EP0211045A1

EP0211045A1 - Method & product for enhancing circulating antibody response

Info

Publication number: EP0211045A1
Application number: EP86900966A
Authority: EP
Inventors: Chin Chuan Chang; Yun-Yen Tsong; Harold A. Nash
Original assignee: Population Council Inc
Current assignee: Population Council Inc
Priority date: 1985-01-25
Filing date: 1986-01-24
Publication date: 1987-02-25
Also published as: WO1986004243A1; AU5359186A

Abstract

Vaccin contenant un antigène et un adjuvant comprenant des cellules de Streptococcus hemolyticus ayant été traitées avec de la pénicilline. L'adjuvant améliore la réaction de l'anticorps en circulation contre l'antigène.A vaccine containing an antigen and an adjuvant comprising cells of Streptococcus hemolyticus having been treated with penicillin. The adjuvant improves the reaction of the circulating antibody against the antigen.

Description

Descrjption

Method &• . Product for Enhancing Circulating Antibody Response

Background of the Invention This application is a continuation-in-part of copending application Serial No. 694,798, filed January 25, 1985.

The present invention relates to a novel method for enhancing circulating antibody response and to a novel vaccine containing cell material of Strepto¬ coccus hemolyticus as an adjuvant.

A defensive reaction called the immune response is evoked when a macromolecule foreign to a given animal gains entrance into its blood or tissues. In one manifestation of the immune response, the foreign macromolecule or antigen binds first to a T lympho¬ cyte, then the immunological information is passed to the B lymphocyte, causing it to undergo development into a plasma cell, which in turn produces an antibody to the antigen. The antibody circulates throughout the blood or tissues until it recognizes and combines with the antigen which elicited its formation, thereby hastening its destruction and elimination and fre¬ quently immediately rendering the antigen inactive. It has been well established that stimulation of such immuno-competent cells will lead to enhanced antibody production. Also, maintenance of the antigen level contributes to prolongation of antibody produc¬ tion. One way of enhancing the immune response is to prepare a vaccine consisting of an antigen together with a substance, which for reasons frequently not well understood, increases and prolongs antibody production.

One particularly well known adjuvant is the water-in-oil emulsion developed by Dr. Freund. When Freund's adjuvant is combined with an antigen and then injected into an animal, the immune response elicited is generally much greater and of longer duration than the immune response elicited from the antigen alone. One of the disadvantages frequently associated with the use of Freund's adjuvant is a pyrogenic response in the subject animal. Another disadvantage of

Freund's adjuvant is that it causes skin eruptions and lesions because of its nonabsorbability by the tissues. Still another disadvantage of Freund's water-in-oil emulsion is the tendency of the emulsion to be unstable upon storage.

The initial exposure of an animal to an antigen usually evokes a primary response, in which circulat¬ ing antibodies are in most cases detectable after 1 to 30 days more. The time needed to attain maximal con- centration of antibodies, and the duration of peak titer vary with the antigens and methods of immuniza¬ tion. The concentration of circulating antibodies may begin to decline within several days after reaching -the maximum level. The adjuvant activity of mycobacteria is now interpreted to be due largely to a complex glycolipid. The immunogenic-enhancing properties of the myco¬ bacteria have been duplicated by a synthesized glyco- peptide called, generally, muramyl dipeptide, and more particularly N-acetyl-muramyl-1-alanyl isoglutamine. Antigens combined with adjuvants of muramyl dipeptide usually have low activity unless introduced into the host animal via a water-in-oil emulsion.

Another widely used procedure for enhancing the immune response of humans involves administration of inorganic gels, such as alum, aluminum hydroxide or aluminum phosphate with adsorbed antigens. The immu- nogenic activity of an alumadsorbed antigen promotes an antibody-producing effect, but one which is signi- ficantly less than that of Freund's adjuvant in terms of maximum titer achieved and duration of circulating antibodies. It has long been recognized in the art that strains of Streptococcus hemolyticus (also known as Streptococcus pyogenes . have anti-tumor activity, but are dangerous to use in cancer therapy because of their pathogenicity. U.S. patent No. 3,477,914 to Oka oto et al. describes processes for treating cells of Streptococcus hemolyticus whereby anti-tumor activity is retained while causing the cells to have no virulence and no hemolytic activity. A product prepared according to the teachings of this patent is available from Chugai Pharmaceutical Co. Ltd. under the trade names OK-432 or Picibanil. The product marketed under the trade names OK-432 and Picibanil contains penicillin used in treating the Streptococcus hemolyticus cells; Banil is a designation for a non- marketed product similar in all respects to Picibanil except that it is substantially penicillin- ee. A number of investigators have reported on studies concerning the mechanism of the anti-tumor action of streptococcal preparations including OK-432 and Picibanil. The literature is varied in its teach¬ ings. Kai, S. et al., Clin Expl. Immunol. 37 (1979) 98-105, reports on experiments showing enhanced cir¬ culating antibody response to hamster erythrocyte or nucleated chicken erythrocytes when Picibanil is given to mice intraperitoneally every other day for a total of 15 doses prior to injection of the antigen. On the other hand, Ohsugi, Y. et al., Seikagaku (biochemis¬ try) 43, 515, 1971, found that OK-432 had no effect on antibody-producing ability in the mouse, and further¬ more, suppressed- the immune response in mice against sheep red blood cells. Also Kiyohashi, A., Kitasato Med., 8: 359-362, 1978, in studying the effect of protein-bound polysaccharide preparation PS-K and streptococcal preparation OK-432 on the immune response of mice to sheep red blood cells concluded that OK-432 did not enhance antibody production in normal ddY mice. On the other hand, Watanabe, T., et al., Microbol. Immunol. Vol. 25(2), 205-208, 1981, found enhanced humoral antibody response in mice against influenza virus by OK-432.

Summary of the Invention It has been found that a vaccine containing

Streptococcus hemolyticus cells treated by cultivation in the presence of penicillin results in enhancement of the circulating antibody response to particular antigens. Preferably, the cells of Streptococcus hemolyticus thus treated are combined with known anti¬ gens and other known adjuvants to form a vaccine which unexpectedly enhances circulating antibody concentra¬ tion by from 3 to 14 fold, as compared with control formulations, based on tests conducted in rabbits. The vaccine of this invention provides the desired enhancement with simultaneous, same site administra¬ tion of the antigen and adjuvant. Furthermore, booster response is enhanced by using the cell prepa¬ ration in the primary injection, irrespective of whether it is used in a booster injection, and pro¬ vides enhancement in the booster.

Detailed Description of the Invention

The preparation of cells of Streptococcus hemo¬ lyticus for use according to this invention is gen- erally in accordance with U.S. Patent No. 3,477,914. Following a preferred embodiment, the cell prepara¬ tions referred to in the working examples of this application were prepared as follows:

The initial innoculum used is lyophilized cells of a nearly avirulent strain Su ATCC 21060 of Strep¬ tococcus hemolyticus preserved in an ampule under reduced pressure in meat-infusion broth. The cells are cultivated serially and used within the fifth or sixth subculture in the broth as seeding material. The cultivation is performed by incubation at 37°C. and the culture is maintained at 2-5°C. during inter¬ vals between transfers.

Two types of media are used. The meat-infusion broth is prepared according to directions for Medium, General Test Procedures in the Japanese Biological

Product Standards. The pH of the broth is adjusted to between 7.3 and 7.5. The broth is sterilized at

2 121°C. for 20 minutes under pressure of 1 Kg/cm .

The yeast extract medium is prepared by dissolv- ing 50 g. of yeast extract in 800 ml of water, with the pH of the solution adjusted to 7.3 to 7.5. It is then heated at 110°C. for 10 minutes, filtered using Toyo Filter Paper No. 5C (Toyo Roshi Kaisha, Ltd.) and sufficient purified water is added to make 1000 ml. The medium is then filtered through a membrane filter with a 0.45 pore size (HAWP, Millipore -Corporation) to remove the fine insoluble materials, and then steri¬ lized at 121°C. for 30 minutes under pressure of 1 Kg/cm². The bacterial strain is innoculated into meat- infusion broth and cultivated at 37°C. for 20 to 24 hours. One volume of the resulting culture is added to 20 volumes of yeast extract medium, prepared as described above, and the mixture is cultivated at 37°C. for 20 hours to obtain a final culture.

The bacterial cells are collected from the final culture by centrifugation and suspended in physiologi¬ cal saline. One volume of 10% (W/V) hydrogen peroxide solution is added to 10 volumes of cell suspension in the saline solution and mixed. The mixture is allowed to stand for 30 minutes at 2°C. and the cells then collected by centrifugation. The cells are resuspended in physiological saline and a cell concentration that will give an absorbance between 0.33 and 0.36 at 390 mμ as determined with a Hitachi Spectrophotometer Model 202-20, using a sample made by diluting one volume of the cell suspension with 19 volumes of physiological saline. Within one hour after the start of resuspending the washed cells in BBM, one volume of a solution of potassium salt of penicillin G (1.6 x 10 units/ml) is added to five volumes of the cell suspension in BBM, the suspension mixed, and then incubated at 37°C. for 20 minutes. The suspension is then heated to 45°C. for 30 minutes, and the suspension cooled.

After cooling, the cells are collected by centri¬ fugation and resuspended in the same amount of a solu- tion made by combining 5 vol. of BBM with penicillin and 1 vol. of physiological saline. An equal amount of 1% DL-methionine solution is added and mixed to obtain a cell suspension for final bulk.

To make a final bulk material, a portion of the above suspension is mixed with 9 volumes of 2.25% maltose solution. The materials for the "Final Bulk" are dispersed into vials in 0.4 ml. amounts and lyo- philized to provide the product known as Picibanil.

The substantially penicillin-free product desig- nated Banil is obtained using a modification of the above process wherein the cells collected after the penicillin treatment and incubation steps are given three additional washing steps using physiological saline. While the procedure described above for preparing Picibanil and Banil produce preferred cell prepara¬ tions of Streptococcus hemolyticus for use according to this invention, other procedures as described in U.S. patent No. 3,477,914 can be used. Thus, the cells can be initially incubated in the presence of penicillin at a temperature in the range of 30-80°C. for at least 10 minutes where the concentration of penicillin is greater than 25,000 units per ml. The temperature of incubation is then increased to within the range of 38-50°C. for an additional time of from 20-60 minutes. Thereafter the culture is cooled.

The present invention will be described in terms of an antipregnancy vaccine based on beta hCG. Inasmuch as this antigen is relatively ineffective in raising antibody response when injected by itself, it is normally conjugated with a subject-compatible immu- nogenic carrier, as described in U.S. patent No. 4,161,519 to Talwar. One suitable carrier for this purpose is tetanus toxoid. For convenience, hereinafter this conjugate will be termed β-hCG-TT. Further, it is beneficial to adsorb the β-hCG-TT on aluminum hydroxide, a conventional adjuvant. While the vaccines of this invention are described in terms of the use of S-hCG as the antigen of interest, as well as rubella and ovine luteinizing hormone, it is recognized that Banil and Picibanil are also useful as adjuvants in other vaccines, such as those for tetanus, diphtheria, influenza, cholera, hepatitis, as well as animal vaccines for diseases such as hoof and mouth disease, distemper, leptospiro- sis, brucella abortus, and animal-vectored diseases such as trypanosomiasis, yellow fever, malaria and plague. Thus, the vaccines and methods of this inven¬ tion include antigens of viral, parasite, bacterial and hormone origin. A preferred group of protein hormone antigens include β-hCG and ovine luteinizing hormone.

Example I

The efficacy of Picibanil as an adjuvant in a vaccine formulation prepared in accordance with the invention, was tested in rabbits with the following procedure: A. Chemical Purification of 8-human ^**

Chorionic Gonadotropin

Purified beta subunit of human chorionic gona¬ dotropin (S-hCG) was prepared from crude hCG by the method of Canfield et al. described in Recent Progr. Horm. Res.. Vol. 27, p. 124 (1971). β-hCG was incu¬ bated at 40°C. for 1 hour with 8 M freshly prepared urea solution that had been purified on a mixed cation-anion exchange resin column, specifically a bed of equal quantities of Dowex-1 X-8 and Dowex-50 X-4, 200 mesh. The column size was 2 x 40 cm. In the presence of urea, the beta subunits were first dis¬ sociated and then separated by the method described by Morgan and Canfield, Endocrinology . Vol. 88, p. 1045 (1971). In accordance with this process, the mixture was passed through ion exchange columns and Sephadex columns repeatedly to obtain the dissociated beta subunit with minimal contamination of the whole human chorionic gonadotropin molecule or its alpha subunit.

B. Preparation of vaccine using l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide as condensing agent

Preparation (for 40 doses) 3.2 mg β-hCG, as prepared in A. above was mixed with 400 Lf tetanus toxoid (1500 Lf/ g protein N) (Wyeth Laboratories, Radnor, Pennsylvania) and 16 mg of l-ethyl-3 (3-dimethyl-aminopropyl) carbodiimide in 6.5 ml of PBS and this solution was incubated at 10°C. for 5 hours with occasional shaking. The molecular proportion of tetnaus toxoid to β-hCG was about 1:10. The reaction product was dialyzed extensively against 1 M phosphate buffered saline, (PBS) (pH 7.5) at a temperature of 10°C. The product, hereinafter β-hCG- TT, was collected in a sterile tube using a Millipore filter and syringe assembly. The product was stored at 4°C. The product was adsorbed on aluminum hydrox¬ ide using sterile 10% alum and then partially neutra¬ lized by adding sterile 10% sodium bicarbonate until a maximum precipitate was obtained. The mixture was centrifuged at 3000 rpm for 10 minutes. The procedure of adding sodium bicarbonate until a maximum precipi¬ tate formed and then centrifuging the mixture was repeated two times. Then the mixture was aspirated and the precipitate was washed three times with PBS. The final precipitate was resuspended in PBS at the required concentration. The β-hCG-TT on aluminum hydroxide was separately mixed with each of the adjuvants identified below in Table I.

TABLE I Adjuvant Identity Source

Corvnebacterium Bacterial suspension Welcome Research parvum Laboratories Beckenham, England

Levamisole l-(-)2,3,5,6-tetra- Ortho Pharmaceutica hydro-6-phenylimadazo- Rahway, New Jersey 2,lb-thiazole

Thymic factor Glu-Ala-Lys-Ser-Gln Dr. Karl Folkers Gly-Gly-Ser-Asn University of Texas Austin, Texas SPLPS Sodium phthalate Dr. G.P. Talwar derivative of All India Institute Salmonella lipopoly- of Medical Sciences saccharides New Delhi, India

OK-432 Lyophilized prepara¬ Chugai Pharmaceutic tion of Su strain of Tokyo, Japan Streptococcus hemo¬ lyticus, treated with penicillin

Avridine™ N,N-dioctadecyl-N' ,N'- Pfizer Pharmaceutic bis [2-hydroxyethyl]- Groton, Connecticut propanediamine

Formulations containing the adjuvants Coryne- bacterium parvum- SPLPS and OK-432, were prepared by adding these adjuvants to the PBS suspension of β- hCG-TT on aluminum hydroxide. Formulations containing Arlacel A, mannide trioleate (Sigma Chemicals, St. Louis, Missouri) and peanut oil, (Table III) were homogenized in a high speed Waring blender to form water-in-oil emulsions. The emulsions contained PBS, Arlacel A and peanut oil in the ratio 50:7:43.

Levamisole and thymic factor were suspended in 2% carboxymethylcellulose in PBS and injected subcuta- neously separately from the vaccine formulations con¬ taining Lipomal, a soy phosphatide preparation (Huhtamaki Oy/Leiras Pharmaceuticals, Turku, Finland) or Intralipid, an oil-in-water emulsion for intra¬ venous use (Cutter Laboratories, Emeryville, California) . Equal amounts of Lipomal and PBS were used. The Intralipid was prepared using 2.5 ml saline, 0.1 ml Tween 80-sorbitan monooleate (Sigma Chemicals) and 4.6 ml Intralipid. β-hCG-TT on alumi¬ num hydroxide was added to the vehicles containing Lipomal and Intralipid to form a suspension.

Immunization Five male New Zealand, Pasteurella-free, albino rabbits from Dutchland Laboratories, Denver,

Pennsylvania were used in each control and test group. Each animal weighed approximately 3 kg and was vac¬ cinated three times with a two week interval between vaccinations. The dose of the antigen at each vac- cination was 100 μg of β-hCG-TT. It was administered in a total volume of 1.6 ml split equally between two sites on the animal's rump. Except as otherwise noted, vaccinations were intradermal.

Antibody Evaluation Blood samples for antibody titration were taken

2, 4, 6, and 8 weeks after the last vaccination. Titrations of sera were conducted using 125I-labeled hCG added to appropriate dilutions of test serum. The initial incubation was for 2 hours in a 37°C. water bath and then the mixture was then refrigerated at 4°C. overnight. Gamma globulin was precipitated by addition of goat antibody to rabbit gamma globulin at 4°C. for at least 18 hours. The bound and unbound labeled hCG was separated by centrifugation at 4°C. for 30 minutes at 3000 rpm. The supernatant was aspirated and the pellet was then counted in an auto¬ matic gamma counter. The antibody titer was expressed as the dilution binding 33 percent of the added 125I- labeled hCG.

Neutralizing Capacity

The effectiveness of the antisera in neutralizing the biological activity of hCG has been determined in the rat uterine weight assay.

Immature female rats of the Sprague-Dawley strain

(19-day old) weighing 45-50 gm were used. A total dose of 0.6 I.U. hCG reference preparation (NIH, CR- 121) in 1% bovine serum albumen (BSA) was prepared as the standard. The anti-serum or the antiserum diluted with 1% BSA was mixed with an equal volume of 0.6 I.U. hCG solution so that each animal received a total volume of 1.5 ml. The solution was incubated at room temperature for 2 hours before initial injection.

Daily, subcutaneous injection (0.5 ml) of the solution was initiated the same day the animal arrived and was continued for 2 additional days. Animals were sacrificed 24 hours following the last injection. Five animals were used for each antiserum or antiserum dilution. The uterus was removed, trimmed, and weighed on a torsion balance. Five animals were used in each treated group and in control groups receiving only the hCG or vehicle. Pyrogen Test

Tests for pyrogenic activity were made by measur¬ ing body temperature of the rabbits rectally with a thermister probe and a Yellow Springs Instrument Company telethermometer one hour before and 1, 2, and 3 hours after dosing. The preparation was judged to be pyrogenic if the average maximum temperature increase over pretreatment was 0.6°C. or more.

The effectiveness of certain of the adjuvants listed in Table I in increasing antibody titers above those obtained in the same experiment with β-hCG-TT on Al(OH), alone is summarized in Table II. Two adju¬ vants, SPLPS and OK-432, appreciably enhanced antibody response when added to an aqueous suspension of β- hCG-TT on Al(OH)₃. Measures of the pyrogenic effect of the 2 adjuvants showed the SPLPS preparation to be a potent pyrogen; however, OK-432 was without pyro- genie effect at doses that gave maximum antigenic response. At doses of 10 mg/rabbit, the pyrogenic threshold was exceeded in some animals. It did not cause local tenderness, swelling or any evident illness. Titer and neutralizing ability of antisera from various formulations of β-hCG-TT on Al(OH)_ plus addi¬ tional adjuvants relative to the response to β-hCG-TT on Al(OH)_ alone were noted. Titers listed in the table are the average of those observed in samples taken 2, 4, 6 and 8 weeks after the last vaccination. Vaccination was intradermal except as noted:

TABLE II

Amount Adjuvant Relative

Additional Used Per Relative Neutralizing

Adiuvant Vaccination Titer³ Activity

Corvnebacterium parvum 1.5 mg 1.7 ND

Levamisole 200 mg 1.3 ND

Thymic factor 200 μg 0.5 ND

SPLPS 1.0, 0.5,

0.5^cmg 7.1 ND

OK-432 0.5, 1, 2 mg^C 10^e, 7.8 6^β 13^d, 4.8^d'^f, ₃d,f 4.8^d

The average titer of the control group receiving β-hCG-TT on Al(OH)_ in the same experiment is taken as "1".

The neutralizing capacity of the control group receiving β-hCG-TT on Al(OH)₃ in the same experiment is taken as "1". The neutralizing capacity was expressed as the reciprocal of the dilution producing 50 percent inhibition of hCG- stimulated uterine weight gain.

Different amounts as indicated were used in the 3 successive vaccinations of each vaccination series.

These results are from animals administered vaccine intramuscularly. Their titers are compared with those of formulation without OK-432 also given by the i.m. route. Titers were higher in both the control and OK-432 groups when vaccination was by the intramuscular rather than by the intradermal route. e,f Determinations made in the same experiment are identified by a common superscript. ND = Not Done. Exploration of the effect of different doses of OK-432 is shown in Figure 1 of the drawings, where antibody titers obtained 2, 4, 6 and 8 weeks after the last of 3 injections separated by 2-week intervals of β-hCG-TT on Al(OH). or the same formulation containing also the indicated amounts of OK-432 are shown. The dose-response curve appears relatively flat from 0.5 to 4.0 mg per injection. __, parvum, levamisole, and thymic factor did not increase titers above those seen with the control preparation in the same experiment.

The results obtained when β-hCG-TT on Al(OH)₃ was formulated in different physical forms are shown in Table III.

TABLE III

Titer and neutralizing ability of antisera from formula tions, of β-hCG-TT on Al(OH)₃ in various relative to β-hCG on Al(OH)₃ as an aqueous suspension

Relati

Continuous Relative Neutrali

Adiuvant^a Formulation Phase Titer

None Arlacel A/

Peanut oil Oil 0.2, 3. .6, 3.4 ND, ND,

None Liposomes Aqueous 0.7 0.5

None Lipomal Aqueous 1.0 0.3

Avridine Tween 80,

Intralipid Aqueous 1.2 1

Avridine Arlacel A/

Peanut oil Oil 1.5 1.2

Avridine Liposomes Aqueous 4.2 2.6

FCA^b 90

^a In addition to Al(OH) 3"

ND = Not Done. b F„reund, ι'—s c -.o-.—m—pl ιe — it-e— a_dj _j;uvant.

Formulation in a water-and-oil emulsion resulted in 3-fold increases in titers over those found fol¬ lowing vaccination with β-hCG-TT on Al(OH)₃ in aqueous suspension. Formulation in liposomes or in an oil- in-water emulsion did not result in increased titers. Also shown in Table III are results obtained when the amine adjuvant, Avridine, was formulated in different ways. No significant enhancement of response over that of β-hCG-TT on Al(OH)₃ alone was obtained by the use of this adjuvant in oil-in-water or water-in-oil emulsions. Moderate enhancement was achieved when the adjuvant was used with β-hCG-TT on Al(OH)₃ incor¬ porated in liposomes.

The above work demonstrates the effectiveness of the streptococcal preparation OK-432 in increasing circulating antibodies in accordance with this inven¬ tion. The increment in titers resulting from OK-432 use is comparable to that observed with use of a muramyl dipeptide analog in a water-in-oil emulsion with the same antigen. It is far less than the incre¬ ment observed with Freund's complete adjuvant (FCA) but unlike FCA does not cause overt local reactions. It has the additional advantages as a vaccine compo¬ nent of not requiring formulation in a water-in-oil emulsion to attain high activity as has proved neces¬ sary with the muramyl dipeptide analogs.

The several variations in formulation of the basic vaccine in the absence of supplementary adjuvant (in addition to aluminum hydroxide) were not notably successful. Formulation in a water-in-oil emulsion gave marginal increases in titer but much less than when muramyl dipeptide analogs were added to the same formulation. The amine Avridine gained in effective¬ ness by incorporation in liposomes. Formulation of β-hCG-TT on Al(OH)₃ in liposomes was not effective in increasing titers in the absence of supplementary adjuvant.

Example II

The efficacy of Banil as an adjuvant in a vaccine based on rubella antigen is tested in accordance with the following procedure: Ten male Pasteurella-free albino rabbits from

Dutchland Laboratories, Denver, Pennsylvania, weighing approximately 2.5 kg at the time of vaccination are used in each experimental group. Each animal receives a single intramuscular injection of 1 ml of either rubella antigen or rubella antigen plus Banil adjuvant split equally between two sites on the animal's rump. The rubella antigen is obtained from Cordis Labora¬ tories, Miami, Florida, and was prepared from Gilchrist strain rubella, grown on baby hamster kidney cells, clone 13 (American Type Culture Collection ATCC CCL 10). The viruses were harvested from the culture supernatant by differential centrifugation. The supernatants were first centrifuged at 8000 rpm for 20 minutes to remove the cell debris. Then they were centrifuged at 40,000 rpm for 1 hour to pellet the virus. The supernatant was decanted and the virus pellet was washed once with 0.1 m tris buffer contain¬ ing 0.1 mM EDTA and centrifuged again at 40,000 rpm for 1 hour. The supernatant was removed and the pelleted virus was resuspended in 1/50 of the original volume in 0.1 M tris buffer containing 0.1 mM EDTA. The viral concentrates were then inactivated with ether and Tween 80 in accordance with the procedure of Norrby, E. Proc. Soc. Biol. Med. 1962, 11, 814. After inactivation the protein content was estimated by absorbance reading at 280 NM. The viral suspension was lyophilized.

Each animal receives 0.5 mg of rubella antigen. The test group also receives 1.0 mg of Banil mixed with the antigen. In either case* the vaccine is brought to 1 ml volume with distilled water.

The animals are bled at 2 and 5 weeks post- immunization. The rubella antibody titers are deter- mined by enzyme-linked immunosorbent assay (ELISA) ,

Engvall E. and Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay for immunoglobulin G. Immunochemistry 8. (1971) 871. It is found that a substantial enhancement in titer is obtained at 5 weeks with the vaccine containing the Banil as com¬ pared with the vaccine without it. Furthermore, there is no local reaction and substantially no temperature increase observed in the rabbits following injections.

Example III

A series of studies were conducted to deter¬ mine whether the primary response to β-hCG is affected by inclusion of OK-432 in a vaccine formulation con- taining β-hCG. In addition, the studies sought to determine the dose-response relationship in the pri¬ mary vaccination series to different doses of OK-432, whether OK-432 enhances the response to booster injec¬ tions, and whether repeated booster injections further enhance the response.

Five rabbits of the type described in Example I were used in each of three groups. Three primary vaccinations were given, spaced two weeks apart. The β-hCG was combined with tetanus toxoid in the manner described in Section B of Example I above, and was adsorbed on Al(OH)... The dose given was calculated to provide 100 μg of β-hCG at each vaccination. The same dosage was used at all primary and booster injections. In the primary vaccination series. Group B received 0.5 mg OK-432, in the first vaccination, 1.0 mg, OK-432 in the second vaccination, and 2.0 mg. in the third. Further, in the primary series, Group C received 0.166, 0.33 and 0.66 mg OK-432 in each of the 3 successive vaccinations. Booster injections to Group A were of same composition as the primary vac¬ cination. The first two booster injections to Group B contained 1 mg of OK-432, and the third booster injec¬ tion to Group C contained 1 mg of OK-432.

The first booster injection was given 4.5 months after the last primary injection, and the interval between booster injections was 2 months. Sera for antibody determinations were taken 2, 4, 6 and 8 weeks after the last primary vaccination. Titers obtained at these time intervals were averaged to determine mean response.

Additionally, sera for antibody determinations were taken one and two months after each booster injection. Titers obtained at one and two months were averaged to obtain mean response following each booster.

Diagram of Protocol Group_A Group B Group C

B- B„ B. B. B. B. B. B. B.

β-hCG-TT + + + + + + + + + + + + A1(0H)₃ + + + + + + + + _+. + + OK-432 + + + + 1/3 OK-432 P = Primary B = Booster

Results

Mean antibody titers following the primary vacci¬ nation and each of the booster vaccinations are shown in Table IV.

TABLE IV

Reciprocal of dilutions of antisera giving 33 percent binding of 125I hCG (+ SEM)

Group Primary Booster 1 Booster 2 Booster 3

A 4,133 35,673 25,225 40,961

(1.977) (12,085) (4,575) (4,965)

-»B 19,503 146,051 104,904 120,571

5,433) (60,238) (27,045) (30,609)

C 8,315 55,190 38,901 66,548

(3,077) (14,413) (4,969) (3,074)

The following conclusions were drawn from this work: 1. The addition of OK-432 to the vaccine used in the primary immunization increased the antibody response in a dose dependent manner.

2. Booster response is enhanced so long as OK- 432 is used in the primary vaccination, irrespective of whether it is included in the booster injection.

3. The inclusion of OK-432 in a booster injec¬ tion is beneficial to the booster response. In Group C, the response to the third booster, which included OK-432, is significantly greater than the response to the second booster; p=0.001. Note, how¬ ever, that the response to the second booster was low in each group, with the difference between responses to the second and third boosters in Group A being significant at p=0.05.

Example IV

A series of tests were conducted to determine if Al(OH)₃ is required for maximum response in the booster series, in accordance with the method of this invention, and to determine if Banil can replace A1(0H)₃ in the booster injections.

Five rabbits of the type used in Example I were used in each group. Three primary vaccinations were given, spaced two weeks apart. Booster injections were given 10 weeks after the last primary injection. Antigen used was β-hCG-TT, as prepared in Example I, in amount to deliver 100 μg of β-hCG in each injection. Except for the booster injections given in Groups M and 0, the antigen was adsorbed on Al(OH)₃, following the procedure outlined in Example I. The diagram given below shows the protocol: Protocol Group N Group M Group 0 Primary Booster Primary Booster Primary Booster Al(OH)₃ + + + +

Banil +

All vaccinations were given intramuscularly. Blood samples for antibody titration were taken 2, 4, 6 and 8 weeks after the last primary immunization and one and two months after booster immunization. Titers obtained_^ at each of the four time intervals after primary immunization were averaged to give the mean response. Similarly, the titers obtained at the two intervals after booster injections were averaged to determine the mean booster response.

The dose of Banil in the booster injection to Group N was one mg per rabbit.

Results

Antibody titers on sera taken after the primary vaccinations and after the booster vaccinations are tabulated below in Table V.

TABLE V Reciprocal of dilutions of antisera giving 33 percent

¹²⁵I hCG (+ SEM) Group Primary Booster

N 3319 (+ 802) 8290 (± 630)

M 3072 (± 479) 5007 (± 763)

0 3520 (± 811) 10,922 ( ± 3272)

It was concluded from the above tests that the inclusion of Banil in the booster injection has increased the booster response as compared with Group M, which similarly to Group 0, received a booster injection which did not contain Al(OH)₃. The difference does not quite reach the 5% level of significance. Example V

A series of tests were conducted to determine if the inclusion of adjuvants supplementary to Al(OH)₃ in the primary immunization series enhances booster response. In addition, the tests were designed to determine if there is advantage in including a murabu- tide analog (MDP) in a water-in-oil emulsion in the booster injection; to determine if there is advantage in including Banil in the booster injection; and to determine if the omission of Al(OH)_ from the booster injection is deleterious to antibody response.

Five rabbits of the type used in Example I above was used in each group. Three primary injections were given, spaced two weeks apart. Antigen used was β- hCG-TT, prepared as in Example I above. In all except for the booster injection given to one group, the antigen was adsorbed on Al(OH)₃ following the proce¬ dure of Example I. Dose was calculated to give 100 μg of β-hCG-TT, and all vaccinations were intramuscular. The urabutide was used as described in Nash et al. (J. Reprod. Immunol. 7 (1985) 151-162) using Span and Tween as emulsifying agents. Dose per rabbit was 250 μg.

The Banil was added to an aqueous suspension of the antigen in amount to deliver one mg per rabbit. Blood samples for antibody titers were taken 2, 4, 6 and 8 weeks following the last primary vaccina¬ tion. Mean response for each rabbit was obtained by averaging the titers obtained on samples taken at these four sampling times.

Blood samples to evaluate booster response were taken at one and two months after the booster and were averaged to obtain the mean; one and two month titers are also detailed in Table VI below. The booster injections were given 10 weeks after the last of the primary vaccinations. Antisera from the first bleeding after booster injection was tested in the rat uterine weight assay described in Example I. A diagram of the protocol followed in this test is shown in Figure 2 of the drawings.

Results

Antibody titers in serum samples taken after the primary vaccinations and after the booster injections are shown in Table VI.

The effectiveness of the antibodies from the first bleeding after booster injection was tested for its ability to neutralize the ability of hCG to stimu- late the uterine weight of immature rats. Results are shown in Table VII.

Conclusions

1. The inclusion of Banil in the primary immuni¬ zation series has enhanced the primary response. The mean response of the 20 animals receiving β-hCG-TT on Al(OH)₃ was 2332 ± 173. That of the 10 animals also receiving Banil in the primary injections was 8666 +.1702. The inclusion of murabutide in the primary series also caused a significant increment increase in the primary response

2. The inclusion of Banil in the primary vaccination caused an increment of increase in the booster response. Comparing:

Group D vs. Group J 1st bleeding p=<0.05

2nd bleeding NS Group G vs. Group K p=<0.05 1st bleeding p=<0.05 2nd bleeding p=<0.02 Note that inclusion of murabutide in the primary vaccination series also caused a significant increment of increase in booster response.

3. The inclusion of Banil in the booster injec¬ tion caused an increment increase in booster response as judged by antibody titers. Comparing: Group D vs. Group G p=<0.1 1st bleeding p=<0.05 2nd bleeding NS Group J vs. Group K 1st bleeding p=<0.1

2nd bleeding NS

4. The high activity in neutralizing the biological activity of hCG (uterine weight assay) of antisera from Group K relative to Group J and of Group G relative to Group D clearly establishes enhancement of antigenicity by including Banil in booster injections.

The higher activity of J relative to D, of I relative to F, of K relative to G establishes the benefit of including a supplementary adjuvant, either

Banil or murabutide, in primary vaccination.

5. On the other hand, the inclusion of murabu¬ tide in the booster has not caused in increment of increase in titers. Compare: Group D vs. Group F NS

Group H vs. Group I NS

TABLE VI

Reciprocal of dilutions of antisera giving 33 percent binding of ^Δ I hCG (±SEM)

Primary Booster

Group i. mo. 2_ mo. mean

D 2,554 16,411 7,451 11,931

(464) (4,272) (1,824) (3,034)

E 1,899 11,986 6,026 9,005

(484) (3,378) (1,770) (2,537)

F 2,591 12,598 5,382 8,990

(222) (1-461) (573) (1,062)

G 2,681 31,851 9,895 20,872

(148) (6,236) (1,737) (3,786)

H 4,417 38,505 19,578 29,041

(188) (6,095) (2,457) (4,034)

I 7,510 37,380 17,943 27,661

(2,545) (2,929) (2,548) (2,687)

J 7,826 39,696 30,091 34,893

(1.978) . (10,184) (15,607) (12,702)

K 9,506 92,658 30,824 60,146

(2,952) (27,101) (7,473) (16,018)

TABLE VII Percent inhibition of hCG stimulation of uterine weight of the immature rat

Group Dilution. of antisera Percent Inhibitio

D 1/1024 5

E 1/1024 11

F 1/1024 18

G 1/1024 74

H 1/2048 52

I 1/2048 36

J 1/2048 17

K 1/2048 75

^a. For method see Nash e_t al. __.. Reorod. Immunol. 7

(1985) 151-162.

Example VI

Five maid Pasteurella-free, New Zealand albino rabbits from Dutchland Laboratories, Denver, PA, weighing 2.5-3.0 kg were used in each group of this experiment. Each animal received 3 vaccination injec- tions, with a two week interval between vaccinations. The dose of antigen at each vaccination was either 100 μg of β-hCG subunit or 100 μg of the β-subunit of ovine luteinizing hormone (oLHβ) linked to tetanus toxoid (TT) carrier; (Tsong, Y.Y., Chang, C.C. and Nash, H.A. (1985) Formulation of a potential antipreg- nancy vaccine based on the β-subunit of human chorio¬ nic gonadotropin (β-hCG). I. Alternative macro- molecular carriers. J__ Reprod. Immunol. 1, 139-149. The vaccine was adsorbed on Al(OH)_. Picibanil at a dose of 0.5, 1.0 and 2.0 mg per animal in the three successive vaccinations was dissolved in saline and mixed with the vaccine in aqueous suspension and injected intramuscularly in a total volume of 1.6 ml, split equally between two sites on the animal's rump (Chang, C.C., Tsong, Y.Y. and Nash, H.A. (1985) Formu¬ lation of a potential antipregnancy vaccine based on the β-subunit of human chorionic gonadotropin (β-hCG). III. Evaluation of various vehicles and adjuvants. J. Reprod. Immunol. 1, 163-169) . Blood samples for antibody titration were taken 2, 4, 6 and 8 weeks after the last vaccination. Titration of sera was carried out using either 0.2 μg of 125I labeled hCG with a specific activity of 82-

101 μCi/μ for determining antibody to β-hCG or 0.2μg of ¹²⁵I labeled oLHβ with a specific activity of 38- 56 μgCi/μ for determining antibody to oLHβ. Detailed procedures used are described in Chang, C.C., Tsong, Y.Y., Rone, J.D., Segal, S.J., Chang, D., Leban, J. and Folkers, K. (1981) A synthetic peptide capable of eliciting antibodies that neutralize human chorionic gonadotropin. Fertil. Steril. 36, 659.-663. The antibody titer was expressed as the dilution bind¬ ing 33% of the added tracer.

Results The effectiveness of Picibanil in enhancing anti- genicity of β-hCG-TT and oLHβ-TT on Al(OH)₃ is summa¬ rized in Table VIII below. The relative titer of β- hCG-TT on Al(OH)₃ containing Picibanil at the doses used increased 4.8 fold over that found following vaccination with β-hCG-TT on Al(OH)₃ alone. The same amount of Picibanil also appreciably enhanced antibody response (3.1 fold) when added to an aqueous suspen- sion of oLHβ -TT on Al(OH)-.

This work demonstrated that Picibanil used as a supplementary adjuvant in an aqueous suspension is effective in increasing the antigenicity of not only β-hCG-TT on A1(0H)- vaccine but also oLHβ -TT on Al(OH)₃ vaccine, although the magnitude of enhancement of antibody titer of the latter was not as high as the former vaccine.

TABLE VIII Relative Antibody Titer of β-hCG-TT or oLHβ-TT to the addition of Picibanil

Vaccine Dose of Picibanil Relative used per Vaccination* Titer** β-hCG-TT 1 β-hCG-TT 0.5, 1.0, 2.0 mg 4.8 oLHβ-TT 1 oLHβ-TT 0.5, 1.0, 2.0 rag 3.1

* Different doses as indicated were used in the 3 successive vaccinations. ** The average titer of the group receiving either β-hCG-TT on Al(OH)₃ or oLHβ-TT on Al(OH)₃ in the same experiment is taken as "1".

Claims

1. A vaccine comprising an antigen and, as an adju¬ vant, penicillin-treated cells of Streptococcus hemolyticus.

2. A vaccine comprising an antigen and, as an adju¬ vant, cells of Streptococcus hemolticus that have been treated at 30-80°C. for at least 10 minutes with penicillin at a level greater than 25,000 units per ml with further incubation thereof at 38-50°C. for about 20-60 minutes.

3. The vaccine of claim 2, wherein penicillin used to treat the cells is retained to form a part of the adjuvant.

4. The vaccine of claim 2, wherein the penicillin used to treat the cells is removed prior to use of the cells as adjuvant.

5. The vaccine of claim 1, wherein the antigen is a protein hormone.

6. The v-ccine of claim 5, wherein the protein hor- mone is the β-subunit of human chorionic gonadotropin.

7. A vaccine comprising the B-subunit of human chorionic gonadotropin linked to tetanus toxoid and adsorbed on Al(OH)₃, as a primary adjuvant, together with an antibody response-enhancing amount of a supplemental adjuvant comprising cells of Streptococcus hemolyticus that have been treated with penicillin.

8. The vaccine of claim 7, wherein the cells of Streptococcus hemolyticus are treated with peni¬ cillin at 30-80°C. for at least 10 minutes with penicillin at a concentration greater than 25,000 units per ml, followed by further incubation thereof at 38-50°C. for from 20 to 60 minutes.

9. The vaccine of claim 8, wherein the penicillin used to treat the cells is removed prior to use of the cells as an adjuvant.

10. The vaccine of claim 8, wherein penicillin used to treat the cells is retained to form a part^"of the adjuvant.

11. A vaccine comprising the B-subunit of human chorionic gonadotropin linked to tetanus toxoid and adsorbed on Al(OH)_, together with an antibody response-enhancing amount of a supplemental adju¬ vant comprising cells of Streptococcus hemolyticus (Su-strain) that have been treated with a peni¬ cillin G solution at about 37°C. for 20 minutes, followed by heating at about 45°C. for 30 minutes, and cooling.

12. A vaccine comprising rubella antigen together with an antibody response-enhancing amount of a supple¬ mental adjuvant comprising cells of Streptococcus hemolyticus that have been treated with penicillin.

13. The vaccine of claim 12, wherein the Streptococcus hemolyticus are treated with penicillin at 30- 80°C. for at least 10 minutes with penicillin at a concentration greater than 25,000 units per ml, followed by further incubation thereof at 38-50°C. for from 20 to 60 minutes.

14. The vaccine of claim 13, wherein the penicillin used to treat the cells is removed prior to use of the cells as an adjuvant.

15. The vaccine of claim 13, wherein the penicillin used to treat the cells is retained to form a part of the adjuvant.

16. A vaccine comprising rubella antigen together with an antibody response-enhancing amount of a supplemental adjuvant comprising cells of Streptococcus hemolyticus (Su-strain) that have been treated with a penicillin G solution at about 37°C. for 20 minutes, followed by heating at about 45°C. for 30 minutes, and cooling.

17. A method for enhancing the antibody response of a vaccine comprising the step of administering a vaccine comprising an antigen and an antibody response-enhancing amount of an adjuvant comprising penicillin treated cells of Streptococcus hemolyticus

18. A method for enhancing the antibody response of a vaccine comprising the step of administering a vaccine comprising an antigen and an antibody response-enhancing amount of an adjuvant comprising cells of Streptococcus hemolyticus that have previously been treated with at least about

25,000 units/ml of penicillin G at 30-80°C. for at least about 10 minutes, followed by an incubation period of about 20- 60 minutes at 38-50°C, and cooled.

19. The method of claim 18, wherein the penicillin used to treat the cells of Streptococcus hemolyticus is retained in the adjuvant.

20. The method of claim 18, wherein the penicillin used to treat the cells of Streptococcus hemolyticus is removed from the adjuvant.

21. A method for enhancing the antibody response of a vaccine comprising the step of administering a vaccine comprising an antigen and an antibody response-enhancing amount of an adjuvant comprising cells of Streptococcus hemolyticus that have been treated with penicillin G at about 37°C. for 20 minutes, followed by incubation at about 45°C. for about 30 minutes, and cooled.

22. The method of claim 17, wherein the antigen is a protein hormone.

23. The method of claim 22, wherein the"protein hormone is the β-subunit of human chorionic gonadotropin.

24. A method for^' enhancing the antibody response of a vaccine comprising the B-subunit of human chorionic gonadotropin linked to tetanus toxoid and adsorbed on Al(OH)₃ which comprises adding thereto, as a supplemental adjuvant, an antibody response-enhancing amount of cells of Streptococcus hemolyticus that have been treated with penicillin G at about 37°C. for 20 minutes, followed by incubation at about 45°C. for about 30 minutes, and cooled.

25. A method for enhancing the antibody response of a vaccine comprising rubella antigen which comprises adding thereto, as a supplemental adjuvant, an antibody response-enhancing amount of cells of

Streptococcus hemolyticus that have been treated with penicillin G at about 37°C. for 20 minutes. followed by incubation at about 45°C. for about 30 minutes, and cooled.

26. The method of claim 17, wherein said step of administering a vaccine comprises a primary injection, and wherein at least one booster injection of the same antigen is given at a desired time interval thereafter.

27. The method of claim 26, wherein said booster injection comprises, as an adjuvant, penicillin treated cells of streptococcus hemolyticus.