanced by genetically modifying the Dictyostelium cells that are used for producing Proteinase 1. For example, genetic modifications can provide Dictyostelium mutants that (1) produce larger amounts of Proteinase 1, (2) produce an altered form of Proteinase 1 that is easier to purify, or (3) produce an altered form of Proteinase 1 that contains a larger number of GlcNAcP residues. These enhancements can be achieved by transfecting Dictyostelium cells with DNA that codes for the synthesis of natural or modified forms of Proteinase 1. Recombinant DNA techniques have been adapted for use in genetic modifications of Dictyostelium (Jenne et al., J. Cell Sci., 111, 61 (1998); Moreno-Bueno et al., Biochem. J., 349, 527 (2000), and Agarwal et al., Differentiation, 65, 73 (1999)), and the use of methods to modify the genome of Dictyostelium so as to enhance either the manufacturing of Proteinase 1 or the carrier functions of Proteinase 1 are within the scope of the present invention.

Conjugation

To conjugate the carrier protein (i.e., the oxidized proteinase) with the PS antigen-hydrazide, the oxidized protein is desalted, suspended in aqueous buffer (preferably at a pH between pH 4 and pH 7), and reacted at a temperature of about 10° C.-30° C. for about 20-30 hrs, with antigen-hydrazide. The resulting mixture is then treated with an excess of sodium cyanoborohydride for about 24-72 hrs at about 0° C. to 20° C. As shown in FIG. 4, these conditions support reactions that generate covalent bonds between aldehyde groups in the carrier protein and hydrazide groups in PS antigen-hydrazide. With some antigens, the conjugation steps could be reversed, so that the ADH is first reacted with oxidized protease, then the free hydrazino group is reacted with an antigen aldehyde.

Upon completion of the conjugation reaction, the conjugate is separated from unconjugated antigen, desalted by dialysis, and filter sterilized. The sterile conjugate may be stored as an aqueous solution, a frozen solution, or as a freeze-dried product.

Vaccine Formulations and Vaccination

Vaccines of the invention are typically formed by incorporating the present PS antigen-carrier conjugates into pharmaceutically acceptable formulations. The formulations may contain pharmaceutically acceptable adjuvants (such as oils, surfactants, alum), immunostimulating agents (such as phospholipids, glycolipids, glycans, glycopeptides, or lipopeptides), and one or more diluents ("excipients"). Examples of diluents suitable for use are water, phosphate buffered saline, 0.15 M sodium chloride solution, dextrose, glycerol, mannitol, sorbitol, dilute ethanol, and mixtures thereof. Pharmaceutically acceptable unit dosage forms of the vaccines can be formulated as solutions, emulsions, dispersions, tablets, or capsules.

For human use, the vaccines are preferably administered parenterally, usually via subcutaneous or intramuscular routes of injection. Alternatively, they may be administered intraperitoneally, intravenously, or by inhalation. Oral dosage forms can also be employed, such as solutions or suspensions. In general, the vaccine of the present invention is formulated so that a dose of vaccine can be administered in a volume between 0.1 ml and 0.5 ml, but if given orally it could be administered in capsule or tablet form. The vaccine dosage, the number of doses given to an individual, and the vaccination schedule depend on the antigenicity and immunogenicity of the antigens in the conjugate and on other known pharmaceutical considerations such as the age and body weight of the individual.

The vaccines of the present invention will provide protective benefits for humans at high risk of developing sepsis and septic shock. These include elderly patients with chronic diseases, patients treated with aggressive chemotherapies or immunosuppressive therapies, patients receiving transplanted organs, and victims of severe traumatic injury. The vaccines of the present invention may also provide protective benefits in humans against one or more kinds of infections involving pathogenic gram-negative bacteria. The levels of protection obtained with the vaccine can correlate with blood titers of anti-LPS antibodies produced in vaccinated individuals. Dosages can also be extrapolated from dosages of toxoid-PS vaccines found to be safe and/or efficacious in humans. See, for example, U.S. Pat. Nos. 4,771,127 and 5,370,872.
 

Claim 1 of 9 Claims

1. An immunogenic conjugate comprising a plurality of deacylated lipopolysaccharide (LPS) molecules from a gram-negative bacterium covalently linked to glucosamine residues of isolated Dictyostelium discoideum Proteinase 1.

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