Cytophaga-Derived Immunopotentiator

The present invention is the field of immunology in general and immunopotentiators or adjuvants in particular. More specifically, it is directed to a pigment-free exopolymer adjuvant derived from Cytophaga strain GB-2.

Immunogenicity is the capacity of an antigen to induce a cellular and/or humoral response. Immunopotentiation is the enhancement of such a response or the development of a response to an otherwise nonimmunogenic material. Agents that have the ability to so enhance or develop an immune response are called "adjuvants" or "immunopotentiators". Adjuvants are used to increase the efficacy of vaccines and in the treatment of immunity-associated diseases such as cancer, immunodeficiency, and certain infective diseases.

Many bacterial components have been investigated as immunopotentiators. Among these are components from a psycrophilic gliding bacterium of the genus Cytophaga. This Gram-negative bacterium was originally isolated from a contaminated lot (designated FBS 762) of fetal calf serum (FCS). Shiigi, S. M. and Mishell, R. I., J. Immunol (1975) 115:741-745. Medium conditioned by the growth of these gliding bacteria was found to have immunopotentiating activity on cultures of murine splenocytes supplemented with deficient FCS. Studies comparing the adjuvant and mitogenic effects of this medium with those of purified lipopolysaccharide (LPS) from S. typhosa indicated the activity of the medium was attributable to substances other than LPS from the gliding bacterium Shiigi, M. S. et al. J Immunol, (1977) 119:679-684. A further report regarding this bacterium describes the preparation of exopolymers (EP) from the medium in which the bacterium (then designated Cytophaga sp) had been cultured by dialysis and lyophilization. The Ep was characterized as being water soluble and containing .ltoreq.0.2% protein and .ltoreq.0.001% LPS. It was found to potentiate immune responses to specific antigens and stimulate murine macrophages and macrophage cell lines to release colony stimulating factors and interleukin 1 (IL-1). Mishell, R. I. et al, in Chemistry and Biological Activities of Bacterial Surface Amphiphiles, Eds Shockman G. D. and Wicken A. J., Academic press, New York (1981).

The present invention provides a method of obtaining the above described Cytophaga exopolymer from the medium in which the bacterium is cultured in a substantially pigment-free form that exhibits unexpectedly high adjuvant activity. The exopolymer is sometimes referred to herein as "GBA" (gliding bacteria adjuvant) and the Cytophaga species that sheds or secretes it as "GB-2".

Accordingly, one facet of this invention is substantially pigment-free GBA.

Another aspect of the invention is a method of isolating substantially pigment-free GBA from medium in which GB-2 has been cultured comprising precipitating the GBA with acetone whereby pigment is extracted from the GBA. In a preferred embodiment of this method, residual protein and lipopolysaccharide are removed from the pigment-free GBA, respectively, by proteolytic digestion and filtration and by affinity chromatography

Yet another aspect of the invention is a method of potentiating an immune response in a subject comprising administering an immunopotentiating amount of substantially pigment-free GBA to the subject.

Still another aspect of the invention is an immunopotentiating composition comprising an immunopotentiating amount of substantially pigment-free GBA admixed with a pharmaceutically acceptable vehicle.

FIG. 1 is a graph of the results of the mitogenicity tests described in the example.

FIG. 2 is a graph of the results of the polyclonal activation tests described in the example.

FIG. 3 is a graph of the results of the in vitro adjuvanticity tests described in the example.

FIG. 4 is a graph of the results of the in vivo adjuvanticity tests described in the example.

FIG. 5 is a graph of the results of the IL-1 induction tests described in the example.

Cytophaga strain GB-2 was deposited at the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md., U.S.A. on 25 Apr. 1986 under accession number 53485 pursuant to the provisions of the Budapest Treaty. This deposit will be maintained and made accessible to others in accordance with the provisions of that Treaty. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The deposit of the strain does not constitute an admission that the written description of this application is inadequate to enable the practice of the any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims.

GB-2 grows only at temperatures below 30.degree. C. and is killed at 37.degree. C., with an optimal growth temperature of 25.degree. C.-28.degree. C. when cultured in mineral salts, 0.5% glucose, 1% lactalbumin hydrolysate media or in media E (J Biol Chem (1956) 218:97-106) supplemented with at least 0.1% glucose. Aeration of GB-2 is essential since cultures in log growth cease to divide once shaking is stopped. The bacteria are able to utilize glucose galactose, cellobiose, xylose, arabinose, saccharose, starch casein, or gelatin but not potassium-acetate, sodium lactate, glycerol chitin, or cellulose as substrates when inoculated onto minimal media agar plates consisting of mineral salts, yeast extract, and agar and cultured under aerobic conditions. No anaerobic growth of GB-2 was observed under these conditions; however the bacteria grew anaerobically on TS-blood agar plates in an atmosphere of nitrogen. GB-2 grows better when cultured in the presence of filter-sterilized glucose than autoclaved glucose. Cultures supplemented with autoclaved glucose grow more slowly, less densely and tolerate a narrower range of glucose concentratlons as compared to cultures grown in filtered glucose.

GB-2 is a gram-negative rod, that does not produce flagella but is capable of gliding mobility on agar supplemented with the media described above. Gliding was not observed when the bacteria were grown on standard Merck agar plates. Light and electron microscopy failed to show the production of fruiting bodies or microcysts by GB-2. By these criteria GB-2 is a member of the order Cytophagales.

GB-2, whether grown in aerobic an anaerobic conditions,synthesizes a yellow pigment which by spectral absorption analysis is believed to be flexirubin. The production of yellow pigment is characteristic of bacteria in the family Cytophagaceae within the order Cytophagales. The presence of the yellow pigment flexirubin is a useful chemosystematic marker for bacteria in the Cytophaga/Flexibacteria group. This noncarotenoid, upon treatment with 20% KOH, undergoes a spectral shift from yellow to red-violet. GB-2, as well as the yellow pigmented substances produced by GB-2, undergo a similar change in color upon this alkaline treatment.

Other characteristics displayed by GB-2 support its classification into the Cytophaga/Flexibacteria group. GB-2 was able to grow anaerobically and produced as major fermentation products succinic, acetic, lactic, propionic, and butyric acids. Most species of Cytophaga and Flexibacteria are aerobic bacteria but some species are facultative producing succinic acetic, lactic, propionic, and butyric acids as the major fermentation products. Furthermore, GB-2 possessed both catalase and oxidase enzyme activities, as do bacteria of these genera. The inability to distinguish clearly between Cytophaga and Flexibacteria genera stems largely from the lack of data on the unique characteristics of these bacteria. Although there are several exceptions, most species of Cytophaga are able to utilize complex carbohydrates like starch as a source of carbon, while species of Flexibacteria are generally able to utilize only simple sugars. GB-2 grows well on starch and therefore is consistent with its tentative classification as a member of Cytophaga.

Substantially pigment-free GBA is isolated from medium in which Cytophaga GB-2 has been cultured in the following manner. The bacteria are first separated from the medium by centrifugation. Preferably the supernatant is then concentrated fifty to one-hundred fold by ultrafiltration. The major contaminants in the concentrated supernatant are yellow pigment (presumably flexirubin), protein and LPS.

The next step in the isolation is the removal of pigment from the GBA by repeated extraction with acetone. Acetone is first mixed with the concentrated supernatant under conditions that cause the GBA to precipitate, leaving the pigment in solution. The precipitation is carried out at reduced temperatures. preferably -40.degree. C. to -10.degree. C., and most preferably approximately -20.degree. C. using an acetone:supernatant volume ratio in the range of about 8:1 to about 4:1, preferably about 5:1. The maximum light absorption by the pigment occurs at 455 nm and its presence in the precipitate may be monitored spectrophotometrically. In order to reduce the amount of pigment in the precipitate to undetectable levels as measured by absorbance at 455 nm, the precipitate is repeatedly dissolved in an aqueous medium and reprecipitated with acetone under the conditions given above.

After the pigment has been extracted protein and nucleic acid contamination may be lowered by enzymatic digestion followed by gel filtration of the digest in the presence of detergent, preferably a nonionic detergent such as Np-40 or a Triton detergent. Depending upon the conditions for enzymatic activity the digestion may have to be carried out in a plurality of steps. The precipitate is first dissolved and optionally dialyzed with water and the pH of the solution is adjusted to that at which the enzyme is active. If necessary, factors necessay for enzymatic activity are added to the solution. Since the purpose of the digestion is to degrade proteins and nucleic acids nonspecifically, it is preferable to use nonspecific nucleases and proteases such as DNase, RNase, pronase and pepsin in the digestion. Following the digestion, gel filtration is used to remove the low molecular weight protein and nucleic acid fragments from the higher molecular weight GBA. Nonionic detergent is added to the digest to promote complexing of the contaminants into micelles that are more easily removed by sizing. Gels in which GBA runs in the void while the contaminants are retarded is used. Preferably the gel has an exclusion point greater than about 200,000 daltons.

LPS may he removed from the purified GBA by affinity chromatography using a ligand that binds LPS, such as a polymyxin-B agarose column. LPS in the purified GBA will be retained by the column.

The resulting substantially pure GBA contains no pigment as determined by absorbance at 455 nm, less than about 0.5% by weight protein as measured by the amino acid analysis method, and less than about 0.01% by weight LPS as measured by the Limulus gelation assay. Electron micrographs of this purified GBA shows that it exists as spherical particles about 200 to 1200 A.degree. in diameter. Its molecular weight is estimated by gel permeation chromatography and electron microscopy at about 10.sup.6 to 10.sup.8 daltons. The extreme water solubility of GBA and its reactivity in the phenol-sulfuric hexose assay suggest it is a polysaccharide. Elemental analysis of purified GBA is consistent with GBA being a polysaccharide. GBA (as measured by biological activity) is stable to treatment with strong base (1.5 M sodium hydroxide), chaotropic agents (potassium thiocyanate and guanidine hydrochloride), and detergent (NP-40).

The following disclosure further illustrates the production and characterization of GBA and its biological activities. The following disclosure is not intended to limit the invention in any manner.

Two hundred liters of a Cytophaga species GB-2 was grown to late log phase in defined, dialyzable medium E. Bacteria were removed by continuous centrifugation and supernatant was concentrated 50-100 fold by ultrafiltration (Pelican, Millipore Inc. Bedford, Mass.). The filtrate was recentrifuged (10,000.times.g for 45 min). The supernatant was frozen until further use.

Aliquots of crude exopolymer were precipitated with -20.degree. C. acetone at a 5:1 v/v ratio (acetone:supernatant) and allowed to stand at 4.degree. C. for at least 2 hr prior to centrifugation (2,000.times.g for 15 min). Precipitates were redissolved with water and reprecipitated, as above, four more times. The final precipitate was dissolved and dialyzed against water and was digested at 37.degree. C. with mixing overnight with DNase and RNase (neutral pH. 5 mM Mg.sup.++, 100 .mu.g of each enzyme/ml). The pH was adjusted to 4 with acetic acid and pepsin (200 .mu.g/ml) was added. After overnight mixing at 37.degree. C., NP-40 was added (0.1% final concentration) and the mixture was neutralized and boiled for 10 min. Aliquots were then passed over a column of Sephacryl-300 gel. Material in the void volume of the gel was collected and dialyzed exhaustively against water and passed over a 10 ml column of polymyxin-B agarose and lyophilized.

Various physical and chemical analyses of the crude and purified GBA exopolymer were carried out. Table 1 below summarizes the results of those analyses.

Based on the chemical analysis of the exopolymer, it is believed to be an amino sugar polymer with no primary amines or cis-hydroxyls.

The following studies were carried out to evaluate various aspects of the biological activity of purified GBA.

Murine spleen cells Were cultured in medium containing various quantities of purified GBA. After 48 hr the cultures were pulsed with tritiated thymidine. The cells were harvested 18 hr after the pulse and tritiated thymidine incorporation was measured with a scintillation counter. FIG. 1 is a graph of the results with incorporation expressed as counts per minute (cpm) plotted against GBA concentration.

Murine spleen cells were cultured in medium containing various concentrations of GBA for four days. Supernatants were then assaYed for murine Ig using a standard enzyme-linked immunosorbent assay (ELISA). FIG. 2 is a graph of the results of the assay with relative Ig concentration plotted against GBA concentration.

Adjuvanticity of GBA was evaluated by the Mishell-Dutton culture assay. In this assay mouse spleen cells were cultured with sheep red blood cells for five days in 1 ml of medium supplemented with 5% FCS (selected lots) and various amounts of purified GBA. FIG. 3 is a plot of the results of this test.

Two pairs of Balb/c mice were injected i.p. with 10 .mu.g of cytochrome C (a suboptimal amount of antigen) with and without 50 .mu.g purified GBA emulsified in PBS containing 0.2% Tween.RTM. 80 surfactant, 2.5% Pluronic.RTM. L121 surfactant, and 5% squalane. Serum antibodY titers were determined 9-11 days post injection by enzyme immunoassay. FIG. 4 is a plot of the assay results.

Murine macrophage cells (WEHI 265) were cultured for three days in media containing various amounts of purified GBA. Culture supernatants were tested for IL-1 content by the thymocyte comitogen assay (measuring incorporation of tritiated thymidine by cultured murine thymocytes). FIG. 5 is a graph of the results with tritiated thymidine incorporation expressed as cpm plotted against GBA content.

As shown in FIGS. 1-5, GBA was effective in potentiating the investigated activities at the nanogram level except for polyclonal activation. Comparison of specific activities for the purified GBA observed in the B cell mitogenicity assay and the IL-1 induction assay with specific activities observed for crude GBA in these assays show that the pure material is approximately 50-100 fold more active than the crude material. Such an increase is much greater than would be expected simply due to removal of inactive substances. In this regard it is believed that the difference in activity is due to inhibition of activity by one or more of the removed substances, most likely the pigment.

Other in vitro tests have shown that GBA induces human peripheral blood monocytes to secrete IL-1.

Two strains of mice, C57B1/6J and C57B1/CR1, were injected i.p. with either saline, 100 .mu.g poly (IC), or 50 .mu.g GBA. Spleens were removed 18 hr after injection and assayed for cytotoxic activity against .sup.51 Cr-labeled YAC target cells. Table 2 presents the results of the assays.

As indicated, GBA induced significant activity in one of the strains of mice.

GBA is nonspecies specific in its biological activity and is, therefore, useful in potentiating immune responses in vertebrate subjects in general, including humans. It will typically be used in prophylactic or therapeutic treatment of humans, domestic animals such as cattle, horses sheep and pigs, or sports and pet animals such as cats and dogs. In a prophylactic setting the pigment-free GBA will typically be incorporated into animal or human vaccines, particularly those considered "weak" vaccines (i.e., those that provide diminished protection in terms of level, extent, and/or duration). Examples of such vaccines are bacterins such as Bordetella bacterin, Escherichia coli bacterins, Haemophilus bacterins, Leptospirosis bacterins, Moraxella bovis bacterin, Pasteurella bacterin and Vibrio fetus bacterin; and attenuated live or killed virus products such as bovine respiratory disease vaccine (infectious bovine rhinotracheitis, parainfluenze-3, respiratory syncytial virus), bovine virus diarrhea vaccine, equine influenza vaccine, feline leukemia vaccine, feline respiratory disease vaccine (rhinotracheitis calici-pneumonitis viruses), canine parvovirus vaccine. transmissible gastroenteritis vaccine, Newcastle disease virus vaccine, and pseudorabies vaccine. The adjuvant may also be used with various recombinant or subunit vaccines.

Accordingly, the vaccines will contain an immunogen that is effective in immunizing a patient against a given pathogen or antigen a pharmaceutically acceptable vaccine carrier, and an immunopotentiating amount of pigment-free GBA. If desired, the vaccine formulation may contain minor amounts of other conventional vaccine additives such as surfactants and preservatives. The particular formulation will depend upon the individual being immunized, the immunogen, and the vaccination regimen. A preferred vaccine vehicle for use in formulating the immunogen and GBA is phosphate-buffered saline (PBS) containing a Pluronic.RTM. nonionic surfactant such as Pluronic.RTM. L121 surfactant (Pluronic.RTM. surfactants are polyoxyalkylene derivatives of propylene glycol), a Tween.RTM. surfactant, such as Tween.RTM. 80 surfactant (Tween.RTM. surfactants are polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides), and squalane. This formulation may be prepared by: mixing the surfactants and squalane in PBS: adding the adjuvant to the mixture with agitation to form a vehicle-adjuvant mix having the consistency of thin milk; and then adding the immunogen in PBS to the adjuvant-vehicle mix. Preferred concentrations of the vehicle additives are Tween.RTM. surfactant, 0.2%, Pluronic.RTM. surfactant 2.5%, and squalane, 5%. Alternatively, again depending upon the individual, immunogen and regimen it maY be desirable to coadminister the GBA as a separate formulation. As yet another alternative it may be desirable to covalently bond the immunogen to the GBA by conventional methods used to couple or conjugate antigens to carriers.

The GBA of the invention may also be used as a prophylactic for potentiating a nonspecific response. Such treatment is used for instance, to protect animals or humans from conditions such as shipping fever in the case of domestic animals, or immunosuppression due to disease, chemotherapy or other causes. Further, the GBA may be used in a therapeutic setting to treat immunodeficiencies such as congenital deficiencies or deficiencies caused bY viral or other infections.

The GBA will normally be administered in amounts ranging between about 1 .mu.g to 1 mg. The particular amount will depend upon the individual being treated, that individual's history the mode of administration, the formulation, and the condition for which the GBA is being administered. The GBA will normally be administered parenterally (e.g., by injection). Oral administration, however may be feasible. Depending upon the purpose of the administration, administration via a sustained release formulation, such as via an implant, may be desirable.

Modifications of the above described modes of carrying out the invention that are obvious to those of skill in the fields of microbiology, biochemistry. immunology and/or pharmacology are intended to be within the scope of the following claims.