AU615016B2 - Immunological adjuvent and process for preparing the same, pharmaceutical compositions, and process - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATICN

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority S Related Art: APPLI.IANT'S REFERENCE: 72-011 COcNT Name(s) of Applicant(s): Frank M. Berger Address(es) of Applicant(s)' 515 East 72nd Street, Suite New fork, 0 New York, UNITED STATES OF AMERICA.

Address for Service is: SPHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: IMMUNCLOGICAL ADJUVENT At PROCESS FOR PREPARING THE SAME, PHARMACEUTICAL CO1 OSITIONS, AND PROCESS Our Ref 119615 POP Code: 1426/94956 T'he following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6003q/i 1 1A

SPECIFICATION

Adjuvants are substances that are capable of increasing the immune response of a wide variety of chemically unrelated antigens. Antigens are substances foreign to the body which on parenteral administration (by some way other than through the digestive tract) induce the formation of antibodies. Antibodies are substances contained in blood, in other body fluids and tissues that bind the antigen and re;nder it harmless to the 0 0 body. Antibodies are one of the natural defense mechanisms o, 10 of the body. When present, they kill or render harinless invading pathogenic bacteria, viruses or protozoa. They also are capable of inactivating or killing cancer cells. All antibodies are highly specific and can kill, bind or render 0 0 harmless only the antigen that has induced their formation.

0a 0 Vaccines and immunizations are administered in order to induce the formation of protective antibodies. This is achieved by administration of killed microorganisms, administration of microbial products or of extracts or portions of microorganisms or by the use of attenuated strains that are no longer capable of inducing disease but still can engender the production of protective antibodies. Utilizing these methods, a substantial number of impjrtart vaccines have been developed which have eliminated or substantially reduced the occurrence of many diseases that in the past endangered the survival of f'

F.

Note: No legalization or-- other witness required er To: The Commissioner of Patents P18l/7i8 PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia IAi 4i4r 1 2 individuals or populations. Examples of vaccines utilizing attenuated or modified living microorganisms are those for the prevention of small pox, poliomyelitis, yellow fever, measles and rubella. Examples of successful vaccines utilizing killed microorganisms are those for the prevention of typhoid, pertussis (whooping cough), pneumonia and cholera. Examples of vaccines produced from bacterial products are those for the prevention of diphtheria and tetanus.

Recovery from certain infectious diseases such as 0 10 smallpox, typhoid fever and many others produce long-lasting 0..0 immunity. It is of interest to note that most of the vaccines S. that are being used at the present time induce protection from diseases that when acquired induce strong and long-lasting immunity. People who survive the first attack of one of these diseases rarely suffer a second attack. The antigenic components of the microorganisms causing these diseases are highly immonogenic, generate antibodies that protect the oo. animal from contracting the disease on a subsequent encounter Swith the specific microorganism.

There is, on the other hand, a large number of diseases against which vaccines have not become available. Attenuated strains of microorganisms that would protect against these diseases could not be obtained because of the tendency of these strains to revert prompbly to their pathogenic, disease- 2i 2 3 inducing-form. An even more important reason for the absence of vaccines that would protect against these diseases is the low immunogenicity of the microorganisms causing these diseases. People who contract a disease caused by microorganisms that are weakly antigenic and of low immunogenicity are not protected against subsequent reinfection and disease by the same microorganism. Examples of diseases caused by weakly antigenic microorganisms where one attack of the disease does not protect against subsequent attacks are malaria, gonorrhoea, syphilis and herpes.

o It has been realized for a long time that a substance that o'o could increase the ability of non'immunogenic microorganisms and soluble proteins and peptides to induce formation of antibodies would be most valuable and would permit the 44 production of vaccines against diseases where protection 4 cannot be induced at the present time. This idea has been on the minds of investigators for many decades.

The first significant advance in this field was made by Glenny, A.I. et al Path. Bact. 29, 31-40, 1926) who showed that absorption of diphtheria toxoid on aluminum phosphate increases the immunological response. Substantial work subsequently carried out with a number of inorganic aluminum and calcium compounds indicated that these can be safely used in humans. Unfortunately, these agents increased the immune 3 4 response only to diphtheria and [tetanus toxoids.. Th'y.did not work satisfactorily as adjuvants with other vaccines and did not significantly increase the immune response t:o weak antigens.

The most effective composition used for augmentation of immunogenicity of antigens is Freund's Complete Adjuvant (FCA). FCA contains killed mycobacteria dispersed in a water-in-oil emulsion (Freund, J. Ann. Rev. Microbiol. 1: 291-309, 1947). Customarily, the antigen is dissolved or 10 dispersed in water and the aqueous phase emulsified in mineral o 0 oil with the help of an emulsifying agent. Freund's Incomplete S" Adjuvant (FIA) does not contain mycobacteria. In addition to their potent adjuvant action, FCA and FIA also induce many .O 00 undesirable and toxic effects and for this reason cannot be used in humans and domestic animals.

a 0 0 0Many efforts have been made to n.inimize or eliminate the toxic side effects by refining the ingredients of FCA.

B 'Attempts to replace the mineral oil which is responsible for granulomatous reactions and an increase in the occurrence of neoplasms in mice by more readily metabolizable fatty substances were unsuccessful.

It was soon recognized that the potent adjuvant action of FCA depends primarily on the presence of mycobacteria in this preparation. The adjuvant active principle of mycobacteria 4 was isolated by Bekierkunst, A.E. et al. (Infect. Immun. 4: 256, 1971) and identified as a diester of mycolic acid. The same substance was also isolated by other investigators and named wax D, cord factor, P3, trehalose diester or trechalose dimycolate (Smith, D.W. et al., Adv. Tuberc. Res. 16: 191, 1968; Lederer, J. Medic. Chem. 23: 819, 1980). However, it soon became apparent that the mycolic acid esters were not only responsible for the immuno-enhancing properties of :O mycobacteria but also induced most of the undesirable side i, o 10 effects caused by FCA. These include pyrogenicity and induction of adjuvant arthritis and of autoimmune diseases (Retzinger, G.S. et al., J. Immunol. 129: 735, 1982).

Several groups of investigators attempted to isolate substances from mycobacteria that were unrelated to mycolic acids and nevertheless capable of replacing mycobacteria in FCA. These studies were successful and led to the isolation of peptidoglycans, also called mureins, mucopeptides or glycopeptides. All these substances consist of a polysaccharide (glycan) linked through muramic acid or similar substitutes D-giucosamine residues to peptides. This work culminated in tl identification and synthesis of muramyl dipeptide (MDP).

This simple peptidoglycan could substitute for whole mycobacteria in FCA (Adam, A. and Lederer, Medic Res.

Rev. 4: 111, 1984). Unfortunately, it soon became apparent 4 6 that MDP and other peptidoglycans were pyrogenic and induced adjuvant arthritis which would make their use as adjuvants unpermissible-in humans and domestic animals (Nagao' and, Tanaka, Infect. Inr ain. 28: 624, 1980; Zikek, Z. et al., Infect. Immun. 3b: 674, 1982; Stewart-Tull, Ann. Rev.

Microbiol. 34: 311, 1980).

Another group of compounds known to possess excellent adjuvant properties are bacterial lipopolysaccharides (LPS), s also known as endotoxins. LPS a component of the cell walls Coo C 10 of gram-negative bacteria possesses a-wide variety of o° biological properties. The biological activities of LPS are Sa o o attributable to lipid A which, like LPS, is pyrogenic and toxic to many biological functions (Berger, Advances in Pharmacology 5: 19, 1967). Detoxification, of LPS or lipid A o 15 by chemical treatment results in the loss of adjuvant properties.

o" o These, however, can be in part restored by addition of trehalose dimycolate and oil (Ribi, E. et al., Cancer Immunol. Immunother.

7: 43, 1979).

It has been shown that with the use of some of the existing adjuvants such as FCA, trehalose dimycolate or MDP it is possible to induce in animals the production of p.otective antibodies against infectious microorganisms that given alone are unable to do so. This was clearly demonstrated by Breund et al., (Am J.

Trop. Med. 28: 1, 1948) who were able to immunize monkeys 6 7 against malaria by means of killed parasites given together with FCA. It has since been shown that with the help of FCA and MDP it is possible to immunize animals or induce the production of antibodies against a number of viral, microbial and protozoal infections, such as herpes virus 1 and 2, cytomegalovirus, H. influenza, N. gonorrhoea, trypansomiasis, leishmaniosis and many others that are not immunogenic enough to induce an immune response when given by themselves.

S-Work carried out up to the present has indicated that 00 o 10 the substances active as adjuvants are also responsible for Sthe toxic and undesirable effects that accompany use of these 6 agents. This has been specifically demonstrated with adjuvants containing mycolic acid esters, muramic acid or lipopolyo, saccharides. As a result of this, it is not possible to administer S 15 adjuvants containing any of these substances to humans or domestic animals.

In accordarie with the invention, an immunological .adjuvant capable of increasing the immune response in animals to antigens and substantially free from mycolic acids, mycolic acid esters and lipopolysaccharides is provided, prepared by an extraction procedure from one or more species of Amycolata, a genus of filamentous branching bacteria known as Actinornycetes. Administration of these immunological adjuvants to animals increases the immune response of antigens without the presence of oil or oily vehicles, 1 I- J 8 and without inducing adjuvant arthritis or other undesirable side effects.

"'he process for preparing such immunological adjuvants in accordance with the invention c:omprises suspending Amycolata bacteria cells in aqueous physiological saline solution; subjecting the Amycolata bacteria cells to extraction with a solvent immiscible with the aqueous saline solution and in which the immunological adjuvant is soluble and/or dispersible; 10 separating the resulting solvent solution of immunological adjuvant :'!rom the bacteria cells and the aqueous saline solution; and recovering the adjuvant, all of the foregoing steps being carried out at a temperature below the temperature at which the immunological adjuvant is inactivated.

To assist in dispersing the bacteria cells in the aqueous saline solution, a wetting or emulsifying agent such as an anionic o a or nonionic surfactant can be added.

"Also in accordance with the invention, pharmaceutical compositions are provided, suitably but not necessarily, in dosage unit form, for administration to animals to which an antigen is also administered, to increase the immune response of the antigen, and comprising an immune-response-increasing amount of the immunological adjuvant, prepared by the above-described process, and a pharmaceutically-acceptable nontoxic carrier or diluent therefor.

8 9 The invention further provides a process for increasing the immune response of antigens which upon administration to an animal induce the formation of antibodies, which comprises administering to the animal an immunological adjuvant prepared according to the above-described process. The immanological adjuvant can be administered together with or separately from administration of the antigen, A preferred embodiment of pharmaceutical composition, also preferably in dosage unit form, comprises the immunological adjuvant and the antigen.

The immunological adjuvants described herein ire novel, and are prepared by a new extraction process from a genus of bacteria that do not ccn.ain mycolic acid and are known as Amycolata, of which some species are new strains S 15 not previously known or described.

Amycolata is the name of a genus of filamentous branching bacteria known as actinomycetes (Order: Actinomycetales).

The new strains of Amycolata disclosed herein as well as those previously known share the following characteristics: 1. Taxonomically, most strains of Amycolata which have been previously described were assigned to the genera Streptomyces or Nocardia, 2. Morphologically, amycolatae belong to the "nocardioform" group of actinomycetes. Nocardioform is a broad descriptive term for actinomycetes which form branching filamehts that r 1tend to break down into smaller, squarish units as a normal stage of their cycle of growth. In this they are similar to members of the genus Nocardia (Prauser, Publ. Fac. Sci.

Univ. Purkyne Brno K40: 196, 1967).

3. Chemically, Amycolata spp. have a peptidoglycan (cell wall) of chemotype IV (Becker, B. et al., Appl. Microbiol.

13: 236, 1965). This is to say that, like nocardiae, the components of the wall include meso diaminopimelic acid, glutamic acid, alanine, glucosamine, muramic acid, arabinose and galactose. Their whole cell sugar pattern is of type A Ir o(Lechevalier, J. Lab. Clin. Med. 71: 934, 1968): o a galactose and arabinose. However, unlike Nocardia spp., the cell lipids contain no mycolic acids (Lechevalier, J.

Bacteriol. 105: 313, 1971; Lechevalier, Can. Microbiol.

19: 965, 1973), the fatty acids are of the branched-chain and normal types (no major amounts of unsaturated ones are present) and their phospholipids are of the PIII type (phosphatidylcholine o as diagnostic constitutent) (Lechevalier, M. Biochem. Syst.

col. 5: 249, 1977; Lechevalier, Zbl. Bact. Suppl. 11: 20 111, 1981). Thus, chemically they differ from members of the S* genus Nocardia.

4. Physiologically, Amycolatae are very diverse; they can be described as mycolateless nocardiae.

Amycolatae have been isolated from many different sources, including air, soil and vegetable matter.

-V I 11 The Amycolata spp. used herein as a source of the immunological adjuvants of the invention conform to the above description: they are nocardioform actinomycetes with a cell composition of chemotype IV, a whole cell sugar pattern of type A, no mycolates and a phospholipid pattern of type PIHI. All occur naturally and were isolated from earth or soil, as indicated below usingthe ollowidng proc dure: Two grams of soil are shaken for 30 minutes at 215 RPM at 28 0 C in 100 cc sterile water, then the suspension permitted to settle out for 10 minutes without further shaking. One ml of the o supernatant is added to a 9 ml sterile water blank, this mixed Sthoroughly, then diluted similarly 10-fold, 100-fold and 1000-fold.

0 o 0. 1 ml of each dilution is spread on an agar medium such as Sdilute glucose-asparagine (glucose, 1. 0 g; asparagine, 0. 05 g; dibasic potassium phosphate, 0. 05 g; agar, 15 g; distilled water, 1 liter, pH 6. 8 before autoclaving) or on tap water agar (technical grade agar, 15 g; tap water, 1 liter) and incubated at 28 0 C for 4 to 5 weeks. Colonies are buff-colored and have filamentous margins.

o 20 In the case of the microorganisms identified as Ml, the soil 0 was a clay soil taken from a tropical garden in Aswan, Egypt. In the case of the microorganisms identified as M2 the soil was a clay soil taken from a field near Mehalla, Egypt, and in the case of M3, the soil was taken from a wheat field in El Arbaeen, Egypt.

11 12 Some of the strains' physiological characteris tics are listed in Table 1.

Table I Test Reactions of Amycolata spp.

LL-371-29, LL-37M-102 and LL-37 0-i 371-29 37M-102 370-4 Degradation/ Transformation of Cas ein Xanthine lypoxanthizie Tyrosine Adenine Production of Amnylase Gelatinase Phosphatase Nitrate Red,,ictase Urease Esculinase Growth on/in NaCl Salicylate Lysozyme Broth Table I (continued) Test Reactions of Amycolata spp.

LL-371-29, LL-37M-102 and LL-370-1 371-29 37M-102 370-1 Ut ilizat ion Acetate Benz oat e Citrate Lact at e Malate Mucate 0Ox~alate Propionate Py ruvat e Succinate Tartrate Growth at 45" C 53 0

C

Acid f iom Adonitc/l Ar-abinose Cellobiose 14 Table I (continued) Test Reactions of Amycolata spp.

LL-37I-29, LL-37M-102 and LL-370-1 00 00 0 0 '0k' 0 00 0 0 040 l~ .0 0 o 0 00 £00 40 0 4 00 0 £0 Dextrin Dulcitol E rythritol Fructose 10 Galactose Glucose Glycerol Inositol Lactose Malt os e Mannit ol Mannos e [ve libios e a -Me -D -GQluc os idE! Raff inose Rhamnose Salicin Sorbitol Sucrose Trehalose Xy lose f-Me-D-Xyloside 371-29 37M-102 370-1 'For methods see: a. Gordon, R. D. A. B~arnett, J. E. Handerhan and C. H. Pang, 1974. Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int. J. Syst.

Bacteriol. 24: 54-63.

b. Gordon, S.K. Mishra and D.A. Barnett, 1978.

Soie bits and pieces of the genus Nocardi N. carnea, N. vaccinii, N. transvalensis, N. orientalis and ae,4ocolonigE.nes. J. Gen. Microbiol. 104: 69-78.

c. Lechevalier, M.P. 1972. Description of a new species, Derskpvixa xanthine olyl ica and em endation of Oerskovia, Prauser et al. Intern. J. Syst. Bacteriol. 22: 260-264.

00 16 Description of the Production, Isolation and Purification of the Active Immunological Adjuvants Amycolata spp. 371-29, 37M-102 and 370-1 were grown at 28 0 C on an agar slant medium such as Bennett's or yeast-dextrose agars (Lechevalier, J. Lab. Clin. Med. 71: 934, 1968) with storage at 40 C. The actinomycete strain to be tested was grown in shaken culture as follows: cells from an agar slant culture, one to two weeks old, were inoculated into a 250 ml Erlenmeyer flask of Bennett's or yeast-extract-dextrose S10 broth dispensed at 50 ml/flask and shaken at 215 RPM for one a week at 28 C. This growth was transferred at 5-1 0 to five to ten similar flasks of the same medium, and these shaken for one week to ten days under the same conditions. The cells were then transferred at 6%0 to 2-liter Erlenmeyer flasks containing 15 200 cc of the same or similar media and shaken at 195 RPM for 0 1 D one week. The cells were then autoclaved, collected by 0 0 centrifugation and either worked up as described below or stored at -20° C until used.

In general, the immunological adjuvants are extracted from the bacteria cells by suspending cells in physiological saline solution at room temperature in the presence or absence of an emulsifier or amphipath. Any temperature below a temperature at which the adjuvant is inactivated can be employed, but there is usually no need to use a temperature above about 28t L 'Ai1 17.

The suspensions are vigorously shaken for 18 hours with an inert water-immiscible organic solvent in which the immunological adjuvant is soluble and/or dispersible.

Hexane and similar aliphatic hydrocarbons having from four to eight carbon atoms are preferred. Solvents which can also be used include cycloaliphatic hydrocarbons such as cyclohexane, petroleum ether 30-40), halogenated oo hydrocarbons such as methylene chloride, aliphatic alcohols such as n-butanol andcchloroform/methanol Emuli ifiers such as Triton-X-100 and Brij 96 can also be used, but Tween oO and SDS are more effective. Hexane alone, without amphipath, was not effective in extracting the adjuvant.

o0 On centrifuging'the sdspension;' a,.number of.'layers form.

.These'are.separated and-the adjuvant recovered from the organic solvet' layer. The remaining layers that show adjuvavnt'.activity 'can bef'irthe'r-puified by re-ext.raction with organic solvents 0 or. enzymatic action.

SBased on the results illustrated in Table III and the results of the tests reported in Examples 1 to 38, a preferred embodiment of thenrmthod of preparation of active and non-toxic adjuvants is as follows: EXAMPLE A Fifty grams of cells, prepared as above, are suspended in 500 ml of pre-cooled (40 C) 0. 9% aqueous NaC1 (physiological saline) for 10 minutes at high speed in an explosion-proof blender,

L-

18.

g of sodium dodecyl sulfate or 2.0 ml of Tween 8 0

R

added, and the whole blended for two minutes. This slurry was further treated by blending with 400 ml hexane fcr five minutes and the hexane-cell slurry further extracted by shaking 18 hours on a reciprocal shaker shaking at 60 strokes/minute.

Following this, the mixture was separated into four phases by centrifugation at 2, 500 RPM for 1/2 to two hours. The first S °o c(top) phase was clear, light amber-colored hexane; the second, oV o emulsion; the third, cells; and the fourth (and bottom), the o0 0o 10 aqueous layer. The emulsion layer was collected and taken to dryness. This dry residue was extracted by grinding with several ml aliquots of hexane, the extracted residue being retained and the hexane discarded. The typical residue is an off-white to beige powder. Typical yields of active material from 50 g 15 of cells were 118.4 mg from iexane-Tween 8 0 R extraction with 873 mg with SDS-hexane.

o o Examples 1 to 38 describe adjuvants that have been So prepared and evaluated with description of the methods of preparation; the results are given in Table II. These represent preferred embodimentsof the invention. In these Examples, three different strains of Amycolata were used. One adjuvant (Example 1) was obtained using similar methods of extraction from the mycolate-containing microorganism M. smegmatic ATCC21732, which is known to possess good adjuvant activity (Adam, A. et al., Infect. Immun. 7: 855, 1973).

Example No.

Control 1 Control 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Microorganism Extract

LPS

AMP

MLO

ML1 Bi B2 B3 B3 Fr I Fr 2 Table 11 Am phipath Tween Tween Tween Tween Tween Tween Organic Solvent Hexane Hexane Hexane Hexane Hexane Hexane Hex ane Hexane Comments Lyz ozym e ins oliible Lysozyme soluble, ether extract Lys ozym e soluble, chlorof orm: methanol extract Lysozyme soluble, residueldeft after above extractions Fr 3 Tween 80 Twveen 80 Example 8 Fr 4 Example 9 Example 10 ML1 Li No. 1 No. 2 Tween Tween Hexane Cyclohexane 0V 0 0 C 0 C 4 0 2 021 C e 00 00 :Examnple No.

Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Exmle1 SExample 18 Example 18 Example 19 Example Microorganism E ztract No. 3 No. 4 5 No. 6 ML1 L2 No. 1 No. 2 No. 3 No. 4 No. 5 ML1 L3 Fr la Fr lb Table 1U (continued) Amphipath Organic SolventC Tween 80 Petroleum ether Tween 80 Methylene chloride Tween 80 n-Butanol Tween 80 Chloroform: methanol (2:1) Tween 80 Hexane Triton X 100 Hexane Brij 96 Hexane SDS Hexane nil H1&h ne Tween 80 Hexane Tween 80 Hexane I ~omments ;,mulsion phase s'muls ion phase mxtracted with hexane lexane extract from Fr lb ,Pmulsion phase Example 22 Example 23 Fr 1c Fr 2a.

Tween 80

SDS

Hexane Hexane

E

I

I

I

I I I 1 *r' Example No.

Example 24 Example 25 Example 26 Example 27 Example 28 Example 29 1 Example 30 Example 31 Example 32 Example 33 Example 34 Example 35 Example 36 Microorganism Extract Fr 2b Fr 2c Fr 3 Fr 4alb ML2 B2 Fr 1 Fr 2 ML2 B3 ML3 B1 ML3 B2 Fr 1 Fr 2a Fr 2b Fr 2c ML3 B3 Fr 1 Table II (continued) Amphipath Organic Solvent SDS Hexane

SDS

Tween 80 nil Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Tween 80

SDS

Hexane n-Butanol Hexane Hexane Hexane Hexane Hexane Hexane Hexane Hexane Hexane Hexane Comments En sion phase extracted with hexane Hexane extract from Fr 2b Hexane phase Emulsion Hexane phase Emulsion phase Residue after hexane extraction of Fr 2a Hexane extraction from Fr 2a Emulsion phase c orD ~il a sor rv r D aii i, r a~ 9 ca r- Jo o~r 7 a- Table HI (continued) Microorganism Extract Example No.

Example 37 Example 38 Am phipath Organic Solvent Comments Fr 2 Fr 3

SDS

SDS

Hexane Hexane Hexane extraction from Fr 1 Residue after hexane e extraction. of Fr 1 Explanations: LPS-lipopolys accharide purchased from Dif co MDP-muramyl dipeptide purchased from Sigma MLO-M. smegmatis obtained from ATCC21732 Extracts designated as ML1. were obtained from Amycolata 371-29; those designated ML2 from strain 37M-102; and those designated ML3 from 37M-102.

SDS-sodium do'iecyl sulfate o 0 0 o 0 00 0~ 0 0 0 008 seC 00 0 00 000 0 0000 0 0 o 0 0 0 o 0 C o t' 0 0 0 0 000 23 Analysis of the adjuvants prepared as shown in Table II showed that no mycolic acid-like materials were present. In contrast, similarly prepared material from Mycobacterium smegmatis ATCC 21732 contained 1% mycolates by weight.

The adjuvant activity of the adjuvants of Examples 1 to 38 was evaluated as follows.

S. o It is well known that there is a direct relationship between 0 00 o 0 0 0 adjuvant activity and the ability to induce non-specific 0 .0 proliferation of B-cells. B-cells are white blood cells occurring S°°S in the blood, spleen and other tissues that are rsponsible for the production and secretion of so called humoral antibodies occurring in the blood and other tissues. Good adjuvants invariably induce a proliferation of B-cells. The test used to 15 measure the extent of this action was previously described by Bona, C. et al., J. Exp. Med. 148: 136, 1978.

In this test splenocytes (spleen cells) from BALB/C mice are incubated with various amounts of the adjuvants and the proliferation of cells associated with DNA synthesis is measured by the incorporation of radioactive 3 H thymidine. The results were expressed as the ~aean counts per minutes (CPM) for triplicate cultures pulsed on day 2 with 1 u Ci (curie) of 3 H-Td (tritiated thymidine). Thymidine is one of the four major nucleotides in DIfA. When tritiated-i. e., containing the hydrogen radioisotope tritium-it is used as a radioactive marker in cell and tissue studies for new formation 23 zo sources, inciuaing air, soi anct vegetable matter.

24 of DNA in which it is incorporated. The cultures were harvested and counted on day 3, 4 or 5. The stimulation index was the ratio of the experimental mean to the blank-control mean in which no adjuvant was used. A S.I. of less than five was nc statistically significant.

In each experiment a positive adjuvant control was also run. Lipopolysaccharide (LPS) was used for this purpose which is known to be the most potent adjuvant for B-cell proliferation O 4 as measured in this test.

4 24 10 The results obtained with adjuvants extracted by various procedures from Amycolata and other microorganisms together with the appropriate controls are 4 0Terslsotandwt duat etatdb aiu E_ 1 (3 U 0 42.3; 4 3 4 ~0 0 0 o cOo 0 4 0~.3 .3 4 -The Mitogeni Example No.

Control 1 Control 2 Example I Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 1"!xample 16 Example 17 E~xample 18 Example 19 Table I] .c Effect of Adjuvants Adjuvant from Microorganism

LPS

MDP

MLO

ML 1 31 B3 Fr 1 Fr 2 Fr 3 Fr 4 ML1 LI No. I No. 2 No. 3 No. 4 No. 5 No. 6 MLI L2 No. 1 No. 2 No. 3 No. 4 No. 5 on SpleenO-Cell Proliferation Mean Counts Stimulation Pe Minute Index 34,2S"7 14.8 3,198 2.6 9,560 4.8 48,331 20.5 42,065 18.0 7,115 8.1 21,858 22.8 482 1,984 4.9 27,759 28.6 7,710 15.2 11, 040 22.8 3,235 '7.4 -173 0.6 603 2.2 183 1.4 2,605 6.1 -419 0.2 -423 0.2 13,301 27.2 348 1.7 Table III (continued) The Mitogenic Effect of Adjuvants on Spleen-Cell Proliferation 4t 4 4 4r 4 5 554 4- 44II .4 4 Example No.

Example 20 Example 21 Example 22 Example 23 Example 24 Example 25 Example 26 Example 27 Example 28 Example 29 Example 30 Example 31 Example 32 Example 33 Example 34 Example 35 Example 36 Example 37 Example 38 Adjuvant from Microorganism ML1 L3 Fr la Fr lb Fr Ic Fr 2a Fr 2b Fr 2c Fr3 Fr 4alb ML2 B2 Fr 1 Fr 2 ML2 B3 ML3 B1 ML3 B2 Fr 1 Fr 2a Fr 2b Fr 2c ML3 B3 Fr 1 Fr 2 Fr3 Mean Counts Per Minute 1,458 15,556 2, 791 25,413 23,247 1, 848 4, 002 2,138 4, 211 1,392 2, 428 7, 0003 2, 016 10, 522 16, 987 1,071 21,731 70, 120 21,300 Stimulation Index 0.8 8.8 1.6 14.4 13.2 1.1 2.3 1.2 2.7 2.4 1.4 3.8 1.1 9.2 0.6 13.2 42.4 12.9 27 f'he results indicate that the Amycolata 371-29 st:-ain yields adjuvants that are equal or superior to those obtained from mycolate-containing organisms. A few of the adjuvants are also more potent than LP,, the most potent adjuvant known, in tests measuring B-cell proliferation, Muramyl dipeptide (M)VDP), which has been recommended as an adjuvant, had only marginal activity in this test.

The action of selected adjuvants on non-specific cell proliferation was also evaluated in other strains of mice.

Among these were the nu/nu BALB/C, also called "nude mice", Swhich do not have T-cells so that the observed antibody formation is exclusively due to a B-cell response. Other mouse strains o investigated were the C3H/HeJ animals which are genetically nonresponsive to the mitogenic and adjuvant effects of S 15 lipopolysaccharides. Another normal strain of mice called CBA/J has been use:d as an additional contrco), The results are given in Table IV..

I rrUI -1T- i-i TT~~i Table IV The Mitogenic Effect of Adjuvants in Different Strains of Mice Example No.

Control 1 Control 2 Example 1 Example 2 Example 23 Example 34 Adjuvant from Microorganism BALB/c Splenocytes BALB/c Thym ocytes nu/nu BALB/c C H/HeJ CBA/J 1, 766 78 78 2, 381 3, 153

LPS

MDP

MlLO 85,112 4, 520 32, 116 25,413 16, 987 250 120 80 196,508 4,450 12,441 4,994 2,299 36,930 11, 216 ML1 L3 Fr 2a ML3 B2 Fr 2b 93 1, 345 86,451 24,448 40,588 82,361 20, 026 21,154 63, 192 32, 012 31, 045 Results are expressed as the mean of countss per minute (CPM) for triplicate cultures pulsed on day 2 with 1 uCi of 3 H-thymidine and harvested on day 3. Dose of all adjuvants is 10 ug.

nu/nu BALB/c are nude mice C3H/HeJ are mice non-responsive to LPS 0. o r 000 oo 00 0 ot i 0 0i o o 00 002 Ci0 0 00r 0 4 3 0 0 0 S 0 29 The results indicate that the adjuvants of Examples 1 to 38 are normally effective in nude mice. This indicates that these adjuvants do not need the presence or assistance of T-cells to be mito'enic, and to induce a proliferative response in B=eells.

When the LPS non-responsive strain of mice C3H/HeJ was used, incubation of their lymnphocytes with LPS did not induce any o" p0.mirtogenic or adjuvant action.. The adjuvant, however, induced a mitogenic and adjuvant response of the same order of magnitude as has been produced by administration of these o° agents to normal mice. These results prove convincingly that the mitogenic and adjuvant action of these adjuvants is not and o cannot be due to the presence of or contamination with lipopolysaccharides or related compounds. This finding is of great 15 importance, since adjuvants containing lipopolysaccharides, because of the toxic effects of these substances, cannot be administered to humans or domestic animals.

The results obtained with splenocytes from CBA/J mice and other strains of mice indicate that the adjuvants of this invention show these effects not only in the BALB/C strain of mice but are also effective in all other strains in which they were tested.

The standard method for the evaluation of the immune response to corpuscular antigens is the Jerne technique, fully i described by Anderson J. et al., J. Exp. Med. 145: 1520, 1977.

With.this technique animals, usually mice, are immunized with a corpuscular antigen such as sheep red blood cells (SRBCs).

At various times after immunization lymphoid cells from the spleen (splenocytes) are mixed with agar and SRBCs that were used for immunization and added to the medium. After addition of complement, each antibody-forming cell in the agar plate o o starts secreting antibody that diffuses from the cell and hemolyses the SRBCs surrounding it. In this manner a plaque is formed.

The number of plaques formed can be counted and indicate the o number of antibody-forming cells that have been produced in response to the administration of the antigen. The antibodies revealed by this technique are of the IgM type. To detect IgG antibodies that bind weakly the complement, the addition of 15 complement must be preceded by the addition into the agar plate of an anti-IgG serum.

With this technique it is possible to evaluate the kinetics of the immune response to corpuscular antigens and to measure not only the amount but also the kind of antibodies formed in response to immunization.

The first antibody that appears soon after the first administration of the antigen is IgM. This immunoglobulin M (M stands for macroglobulin) is important as one of the first lines of defense in the process of protection against bacterial and viral invaders. The other antibody, called immunoglobulin G L- saline) for 10 minutes at high speed in an explosion-proof blender, "31 or IgG, begins to appear only a few days after the administration of the antigen and is particularly important for the secondary response. The secondary response occurs when the antigen is injected for a second time and is followed by immediate rise of the serum level of IgG. IgG plays an important part in rapid killing or neutralizing bacterial or viral invaders with which the body has been previously in contact. It is the high affinity antibody that combines avidly with an invader immediately after its entry into the body. The anti-SRBC response constitutes one of the 10 best models for measuring the immune response to bacterial o" and viral invaders.

The classic adjuvant that has proven most effective in this system is lipopolysaccharide (LPS), also called endotoxin, which induces a powerful response on both IgM and IgG formation. In S 15 the same series of tests we also evaluated muramyl dipeptides that recently attracted a lot of attention as a possible adjuvant and MLO, a mycolate-containing organism extracted by similar o o 0" methods of extraction as the Amycolata strains described in this document.

Table V gives some of the results obtained in two experiments to give typical examples of the results that were obtained.

Table V The Effect of Adjuvants on the Anti-SRBC Plaque-Forming Cell Response Example No.

Exp. 1 Control 1 Control 2 Example 1 Example 3 co Example 4 Example 8 Exp. 2 Control 1 Example 11 Example 13 Example 15 Example 18 Adjuvant Micror Nil

LPS

MDP

MLO

ML1 B2 ML1 B3 ML1 B3 Nil

LPS

ML1 LI ML1 L ML1 L2 ML1 L2 from 4 days ganism IgM 21, 802 64,840 25,190 102,700 85,100 65,830 Fr 4 111,530 2,900 2, 880 No. 3 4,400 No. 5 19, 600 No. 1 3,840 No. 4 15,280 7 days IgM 16,130 29,320 12,260 18,690 16,940 15,590 15,500 11,050 52,200 63,360 18,400 18,100 68,000 7 days IgG 7,770 17,180 6, 850 22,050 20,370 14,770 19,000 4,550 20, 850 17, 600 18,100 21,900 17,900 000 6-

.I

33 The pqtent adjuvant action of LPS is clearly evident in both Experiments. In Experiment 1 the peak level of IgM was reached after four days, whereas in Experiment 2 it took seven days to reach the peak response. In both experiments a significant elevation of immunoglobulin G was not reached until the seventh day. Several adjuvants that are the subject of this invention, such as ML1 B2, ML1 B3, ML1 L2 No. 1 and ML1 L2 No. 4, Examples 3, 4, 8, 15 and 18 were equal and usually superior in the Sadjuvant activity to LPS, which is generally recognieed as the most active adjuvant. It will also be noted that these adjuvants are not only free from mycolic acids but also are at the same time equal or superior to an adjuvant prepared from a mycolatecontaining strain (MLO).

It is well recognized that polysaccharides are much poorer immunogens than protein antigens. This has been a problem, particularly in the preparation of vaccines for the prevention of infections by N. meningitidis, influenzae and S. pneumoniae. The problem with development of these vaccines is particularly acute in infants who do not form antibodies to these vaccines during the first 18 months of their lives. It has now been determined that infants have a natural ontogenic delay in the formation of B-cell populations, responding to polysaccharide antigen TI2, and that this is the cause of ti high incidence of meningitis produced by N. meningitidis and H. Influenzae in infants.

1 a 9& -4 The antibody formation to polysaccharide antigens can be best measured by utilizing the trinitrophenyl-Ficoll conjugate (TNP-Ficoll) as the antigenic stimulus. TNP-Ficoll is a soluble antigen which serves as prototype for all soluble polysaccharide antigens. The adjuvants of this invention were injected with TNP-Ficoll intraperitoneally to groups .oO of five mice. After five days the antibodies to TNP-Ficoll were 0 00 oX measured by the plaque forming cell response and the number of a 00 o plaque-forming cells per spleen of animals receiving the antigen o o 10 alone compared with those that received various adjuvants in o o 0 o addition. The technique used has been fully described by Mond, 0o J. J. et al., J. Exp. Med. 158: 1401, 1983.

0a As a rule, 10 ug TNP-Ficoll was administered with 10 ug o of the adjuvant, but in a few cases the dose of TNP-Ficoll was °o 15 reduced to 1 ug, and the dose of the adjuvant increased to 100 ug.

o Table VI gives some of the results obtained in three separate experiments.

i

I

Table VI of the Immune Response Induced by the Polysaccharide Antigen T12 Enhancement Example No.

Control 1 Control 2 Example 1 Example 2 Example 3 Exmpe SExample 4 Example 6 Example 6 Examnple 7 Example 8 Example Adjuvant from Microorganism Nil

MDP

MLO

ML1 B1 Experiment 1 17, 640 23, 830 29, 770 13, 040 24,480 33, 467 34,9275 Experiment 2 11, 500 19, 400 Experiment 3 9, 000 10, 620 B3 Fr I Fr2 Fr 3 Fr 4 ML1 Li No. 1 No. 2 29, 850 14,940 29,480 23, 880 14, 850 0 t 0 0 ac..

o o C, 0 0 0 0 00 00 00 0 0 0 0 0 0 00 000 000 00 000 0 0 0 0 00 0 0 C

C

~2 .3 Fr I Examp] Examp Examp Examp Examp Examp t Exam CO Examp Examp Examp Examp Examp Examp Examp Examxp Table VI (continued) Enhancement of the Immune Response Induced by the Polysaccharide Antigen T12 Adjuvant from Le No. Microorganism Experiment 1 Experiment 2 Expar lel11 No. 3 17, 85 le 12 No. 4 15,54 le 13 No. 5 19, 26 le 14 No. 6 15, 78 le 15 ML1 L2 No. 1 20, 52' le 16 No. 2 23, 761 le 17 No. 3 17, 764 le 18 No. 4 23, 88' le 19 No. 5 18,37 le 23 ML1 L3 Fr 2a 28, 500 le 28 ML2D2 Fr 1 38,080 le 29 Fr 2 17 AA iment 3 0 0 0 0l 0l 0l 0l 0l le 31 le 34 ML3 BI ML3 _B2,F 11 TS 000 000 00" 36, 530 0' I-G 37 The results indicate that many adjuvants of this invention more than double the antibody formation. This is remarkable, because no known adjuvant is capable of achieving such an increase with the poorly immunogenic polysaccharide antigens.

Keyhole limpet hemocyanin (KLH) coupled with TNP is a T-dependent protein of low immunogenicity. It is arsoluble antigen that requires the presence of both B-cells and T-cells to induce an antibody response. Because of its very low immunogenic o capacity, antibodies to TNP usually do not appear until a 0o o S 10 second "booster" injection has been given.

To evaluate the ability of adjuvants of this invention to oo increase antibody formation, groups of mice were immunized with trinitrophenyl-keyhole limpet hemocyanin conjugate o (TNP-KLH) 100 p g with and without the adjuvants in graded doses from 1 pg, 10 pg and 100 /pg. Control animals were treated o 0 with Freund's Complete Adjuvant or MDP 100 pg, Five weeks later all mice received the second injection of TNP-KLH 10 pg without the adjuvants and the number of antibody-forming cells determined by the plaque assay performed seven days after the second injection. The details of the technique used were described by Mond, J. J. et al, J. Exp. Med. 158: 1401, 1983.

The result of one of the tests is illustrated in Table VII.

I Y Table VII Anti-TNP Plaque-Forming Cell Response to Mice Immunized with TNP-KLH Concentration of Adjuvant Used in the First Injection IgM Direct Anti-TNP PFC/Spleen IgG Indirect AnFi-TNP PFC/Spleen Example No.

Nil 480 124 220 73 Control 1 FCA Control 2 Example 23 Example 33 Example 34 Example 35 100 pg MDP 1gg ML1 L3 Fr 2a 10 p.g ML1 L3 Fr 2a 100 ML1 L3 Fr 2a 1 g ML3 B2 Fr 2a 10 -g ML3 B2 Fr 2a 100 pg ML3 B2 Fr 2a 1 pg ML3 B2 Fr 2b 10 pg ML3 B2 Fr 2b 100 pg ML3 B2 Fr 2b 1 jg ML3 B2 Fr 2c 10 pg ML3 B2 Fr 2c 100 pg ML3 B2 Fr 2c 3, 760 2 i80 1, 860 1 545 1,140 199 640 1 157 920 289 1, 220 1 499 1, 500 1286 620 156 900 1 243 940 277 740 -374 1, 180 t 414 1, 040 216 1, 480-1-994 2; 520 1,200 6501 302 980 338 380 180 800 1.336 3, 620 407 780 183 1, 100 1-436 800 1 288 480 -120 360 1 175 720 t 262 520-1 198 840 +-314 Results are expressed as the mean SEM for 5 mice in each group. All mice received 100 pg TNP-KLH along with the adjuvant in the first injection. Five weeks later they were given the second injection and the plaque assay was performed 7 days after the second injection, o a 0* C *0 0 0, C 0 0 0 0 -I D O C, 0, 0 G F0 00 0 0L 0 0 000 000 0 00*0 0 't 39 FCA caused the greatest increase in IgM as evaluated by the average number of anti-TNP-KLH plaque-forming cells, However, this increase was not stAtistically significant, as is evident from the large standard deviation, and occurred only in two out of five mice. It will be noted that several adjuvants of this invention caused a significant increase of both IgM and IgG antibodies to the weakly immunogenic TNP-KLH antigen.

The immune system in humans and animals can be divided into two major parts-cone involving humoral immunity and the other immunocompetent cells. The preceding shows that the adjuvants of this invention strongly increase the formation of humoral antibodies. Cellular immunity is of particular importance in counteracting intracellular infections, in immunity to tumors, and in transplant rejection.

Despite the development of a multitude of complex procedures for the assessment of cellular immunity, the relatively simple test for the measurement of the delayed-type hypersensitivity remains the most accurate and reliable. In this test guinea pigs were injected into each hind foot pad with keyhole limpet hemocyanin (KLC). An antigen of low immunogenicity was given alone or in addition to adjuvants of this invention. Twenty-eight days later the animals were injected intracutaneously with KLH and the surface area of the erythema measured after challenge. A dose of purified protein derivative (PPD) obtained from M. tuberculosis was IL_ injected into a separate area of the skin at the same time. The details of the technique used were previous described 'y Adam et al., Proc. Nat. Acad. Sci. USA 69: 851, 1972.

The results are given in Table VIII.

Table VIII Increase of Cellular Immunity of Adjuvants as Measured by the Development of Delayed-Type Hypersensitivity Example No. Immunization PPD (20 pg) KLH (100 pg) Control I KLH/saline 0 15.86 t 5.28 Control 2 KLH/FCA 344.16 63.96 327.36 t 61.02 Example 23 'KLH/ML1 L3:En2a 0 63.14 27.45 Example 34 KLH/ML3 B2 Fr2b 0 123.15 30.79 Example 35 KLH/ML3 B2Fr2c 0 60.32 t 15.97 Animals received an injection of 1 mg KLH/adjuvant divided equally into each hind foot pad. The dose of adjuvant was 100 ug.

The animals were challenged 28 daysafter immunization and the surface area (MMa) of erythema measured 24 hours after challenge.

The results indicate that these adjuvants are able to substantially increase cellular immunity as measured by the intensity of the delayed-type hypersensitivity. All products tested were devoid of tuberculin-like activity as determined by the absence of any reaction to PPD. It is particularly remarkable that these adjuvants may increase cellular immunity when administered in aqueous solution. Other standard adjuvants such as FCA or MDP increase cellular immunity only when

'I

41 suspended in an oily emulsion such as Freund's Incomplete Adjuvant. The observation that these adjuvants can be administered in aqueous solution is of great importance because administration of existing adjuvants in oily emulsions results because of the presence of oil in the formation of granulomatous reactions at the site of injection aich may lead to the formation of severe local reactions in the muscle such as fluctuant nodules or abscess formation. Liberation of free fatty acids from the oil in the body tissues has led to toxic reaction. It is therefore of great practical importance that the adjuvants of this invention can be administered and are effective when given in aqueous solution without the addition of any oil.

It is well known that the principal reason why adjuvants are not used in human and veterinary vaccines is their proclivity to induce adjuvant arthritis. Thus it is important to determine whether the adjuvants of this invention produce adjuvant arthritis.

The technique used to determine this was described by Waksman, B. H. et al., J. Immunol. 85: 403, 1960 and Pearson, C. M., J. Chron. Dis. 16: 863, 1963. To induce adjuvant arthritis, male Sprague Dawley rats weighing 160-190 grams, receive 0.2 ml of Freund's Complete Adjuvant in the left hind foot pad. This invariably induces arthritic changes in all limbs within 21 days.

Five rats received either FCA or adjuvants of this invention. The rats were.examined five times weekly and the right hind limb, left I severe The animals were weighed on days 0, 7, 14 and 21.

42 fore limb and right fore limb were carefully inspected and graded for edema, erythema and mobility. Each limb was assigned a score of 1 to 4 based on the severity of a the impairment- 4 being the most severe and 1 being the least severe. The animals were weighed on days 0, 7, 14 and 21.

Freunds Complete Adjuvant served as a control ind ach experiment Other controls used were commercially available vaccine and DPT (diphtheria, pertussis, tetanus) vaccine.

Other compounds tested were Freund's Incomplete Adjuvant and 10 MDP.

All the adjuvants of the Examples were suspended in sterile phosphate buffered saline solution pH 7.2. Freund's Complete Adjuvant and Freund's Incomplete Adjuvant and the typhoid and DPT vaccines were injected as commercially available. MDP was evaluated in Frund's Incomplete Adjuvant.

Example No.

Control 1 Control 2 Control 3 Control 4 Example 23 Example 33 Example 34 Table IX Induction of Adjuvant Arthritis in Rats by Adjuvants and Vaccines Score Relative Scores Adjuvant- day 21 FCA DPT Typhoid Freund's Complete Adjuvant 46 1.0 2.7 3.3 MIDP 100pjg 36 0.8 2.1 2.6 DPT 17 0.4 1.0 1.2 Typhoid vaccine 14 0.3 0.8 ML1 L3 Fr 2a1pg 12 0.3 0.7 0.9 l0 Pg 12 0.3 0.7 0.9 l00 [g 10 0.2 0.6 0.7 ML3 B2 Fr 2a 1pg 112 0.3 0.7 0.9 4O Mg 12 0.3 0.7 0.9 100 Pg 11 0.2 0.6 0.8 ML3 B2 Fr2b Ipg 12 0.3 0.7 0.9 10 pg 12 0.3 0.7 0.9 100 pg 10 0.2 0.6 0.7 ML B3Frl1l10pg 14 0.3 0.8 MLI B3 Fr 4 10jg 11 0.2 0.6 0.8 Example Example and viral invaders. The other antibody, called immunoglobulin G t 44 As noted in Table IX, the adjuvants of this invention did not induce adjuvant arthritis. The scoring obtained with them in the adjuvant arthritis test was of the same order of magnitude or lower than those produced by standard, commercially available and widely used vaccines such as DPT (diphtheria, pertussis, tetanus) or typhoid vaccine which are known never S' to have produced arthritis in man. It is of particular interest that these adjuvants did not increase their proclivity to induce adjuvant arthritis with increasing doses, indicating the absence 0 a 10 of adjuvant-arthritis-inducing substances in these preparations.

MDP, on the other hand, as previously reported, induced marked oo adjuvant arthritis (Nagao and Tanaka, Infect. Immun. 28: 624, 19?0) and confirmed that MDP can substitute for mycobacteria when 4, used in Freund's Incomplete Adjuvant (Adam, A. and Lederer, s 15 Medic. Res. Rev. 4: 111, 1984).

Lipopolysaccharides (LPS) or endotoxins are substances isolated from gram-negative and certain other microorganisms that have many interesting biological properties. Among' these is their ability to act as adjuvants, but they ,nnot be used for this purpose because of the many toxic manu ations they elicit, such as fever, leucopenia, increased sensitivity to catecholamines, hemorrhagic necrosis and the local and generalized Shwartzman reaction, profound vasomotor disturbances and shock (Berger, F. Advances in Pharmacology 5: 19, 1956). It is therefore 44 i .0 0 f 0 3 .f 0 tB O f D 9 ii C 8 I B f

I

i of great importance that adjuvants and vaccines for human or veterinary use be free from LPS.

The Limulus Amebocyte Lysate method accurately and quantitatively determines the amount of LPS present in a sample.

The test is of extreme sensitivity and will detect as little as 0. 125 EU/ml, which could be called a faint trace of LPS that would be undetectable by any other method.

Table X gives the results of the tests which indicate that there are no detectable traces of LPS in adjuvants of this invention.

10 Table X Liposaccharide Content of Adjuvants as Determined by the Limulus Amebocyte Lysate Test Example No. Adjuvant Result Control 1 LPS Clot formation:- Control 2 Negative control Less than 0.125 EU/mL Example 23 ML1 L3 Fr 2a Less than 0.125 EU/ml Example 33 ML3 B2 Fr 2a Less than 0.125 EU/ml Example 34 ML3 B2 Fr 2b Less than 0.125 EU/ml Control Typhoid vaccine Less than 0.125 EU/ml The complete freedom from LPS in the adjuvants of this invention is further confirmed by the observation that our compounds show marked adjuvant action in mice of the C3H/HeJ strain that is unresponsive to LPS.

1 It is known that certain substances, such as LPS, produce polyclonal B-cell activation- produce plaque-forming cells to an antigen, such as SRBCs- by themselves in the absence of the antigenic stimulus. If a product possesses a high polyclonal Bcell activating effect, it is difficult to evaluate the anti-SRBC immune-enhancing effect because one cannot be sure whether the increased number of plaque-forming cells is due to a true enhancement of the specific immune response or to a non-specific polyclonal B-cell activation.

10 Table XI gives data for a number of adjuvants of the invention.

o 0.

044 0 01 04~r 4,~ 0Z 4 40a 0o 4 04 0 4 44 4 i i i Table XI Polyclonal B-cell Activation by Adjuvants Example No.

Example 28 Example 29 Example 30 Example 28 Example 29 Examnple 31 Adj uvant ML2 Fr I ML2 Fr 2 MU3 ML2 Fr 1 ML2 Fr 2 ML3

SIRBC

none none none 107 107 107 Stimulation Index 1.1 0. 8 0.4 32.0 28.7 73.3 000 0.0 00 COO C K- 48 As is apparent from Table XI, adjuvants of this invention when given alone do not increase the number of plaque-forming cells, but do so very strongly when given together with SRBCs.

The immunological adjuvants in accordance with the invention can be administered to animals as a class, including i man, and both large and small animals, by any conventional oo administration procedure, including, for example, oral administration, transdermal administration, transnasal 0 o administration, sublingual administration, rectal administration, and parenteral administration.

The immunological adjuvants can be administered per se, but are usually given together with one or more of the specific antigens. Specific antigens that can be used for this purpose include: Bacillus anthracis Bordetella pertussis abortus Brucella melitensis Brucella suis Cholera toxin Cholera toxin beta subunit Cholera toxin alpha subunit Cholera vaccine Clost. botulinum alpha and beta toxins Clost. Novyi toxoid 48 -c 1. 1 11~-~111 101- 49 Ciost. aedematiens toxoid Clost. perfringens Clost. perfringens toxoid Clost. tetani Coryn. diphtheriae Coryn. equi Coryn. parvum Diphtheria toxin Diphtheria toxoid Esch. coli Francisella tularensis Ilemophilus influenzae Hemophilus influenzae group b antigen Legionella Legionella pneumophila group 1-6 antigens Leprom in Leptospira antigens Xlebsiella pneumoniae Pseudomonas aeruginosp Saint abortus equi Serr. marcescens Shig. flexneri Shig. dysenteriae group A, Al1, B, C, Cl1, C2, D antigens Vibr. Cholerae Yersinia enterocolitica Listeria Micrococcus Mycobacteria M. :eprae glycolipids I, I and III M. vaccae Mycoplasma Mycoplasma pneumoniae CF antigen Neisseria meningitidis Neisseria meningitidis polysaccaride vaccine groups A, C, Y, W-135 Pertussis vaccine 8 B. Pertussis lymphocytosis-promoting factor Pertussis toxin (islet-activating protein) N. gonorrhoeae o N. gonorrhoeae pilus protein N. gonorrhoeae glycoprotein N. gonorrhoeae principal outer membrane protein Chlamydia Protein A Proteus 0 Pseudomonas aeruginosa Reiter treponema antigen Salm. N antigens a, b, c, d, eh, g, i Salm. O antigens A through I Vi SaTilm. enteritidis H antigen Salm. enteritidi. Oantigen Salm. paratyphi H antigens a, b, c Salm. paratyphi O antigens A, B. C Salm. typhi H antigen Salm. typhi O antigen Salm. typhi Vi antigen Salm. typhi vaccine Salm. typhimurium Staph. alpha toxin Staph. enterotoxins A, B, C1, C2, D, E Staph. protein A Strep. antigens groups A, B, C, D, F, G Strep. faecolis Strep. pneumoniae polysaccharide antigens 14 types Streptolysin

O

Swine crysipelas vaccine S. Syphilis antigens FTA-ABS sorbent, RPR, VDRL T. pallidum Tetanus toxoid Typhoid vaccine Mycoviruses Adenoviruses types 1-35 7a Adenoviruses human group CF antigen Adenoviruses pig Avian leukosis sarcoma 22 strains Avian myeloblastosis Avian reticuloendotheliosis 4 strains Avian tumor viruses Bovine leutemia (BLV-FLK purified)

I

52 Bovine papilloma Bovine polular stomatitis Bovine parvo Bovine resp. syncytial Bovine biral diarrhoea Burkitt's lymphoma Canarypox Canine distemper Canine parainfluenza Colorado tick fever Corona OC-43 and 229E Coronaviruses from cat, chicken, dog, human, mouse, pig, rat Coxsackie Al through A Coxsackie B1 to B6 Creuzfeld-Jacob virus Cytomegolo virus and CF antigen Dengue types 1-4 DuCk hepatitis ECHO virus including types 1-9 and 11-33

EMBU

Encephalitis virus, California, Eastern, equine, Japanese B, Russian spring-summer, St. Louis, Venezuela equine, Western equine Enterovirus types 68-71 Ep,tein-Barr virus and copsid Ag, early AgD, AgR, nuclear Ag, DNA Equine arteritis Equine herpes, all types 44 153 Equine infectious anemia Feline leukemia Feline panleukopenia Feline picornoviruses 8 strains Fowlpox 5 strains GD-*VIrI virus (Theiler) Goose hepatitis Hepatitis A Hepatitis B vaccine and surg Ag (adr), (adw Herpes saimiri Herpes simplex' 1 and 2 vaccines and CF Ag, MS Hog cholera Influenza (avian) r) 4 (ayw) Influenza A 11 strains Incluc-nza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Intluenza A Influenza A Influenza A 2 5 Influenza A Influenza A vaccine CF Ag and AlCHl/2/68 equine 1 and 2 (FM- 1/47) (Japan/170/62) (Japan/305/57) (P 8/34) (Swine/1976,/3 1) (Taiwan/1/64) (USSR/90/77) (Victoria/3/75) HA Ag (WS/33) d 1~ 54 Influenza A Various strains Influenza A2 various strains Ynfluenza B various strains Influenza A vaccine Influenza B vaccine Influenza B (Great Lakes/1739/54) Influenza B (Hong Kong/8/73) HA Ag Influenza B Influenza B (Maryland/1/59) Influenza B (Mass/3/66) HA Ag Influenza B (Singapore/3/64) Influenza B (Taiwan/2/62) Influenza B (Taylor/1233/47) Influenza C Kuru virus K virus LDH virus Lymphocytic choriomeningitis Marek's disease Measles vaccine Measles CF and HA Ags

MMTV

Monkeypox Mouse hepatitis IMumps vaccine Mumps CF ag, HA Ag 1; II~-L--LI rats were.examined five times weekly and the right hind limb, left

I,

Murine Ileukemias Murine sarcomas Mycoviruses 38 rains Newcastle disease vaccine Oncogenic viruses Parainfluenza 1-5, 1 CF and HA Ags Picorna virus Polio virus vaccine strains 1, 2 and 3 Polyoma vaccine Rabbit fibrornma Rabbit myxoma Rabbitpox Rabbies 6 strains Rabbies vaccine Raccoonpox Rauscher leukemia virus Reovirus from birds, cow, man, monkey Resp. syncytial vaccine and CF antigen Betroviruses 14 strains Retrovirus vaccine R otavir!s Rous sarcoma virus Rubella Vaccine Rubella CF and HA antigens 56 Semliki forest virus Sendai virus Simian adenovirus hyper 1-26 Simian cytomegolovirus Simian foamy Simian herpes Simian oncorna SSimian paramyxo Simian picorna Simianpox SSimian reo Sim'ian rota Simian sarcoma Sindbis virus Smallpox vaccine Swinepox Vaccinia virus Varicella vaccine Varicella Zoster vaccine Vesicular stomatitis virus West Nile virus Yaba tamor poxvirus Yellow fever vaccine Chlamydia CF Ag Bovine encephalomyelitis chlamydiae 57 Inclusion conjunctivitis chlamydiae Lymphogranuloma venereum Meningopneum onitis Ornithos is Ovine chiamydial abortion Ovine polyarthritis Psittacosis Sporadic bovine encephalomyelitis T rachomna Coxiella burnetiL Rickettsia akari Rickettsia canada R ickettsia montana.

Rick1ettsia prowazeki Rickettsia rickettsu.

0 Rickettsia tsutsugarnushi Tha tick typhus Fungus extracts Aspergillus (various strains) Blastomyces Candida albicans Cocc idioides Histophasma Micropolyspora Neurospora (various strains) 58 Saccharom onospora Saccharomyces Trichophyton.

Algae Acanthamoeba Amoebae Babes ia Chiamydom onas Crithidia Entamoeba (various strains) Herpetomonas Hyrpotri chononas Leishmania (various strains) Leptom onasi Naeglerila Phytom onas Plasmodium (various strains) Rhizopods Toxoplasma gt. idi Trichomonas (various strains) Trypanosoma (various strains) Echinococcus granulosis Fasciola hepatica 59 Hymenolepis diminuta Schistosoma hematobiam Schistosoma Japonicum Schistosoma mansoni Ascaris (various strains) Nematodes (various strains) Trichinella spiralis o o All of these are commercially available and are listed in aS Linscott's Directory of Immunological and Biological Reagents o 0' o.n 10 3rd Edition 1984 1985. These include vaccines, intact microo S° organisms such as viruses, chlamydiae, rickettsiae, bacteria and bacterial antigens, fungi, protoza, algae and slime molds, platyhelminthes, nemathelminthes, annelida, anthropoda, molusca and others, as well as extracts, purified fractions and subunits.

The specific antigen administered with the adjuvant may be oo o the killed or inactivated intact microorganism, an extract prepared from the microorganisms or a small portion of the microorganism responsible for the induction of protective antibodies (the subunit).

The specific antigen in any of the above-mentioned forms may be physically mixed with the adjuvant or chemically coupled or covalently linked with the help of coupling reagents in accordance with standard procedures as described by K. Lynn Cates et al., Infect. Immun. 48: 183, 1985 or Chiayung Chu et al., Infect. Immun.

245, 1983. The antigen may be used in its native form or chemically attached to a protein or fatty acid carrier.

ii 'l L I The immunological adjuvant administered jointly with one or several of the specific antigens in a manner mentioned in the previous paragraph is usually given in association with a pharmaceutically acceptable diluent or carrier. The invention accordingly also provides a pharmaceutical composition in dosage unit form comprising from 0. 001 to about 50 mg of immunological adjuvant, per dosage unit, together with a pharmaceutically acceptable nontoxic inert carrier or diluent therefor.

The immunological adjuvant can be administered to the 00 4 10 animal in an amount sufficient to increase the immune response of Pntigens, and will depend upon the species of animal, and the weight of the animal. For example, in human administration, a dosage of immunological adjuvant within the range from about 0. 001 mg/kg to about 50 mg/kg per day is therapeutically effective.

In the treatment of lower test animals, a similar dosage range is therapeutic. The upper limit of dosage is that imposed by toxic side effects, and can be determined by trial and error for the animal to be treated, including humans.

To facilitate administration, the immunological adjuvant can be provideu in composition form, and preferably in dosage unit form. While the compound car be administered per se, it is normally administered in conjunction with a pharmaceutically

I

acceptable carrier therefor, which dilutes the compound and facilitates handling. The term "pharmaceutically acceptable" 61 means that the carrier (as well as the resulting composition) is sterile and nontoxic.

The carrier or diluent can be solid, semisolid, or liquid, and can serve as a vehicle, excipient, or medium for the immunological adjuvant. Exemplary diluents and carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl- and propylhydroxybenzoate, talc or magnesium stearate.

For convenience in handling, the immunological adjuvant and carrier or diluent can be enclosed or encapsulated in a capsule, sachet, cachet, gelatin, paper or other container, especially when intended for use in dosage unit form. The dosage units can for example take the form of tab-sts, capsules, suppositories or cachets.

The following Examples illustrate various forms of dosage units in which the immunological adjuvant can be prepared: Example I Tablet formulation Mg/tablet Immunological adjuvant Lactose 86 Corn starch (dried) 45.5 Gelatin Magnesium stearate 62 The immunological adjuvant is powdered and passed through a mesh sieve and well mixed with the lactose and 30 mg of the corn starch, both passed through a sieve.

The mixed powders are massed with a warm gelatin solution, prepared by stirring the gelatin in water and heating to form a 10% W/w solution. The mass is granulated by passing through a sieve, and the moist granules dried at 40 0

C.

The dried granules are regranulated by passing through ao.

S a sieve and the balance of the starch and the magnesium stearate is added and thoroughly mixed.

The granules are compressed to produce tablets each weighing 150 mg.

0 Example II Tablet formulation Mg/tablet Immunological adjuvant 100 0 Lactose 39 Corn starch (dried) Gelatin Magnesium stearate The method of preparation is identical with that of Example I except that 60 in of starch is used in the granulation process and 20 mg during tabletting.

Example III Capsule formulation Mg/capsule Immunological adjuvant 250 Lactose 150 The immunological adjuvant and lactose are passed through a sieve and the powders well mixed together before filling into hard gelatin capsules of suitable size, so that each capsule contains 400 mg of mixed powders.

Example IT Suppositories Mg/suppository 0 r 00 0 u 10 ~t.'7 Immunological adjuvant Oil of Theobroma 950 The immunological adjuvant is powddr d and passed through a sieve and triturated with molten oil of theobroma at 45°C to form a smooth suspension.

The mixture is well stirred and poured into molds, each of nominal 1 g capacity, to produce suppositories.

Example V Cachets Mg/cachet Immunological adjuvant 100 Lactose 400 The immunological adjuvant is passed through a mesh sieve, mixed with lactose previously sieved and fitted into cachets of suitable size so that each contains 500 mg.

.1 j 64 Example VI Intramuscular injection (sterile suspension in aqueous vehicle) Mg Immunological adjuvant Sodium citrate 5.7 Sodium carboxymethylcellulose (low viscosity grade) Methyl para-hydroxybenzoate o Propyl para-hydroxybenzoate 0.2 10 Water for injection to 1.0 ml oo Example VII o° 0 Intraperitoneal intraveneous or subcutaneous injection (sterile solution in aqueous carrier system) Mg Immunological adjuvant Sodium citrate 5.7 Sodium carboxymethylcellulose (low viscosity grade) Methyl para-hydroxybenzoate Propyl para-hydroxybenzoate 0.2 Water for injection to 1.0 ml -4 Broadly speaking, the preparation of new vaccines has three aims: 1. To prepare vaccines that would protect against diseases against which protection is not possible today.

2. To purify existing vaccines so that they would not induce adverse reactions or unpleasant side effects.

3, To produce vaccines more cheaply so that the existing vaccines could be made available to more people.

These three aims are attainable with the use of non-toxic adjuvants.

It is now known that only a small portion of the pathogenic microorganism represents an antigenic entity against which, under appropriate conditions, protective antibodies are produced. These small portions are often called subunits and represent specific segments of the peptide chain which when administered together with a suitable adjuvant induce specific protective antibodies. The use of subunit vaccines will make it possible to eliminate from existing vaccines that part of the molecule that is responsible for the side effects and adverse reactions that vaccines may induce.

Other vaccines, such as hepatitis B, are very expensive. An addition of adjuvant to such a vaccine would permit the utilization of much smaller amounts of the specific antigen and thus substantially decrease the cost of the vaccine.

Claims (21)

1. A process for preparing an immunological adjuvant that when administered to animals increases the immune response to antigens, and that is substantially free from mycolic acids, mycolic acid esters, and lipopolysaccharides, which comprises suspending Amycolata bacteria cells in aqueous saline solution; extracting the cells with an inert organic solvent in which the immunological adjuvant is soluble or dispe-'sible; separating the organic solvent solution from the bacteria cells and the aqueous saline solution; and recovering immunological adjuvant.

2. A process according to claim 1 in which the extraction is carried out in the presence of an emulsifying agent.

3. A process according to claim 2 in which the emulsifying agent is an organic surfactant.

4. A process according to claim 3 in which the organic surfactant is sodium dodecyl sulphate. A process according to claim 4 in which the organic surfactant is Tween 80 R

6. A process according to claim 1 in which the organic solvent is an aliphatic hydrocarbon having from about four to about eight carbon atoms.

7. A process according to claim 6 in whtdh the aliphatic hydrocarbon is hexane. 66 t 67

8. A process according to claim 1 in which the organic solvent is a cycloaliphatic hydrocarbon.

9. An immunological adjuvant substantially free from mycolic acids, mycolic acid esters, and lipopolysaccharides, and capable of increasing the immune response of an antigen upon administration to an animal body, prepared in accordance with the process of claim 1. An immunological adjuvant according to claim 9, in which the extraction is carried out in the presence of an emulsifying agent.

11. An immunological adjuvant according to claim in which the emulsifying agent is an organic surfactant.

12. An immunological adjuvant according to claim 11, in which the organic surfactant is sodium dodecyl sulphate.

13. An immunological adjuvant according to claim 11, in which the organic surfaccant is Tween 80 R

14. An immunological adjuvant according to claim 9, in which the organic solvent is an aliphatic hydrocarbon having from about four to about eight carbon atoms. An immunological adjuvant according to claim 14, in which the aliphiatic hydrocarbon is hexane.

16. An immunological adjuvant according to claim 9, I in which the organic solvent is a cycloaliphatic hydrocarbon. 67 68

17. A process for increasing the immune response of antigens which in animals induce the formation of antibodies, which comprises administering to the animal an immunological adjuvant prepared according to the process of claim 1, and an antigen.

18. A process according to claim 17 in which both the o c no 0 1 immunological adjuvant and the antigen are administered together.

19. A process according to claim 17 in which both the 00, immunological adjuvant and the antigen are administered S l o separately. A process according to claim 17 in which the o o immunological adjuvant is administered parenterally.

21. A pharmaceutical composition for increasing the immune response of antigens which in the animal body induce the formation of antibodies, comprising an immunological adjuvant prepared according to the process of claim 1, and a pharmaceutically-acceptable nontoxic carrier or diluent.

22. A pharmaceutical composition according to claim 21 which also comprises an antigen.

23. A pharmaceutical composition according to claim 21 in dosage unit form.

24. A pharmaceutical composition according to claim 23 which also includes an antigen. A¥E+-3O-Deeemb j PHILLIPS ORMONDE FITZPATRICK Attorneys for: FRANK M, BERGER 7l il- i.i, I: 69 A process according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

26. An immunological adjuvant according to claim 9 substantially as hereinbefore described with reference to any one of the examples. DATED: 20 May 1991 PHILLIPS ORMONDE FITZPATRICK Attorneys For: FRANK M BERGER ~O~A~4 t4 t 4.0 I i tr I (9961h') 7Xt 171 4 JC