AU629103C - Vaccine and method of preparation - Google Patents

Description

N, A Commnonwealth of Australia The Patenits Act 1952 DECLARATION IN SUPPORT In support of the (eonventimnj Application made by: EMORY UNIVERSITY o~f 1380 South Oxford Road, Atlanta, GA 30322 United States of America for a patent for an invention entitled: "VACCINE ANDL METHOD OF PREPARATION"

I{Wej Ann R. Stevens of and care of the applicant company do solemnly and sincerely declare as follows:

a-Iam (148ee th's opplicant(s) for the patoont or b) I am (W~e authorised by the applicant(s) for the patent to make this declaration on its behalf. Dee a lloWing If not a Convention Application.

:The basic ication(s) as defined by section 141 (142) of the Act was (were) made on in

Thebasc aplcation(s) referred to in this paragraph is (are) the first application(s de in :a Convention country in respect of the invention the subject of the application. 1)am (Wec arc) the actual iniventor(a) of the i''n~n

or b) Robert L. Hunter of 3640 Churclhwell Court, Tucker, Atlanta GA 30322, United States of America

is r) th& actual inventorf4A of the invention and the facts upon which The Applicant is (j4entitled to make the, application are as follows:

The applicant is the assignee of i,:;he invention from -the said actual inventor.

Declared at.. A41an,tAL', thi 2. day 199.1.

Associate Vice President for Acadevtc SindX 4 Stau Affairs and Sponsotad Research

Declarant's Name ,Ann R. Stevens, Ph.D., on behalf of EMORY UNIVERSITY

F.B. RICE CO PATENT ATTORNEYS rbIis ffirm I iqutab for any ty pootf iltnt AppI Ica Ifun, No legalisatloa WqlIted,

(12) PATENT ABRIDGMENT (11) Document No,AU-B-71131/91 (19) AUSTRALIAN PATENT OFFICE (10) Acceptance No. 629103 (54) Title VACCINE AND METHOD OF PREPARATION International Patent Classification(s) 5 (51) A61K 047/32 (21) Application No. 71131/91 (22) AppIllcation Date; 15.02.91 Priority Data (31) Number (32) Date (33) Country 909964 22.09.86 US UNITED STATES C>F AMERICA 075187 16.07.87 US UNITED STATES C)FAMERICA (43) Publication Date 11.07.91 (44) Publication Date of Accepted Application :.24.09.92 (62) Related to Division(s) 80232/87 (71) Applicant(s) EMORY UNIVERSITY (72) Inventor(s) ROBERT L. HUNTER (74) Attorney or Agent F B RICE CO, 28A Montague Street, BALMAIN NSW 2041 (57) Claim 1. A co polymer with the following formula:

(C 3 H 6 0 b(C2H40 )a\ (C 2H40)a(C3H6 0 )b

N-H 2 C-CH N

1a (C 2H40)a(C3H60)b C 3 E0 6 )b(C2H40

wherein: a is a number such that the hydrophile portion represented by polyoxyethylene (C2H40)a (POE) constitutes between approximately 10% to 40% of the total molecular weight of the compound; the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene,(C3H60) b (POP) is between approximately 5000 and 7000 daltons; and b is a number such that the polyoxypropylene (C3H60)b (POP) portion of the total molecular weight of the octablock copolymer constitutes between

/2

AU-B-Ti 131/91 -2- 629103

approximately 60% and 90% of the compound. When used as a vaccine adjuvant. 2. The vaccine ad(juvant of claim 2 where- the adjuvant has the following formula:

(C3H6O)b(C2H4O)a (C2H40)a(C3 H6O)b

(C3 H6O)b(C2H4O)a /NHCH \(C2H40)a(C3H60)b

wherein a is equal to 5 and b is equal to 32.

629 103 COMMONWEALTH OF AUSTRALIA

Patent Act 1952

COMPLETE SPECIF CAT TON (ORIGINAL)

Class Int. Class

S Application Number 00

0 Lodged

Complete Specification Lodged Accepted Published

S Priority:

Related Art C **CC

Name of Applicant S EMORY UNIVERSITY

Address of Applicant 1380 South Oxford Road, ATLANA, GA. 30322. U.S.A. CS

Actual Inventor HUNTER Robert L. of, 3640 Churchwell Court TUCKER, GA. 30084. U.S.A.

Address for Service F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN. 2041.

Complete Specification for the invention entitled:

"VACCINE AND METHOD OF PREPARATION"

The following statement is a full description of this invention including the best method of performing it known to us:- it1

CGross refcrenee !o Related ARpIORation TWapi tion is continuation Wnprto U.S. Applintion 0 cc &Utltfdv VRdIW'VW"V 0

0* SO S. 0 S

0 Technical Field The present invention relates to a viccine and m ore 0

0 particularly to a vaccine comprising a bacterial flagella which, 0O

*5@ when conjugated with an antigen moiety, amnplifies the immune response to the antigen.

Background Art 0 .00600 0 10 A vaccine is defined herein as a suspension of antigenic 0000

S. 0* 0 moieties, ivsually consisting of infectious agents, or some part of the infectious agents, that is injected ito the body to produce active SO S S S immniy. The antigenic moiety making up the vaccine can be either a nauzral product purified from a microorganism, a synthetic product or a genetically engineered protein peptide or similar 6S 0 5S 00 product. An adjuvant is defined herein as any substance whose @6 00 50 0 admixture with an injected irnunogen increases the immune response. A hapten is defined herein as a substance that reacts selectively with appropriate antibodies but the hapren itself is usually not immunogenic. Most haptens are small mole"e Ias, but some macromolecules can also function as haptens. Conjugation is defined herein as the covalent or other form of linking of two or more molecules. Sixty years ago it was demonstrated that it was possible to augment the antitoxin response to diphtheria and tetanus by administering vaccines as a mixture with pyogenic bacteria or with various additional compounds. Since that time, clinicians and immunologists have sought to potentiate the immune response,,with adjuvants while attempting to minimize the often-present side effects. Biosynthetic and recombinant DNA technology is permitting development of vaccines possessing antigenic epitoms that were previously impossible to produce. Current vaccine candidates, by way of example, include synthetic peptides that immunogenically 0 mimic streptococcal, gonococcal, hoof and mouth disease, AIDS *0 0 (HV- virus) and malarial antigens. The work on the parasitic disease malaria is especially 0 0

0 15 0 important. This disease affects in excess of 200,000,000 people per year worldwide and is the most important disease in the world in S 000600 0

06 Si 005 terms of morbidity and loss of work. The techniques of genetic engineering have been used to identify, and now to produce in substantial quantities, several proteins of malarial parasites. In particular, a twelve amino acid peptide from the sporozoite stage has been determined to carry an important antigenic site. *O S Antibodies against this particular peptide can kill the parasite immediately after it is injected. Unfortunately, this peptide, by 25 itself, does not produce an adequate immune response. In an effort to induce an effective immune response to the sporozoite peptide, the peptide has been 'Idministered with 0 adjuvants. To date, however, the adjuvants used with the peptide a o have not produced satisfactory results. Thus, interest has arisen in the development of potent, nontoxic adjuvants that will enhance the immunogenicity of haptenic epitopes. In addition, adjuvants are needed for use with conventional vaccines to elicit an earlier, more potent, or more prolonged response. Such an adjuvant would also be useful in cases where antigen supply is limited or is costly to produce.

The development of adjuvants has, until recently, been empirical. An enormous number of compounds have been found to modulate the immune, response. These compounds have been notably diverse in both substance and function, a fact that has complicated attempts to discover the unifying mechanisms of adjuvant action. The elucidation of these mechanisms has lagged behind recent advances in the understanding of the immune system. This diversity of adjuvants has presented difficulties in their classification. Adjuvants are occasionally grouped according to their origin, be it mineral, bacterial, plant, synthetic, or host product. The first group under this classification are the noribacterial adjuvants, such as aluminum compounds. The first use of aluminum compounds as adjuvants was described hai 1926. Since that time antigens precipitated with aluminum .mlts or antigens mixed with or adsorbed to performed aluminum compounds have been used extensively to augment immune 565fee responses in animals and humans. Aluminum compounds and 20 similar adjuvants appear to work through the following mechanism: excretion of the antigen is slowed, thus prolonging the time of interaction between the antigen and antigen-presenting cells such as macrophages or foilicular-dendritic cells. In addition, immunocompetent cells are attracted to the area of injection. Aluminum particles have been demonstrated in regional lymph 5 nodei of rabbits seven days following immunization, and it may be that another significant function is to direct antigen to T cell- containing areas in the nodes themselves. Adjuvant potency has S been shown to correlate with inflammation of the draining lymph S.

0S 5*

S nodes. While many studies have confirmed that antigens SO 5* administered with aluminum salts led to, increased humoral immunity, cell mediated immunity appears to be only slightly increased, as measured by delayed-tq;pe hypersensitivity. Aluminum hydroxide has also been described as activating the complement pathway. This mechanism may play a role in the local inflammatory response as well as immunoglobulin production and B cell memory.

Primarily because of their excellent record of safety, aluminum compounds are presently the most commonly used adjuvants in humans. They are, however, not without problems. Aluminum containing vaccines occasionally cause local reactions. Although allergic manifestations are not usually a clinical problem, aluminum compounds have been also said to attract eosinophils to the area of injection via a T cell-dependent mechanism, to induce an IgE response if injected after antigen priming, and to elicit a carrier-specific cell population with helper function for IgE response. In addition, aluminum-containing vaccines cannot be lyophilized, thus necessitating refrigerated transport and storage with the rsulting risk of contamination. Finally, and most importantly, aluminum compounds are not always successful in inducing sustained protection from disease. S 15 Thus, while aluminum salts have been a sufficient adjuvant for strong immunogens that reqqir antibody responses only to elicit protection, they are not effective when used with weak immunogenic-like synthetic peptides of malaria for introducing 2O cell-mediated immune responses of the type required for many infections. Another large group of adjuvants are those of bacterial 0 origin. Adjuvants with bacterial origins have recently been 6 O purified and synthesized muramyl dipeptides, lipid A) and host mediators have been cloned (Interleukin 1 and providing chemically characterized products for study. The last decade has brought significant progress in the chemical purification of three adjuvants of active components of bacterial origin: Bordetella perrtusis,lipopolysaccharide and Freund's complete adjuvant. So 00 B. pertussis is of interest due to its ability to modulate cell- mediated immunity through action on T-lymphocyte populations. For lipopolysaccharide and Freund's complete adjuvant, adjuvant- active moieties have been identified and synthesized, which permit study of structure-function relationships and the possibility of modifying the original adjuvant to create a more beneficial toxic- therapeutic ratio.

Lipopolysaccharide and its various derivatives, including lipid A, have been found to be powerful adjuvants in combination with liposomes or other lipid emulsions. It is not yet certain whether derivatives with sufficiently low toxicity for use in humans can be produced. Freund's complete adjuvant is the standard in most experimental studies. However, it produces severe local and systemic inflammatory reactions which may be severe enough to cripple or kill the host. It cannot be used in humans. Adjuvants have also been categorized by their proposed mechanisms of action. This type of classification is necessarily somewhat arbitrary because most adjuvants appear to function by more than one mechanism. Adjuvants may act through antigen OOSQgO localization and delivery, or by direct effects on cells making up the immune system, such as macrophages and lymphocytes. Another 15 mechanism by which adjuvants enhance the immune response is by creation of an antigen depot. This appears to contribute to the 0 0. adjuvant activity of aluminum compounds, oil emulsions, liposomes, and synthetic polymers. The adjuvant activity of *0 S lipopolysaccharides and muramyl dipeptides appears to be mainly mediated through activation of the macrophage, whereas B. perrusis affects both macrophages and lymphocytes. Recent and speculative approaches to immunopotentiation, such as the SI utilization of monokines and lymphokines, and the manipulation of the antigen, carrier, and adjuvant to augment the immune response are currently fashionable. Small immunogens, such as the synthetic peptide of malaria, can be attached to larger proteins or other carriers to increase the immune response. The relationship between molecular size and :355 complexity of an antigen relative to immunogenicity reflects the availability of antigenic determinants on the molecule. This relationship was first noted by Landsteiner when he demonstrated the need to complex small radicals with larger (carrier) molecules to stimulate an immune response. However, the mechanistic basis 2 for the requirement was to await experiments that demonstrated the carrier effect and the need for a minimum of two antigenic determinants on a molecule to express immunogenicity. These determinants represented the carrier and haptenic determinants that interact with T and B lymphocytes, respectively. However, the influence of the carrier moiety extends beyond simple antigenicity through activation of T cells in T-dependent humoral responses. The combination of determinants on an antigen molecule can influence the immune response by differential activation of helper and suppressor T cells. A model system demonstrating this effect is the genetically controlled humoral response of responder (C57B1/6) and nonresponder (DBA/1) mice to the synthetic terpolymer 1-glutamic acid6 0 -L-alanine 3 0 -L-tyrosinel0 (GAT). While C57BI/6 mice respond to this polypeptide, DBA/1 mice will O O0 respond, only if the GAT is coupled to methylated bovine serum C albumin (MBSA). However, if the mice are injected with GAT 15 prior to immunization with GAT-MSBA, a detectable antibody response to GAT does not occur. The explanation for these 0 observations is that GAT stimulates helper T cells in the responder mice but preferentially activates suppressor T cells in nonresponder mice. This predominance of suppressor cells prevents a response to GAT even when coupled to MBSA. However, if primary immunization is with GAT-MBSA, activation of helper T cells by the carrier moiety provides help that overrides the effect of any suppressor cells activated by GAT. Determinants associated with a native protein molecule have SO also. beef demonstrated to contribute differently to help and suppression. Conjugation of an immunogenic carrier to an antigen C can change the isotype of antibodies produced in response to that *9 antigen. Purified polysaccharides from a variety of encapsulated S

S. 00 bacteria are thymus-independent antigens, due to their polymeric nature with multiple repeating antigenic determinants. While they represent protective antigens of these bacteria, the IgM antibodies produced have limited efficacy in preventing disease. Therefore, polysaccharides from Neisseria meningitidis and Haemophilus influenza type b have been conjugated to proteins, such as tetanus toxoid. These conjugated preparations act as thymus-dependent

1/ antigens and induce IgG responses to the polysaccharide moiety as well as immunologic memory. Likewise, the thymic-independent polysaccharide carriers have little potential for enhancing the immunogenicity of small peptides, such as those involved with malaria which require thymic-dependent IgG immune responses. Publications by Feldmann and Lee state that flagella antigens of Salmonella organisms are typical thymic-independent antigens which stimulate strong IgM antibody responses.(See Feldmann, M, et al., "The Relationship between Antigenic Structure and the Requirement for Thymus-derived cells in the Immune Response", J. Exp. Med., VoL 134, pp 103-119, 1971; and Lee, et aL, "Decline and Spontaneous Recovery of the Monoclonal Response to Phosphorylcholine during Repeated Immunization", J. Immun., VoL 113, pp 1644-1646, 1974) This published data would lead one 15 to believe that they have little potential as adjuvants or carriers for 0 malaria peptides or other small antigens wich require thymic- dependent IgG antibody responses. There probably is no precise point of transition that distinguishes a carrier from an adjuvant Obviovsly, the carrier moiety is contributory to a property of antigens that has been termed i insic adjuvanticity. The capacity of certain materials to convert tolerogen to an immunogen has been termed as extrinsic adjuvanticity. Adjuvanticity can be enhanced by increasing the size of the-antigen through aggregation of proteins or adsorption to S immunogenic or inert carriers. Thus materials, such as aluminum hydroxide, latex particles, bentonite, or liposomes that adsorb *5 5 antigen and enhance the immune response, are termed adjuvants. o; 0O

55 0* However, this observed effect of aggregation of antigen represents .5 S OS only a limited view of adjuvant actions which are now recognized as being extremely complex. Small peptides and other haptens are incapable of evoking a strong immune response without the use of an adjuvant. Most adjuvants that are currently available do not evoke an immune response that is effective in protecting the animal or human against infection with the infectious agent. Thus, what is needed is a vaccine which can be administered to an animal or human and will cause the immune system to mount a prolonged and potent immune response against the peptide or other hapten that is capable of protecting the animal or human against infection.

Summary of the Invention In accordance with the present invention, a vaccine that is especially effective for vaccinating a human or animal against a peptide or other small hapten is provided. The improved vaccine provides a prolonged and potent immune response against the peptide or other small hapten. The present invention comprises a bacterial protein conjugated to small antigenic dete.ninants such as peptides or other small haptens. The bacterial protein that is used in the present 15 invention is bacterial flagella. The flagella may be derived from 6*eQe2 S00 any flagellated microorganisms; however, those from Salmonella species are preferred. However, it is to be understood that the 0 preferred bacterial species from which the flagella are derived for any particular application is dependent upon the particular antigen requiremeilts of the application and is not critical for this invention. The bacterial flagella can be in the native polymerized form or can 0 S be repolymerized flagellin. OS 0B 0 The present invention also includes administration of the OS Be conjugated flagella axd peptide with an adjuvant, such as a block copolymer. The preferred adjuvant that can be used with the 0 S 5

vaccine of the present invention is a block copolymer that *5 0 comprises a polymer of hydrophilic polyoxyethylene (POE) built on an ethylene diamine initiator. Polymers of hydrophobic OB C. SO 0 U 0S polyoxypropylene (POP) are then added to block of hydrophilic polyoxyethylene (POE). This results in an octablock copolymer with the following general formula:

Hydrophobe-- Hydrophile -IHydrophobe (C 0)(C2H4 0) (C2 H4 0 )a(C3 H )b N-H.(-CH-N 2 (CH 6 0)(CH 4 0 (C2 H4 0)(C H6 0)b POP POE POE POP

wherein: a is a number such that the hydrophile portion represented by polyoxyethylene (C2H40)a (POE) constitutes between approximately 10% to 40% of the total molecular weight 'of the compound; the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene 0.10 (C3H60)b (POP) is between approximately 4000 and 8000 daltons; and b is a number such that the polyoxypropylene (C3H60)b 0

0* (POP) portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound. Flagc!. can be used as a very effective adjuvant and carrier for inducing antibody responses which are long-lasting, high titer, 0 5505 and of high avidity against small antigenic determinants, such as 55 haptens, drugs, peptides. The peptides can be either synthetic or 0 genetically engineered. Examples of a genetically engineered S peptides are those currently available for malaria. The improved vaccine comprising a conjugate of a small antigenic determinant 00* and flagella can be used to induce strong and prolonged thymic- 05 dependent IgG antibody responses. "'25 5

Accordingly, it is an object of the present invention to provide a vaccine that is particularly effective in providing a prolonged and potent immune response to small immunogenic determinants. Another object of the present invention is to provide a effective vaccine that can utilize a synthetic peptide, such as malaria, to produce a sustained immune response capable of protecting anindividual from infection by the malaria parasite. Another ibject of the present invention is to provide an effective vaccin that can utilize a synthetic peptide of the AIDS virus to produce an immune response that is effective in preventing the disease. Yet another object of the present invention is to provide a vaccine that is capable of stimulating the immune system of an animal or human to produce a potent and prolonged IgG response to a small immunogenic deterrnnant, such as a p~eide or hapten. Another object of the present invention is to provide a vaccine which has very low toxicity for humans or animals. Yet another object of the present invention is to provide a ee vaccine which causes little or no local allergic reaction. P* 1 A further object of the present invention is to provide a vaccine which can be lyophilized. Another object of the present invention is to provide an adjuvant that can be used with a vaccine preparation. These and other objects, features and advantages of the S* 0 20 present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.

C Vt.; Brief Description of the Drawings 2300 SFig. 1 is a graph illustratng the antibody titer in a mouse immunized with trinitrophenol (TNP) conjugated to flagella protein from Salmonella. ig. 2 is a graph illustrating the dose response of a mouse tnxiziuit with TNP conjugated to flagella protein from Salmonella. Fig. 3 is a graph comparing the immune response of a mouse immunized with TNP conjugated to hen egg albumin (hEA)w .f TNP conjugated to flagella protein from Salmonella. The _,aph also compares using the two compounds with Li, withov. the adjuvant Polyphore 32:5 (CytRx Corporation, Atlanta, Georgia).

Detailed Description The present invention comprises a vaccine that is especially useful for immunizing an animal or human against a small peptide or other hapt n. According to the present invention, the small peptide or hapten is conjugated to flagella that is derived from a microorganisnt. The flagella may be derived from any flagellated microorganism; however, those from Salmonella species are preferred. It is to be understood that the preferred bacterial species irom which the flagella are derived for any particular application is dependent upon the particular antigen requirements of the application and is not critical for this invention. Some bacteria possess a single flagellum while others have a 15 tuft of flagell-: and still others have flagella distributed over the entire cell surface. Bacterial flagella are between 10 and 35 nm in diameter and may sometimes exceed 10 to 15 gm in length, or many times the diameter of the cell. Most bacterial flagella show a regular and uniform curl with a wavelength of about 2.5 gm. When bacterial flagella, which are protein in nature, are acidified to pH3, they dissociate into identical monomeric subunits O called flagellin, which has a molecular weight of approximately 40,000 in most species. Under appropriate conditions of pH and 6 salt concentration, flagellin monomers will spontaneously S reaggiegate to form structures that appear to be identical with intact flagella possessing periodic curls of the same wavelength as the native flagella. 00 S S S 6e IntaCt bacterial flagella in the native form or fixed with a 6O 6 *i S number of fixative agents can be used in pra icing the present invation. Additionally, repolymerized flagellin is satisfactory in practicing the present invention. It is believed that an essential component of the present invention is tlat the preparation consists of a polymer composed of flagellin molecules regularly spaced in a geometric pattern to produce the elongated flagellar structure typical of the particular microorganism. Antigens are compounds which, when introduced into a mammal, will result in the formation of antibodies. Representative of the antigens that can be used according to the present invention are proteins, glycoproteins and nucleoproteins, such as peptide hormones, serum proteins, complement proteins, coagulation factors, and viral or bacterial products. T1he viral or bacterial products can be components which the organism produced by enzymatic cleavage or can coimponents of the organism that were produced by recombinant DNA techniques that are well-known to those of ordinary skill in the art. The following is apartial list of representative antigens: proteins glycoproteins nucleoproteins peptide normones serum proteins complement proteins coagulation factors microbiocidal products viral products bacterial products 0 fungal products specific immunogens albumin angotensin bradykiniin calcitonin carcinoemnb~yonic antigen choriomamotropin choriogonadotropin corticotropin 1% 0 erythropoietin Factor VII fibrinogin alpha-2-H globulin follitropin Gastrin a 00 gastrin sulfate glucagon gonadotropin haptoglobin Hgpatits B surface antigen irnmunoglobulins insuin lipotropin melanotropin oxytocin pancreozymin placental lacogtn Iprathryin proangiote sin p17olactin somatotropin sornatomadin somatostatin thryrotropin vasotocin .4 thyrfopoietin vasopressin alpha-i -fetoprotein alpha-2-H globulin mhvelin mvelin basic protein

Haptens are compounds which, when bound to an immunogenic carrier and introduced into a chordate, will elicit formation of antibodies specific for the hapten. Representative of the haptens are steroids such as estrogens and cortisones, low molecular weight peptides, other low molecular weight biological compounds, drugs such as antibiotics and chemotherapeutic compounds, industrial pollutants, flavoring agents, food additives, and food contaminants, and/or their metabolites or derivatives. A number of procedures for preparing flagella from bacterial cultures have beenfdeveloped and are well-known to those of ordinary skill in the art The preferred procedure. is a modification of the procedure of Kobayashi, Rinker, and Koffler 0, Arch. Biochem. Biophys. 84, 342-362 (1959) as described herein. f 0 15 Salmonella typhi organisms of strain of TY2 are grown in motility agar. The highly motile organisms should be selected because they produced the most flagella. Organisms are then inoculated in 20 liters, of trypticase soy broth and ncubated at 370C for approximately 30 hours until the end of the log phase of 0 growth. The organisms may be killed at this time by the addition of formaldehyde to produce a 0.3% suspension. The organisms are preferably collected by centrifugation; however, care should be taken to avoid production f excessive shear force. The flagella are 00 0 then removed from the organisms by shaking vigorously for 0 0 S minutes in a shaker. Other mixes and devices which produce a shear force to break off the flagella without disrupting the organism are equally satisfactory. .00..0S0 The flagella are then separated from the cell bodies by *0 0 differential centrifugation. The cell bodies are removed by 06 centrifuging at approximately 2000 rpm in a standard laboratory centrifuge. The flagella are then collected by ultracentrifugation at 30,000 rpm. The flagella are then resuspended and recentrifuged in an ultracentrifuge, and soluble contaminating materials are poured off. Large contaminating materials will form a black spot at the bottom of the transparent flagella pellet. This material is physically removed and discarded. The end product derived from liters of bacterial culture will be approximately 100 mg of purified flagella. Flagellin may be produced by acidifying unfixed flagella at a pH of approximately 2 overnight. This treatment disassociates the flagellar proteins to produce the monomers of flagellin which have a molecular weight of approximately 30,000. The monomers reassemble into the polymerized flagella when allowed to stand at neutral pH for a period of at least 24 hours. The repolymerized flagellin is nearly as effective as the native flagella as an adjuvant and carrier for small antigen moieties. The monomeric flagellin or proteolydic cleavage fragments of flagUiin protein are very much less effective. The antigen hapten or peptide moieties can be chemically too**: conjugated to the flagella by any one of the standard means well- c 15 known to those of ordinary skill in the art. One of the simplest and most effective means is by using gluteraldehyde. Gluteraldehyde is a divalent cross-linking compound which covalently attaches the peptide to the flagella and further fixes the flagella preparation. 6 Other chemical cross-linking reagents or chemical antigen 0S derivatives, such as dinitrofluorobenzene, are effective. 5 The amounts of antigen attached to the flagella varies with the particular application and is not a critical component of this *0 S S. 0. 0 invention. Preferably, between 2 and 10 peptide or hapten units per flagellin monomer in the flagella preparation is sufficient. 2.0 -The conjugated flagella peparation is purified by dialysis, 5 centrifugation, or any other standard method. The material is then resuspended in saline at a concentration approximating 100 gg/ml. This preparation is effective in low doses between 1 and 100 gg per injection. A dose of 10 pg produces a satisfactory response in many situations. The material can be injected by any convenient route, intravenous, subcutaneous, intramuscular, or intraperitoneal. The subcutaneous or intramuscular route is usually the most convenient for many vaccine purposes. As an example, injections of 20 jgg of Salmonella typhi flagella conjugated with dinitrophenol resulted in IgG antibody titers specific for the hapten DNP which rose at the end of the first week after injection and persisted for several months. Persistence of the immune response to flagella and to antigenic moieties conjugated to flagella is unusual and unexpected. The material does not form a local depot of antigen at the site of injection. Approximately 90 to 95% of the injected dose of flagella is broken down and excreted within 24 hours. A portion of the material is retained for a prolonged time in germinal centers within local lymph nodes. It is believed that the presence of this antigen in germinal centers is responsible for the prolonged antibody production. This invention has numerous advantages over other available adjuvant preparations. It produces very little inflammation at the site of injection and is entirely biodegradable. This contrasts 15 sharply with oil emulsions or mineral salts, such as aluminum. Very small doses of antigen are required to produce prolonged i immune responses. A significant portion of the antibody is complement-fixing IgG which is the type required for protection against malaria, sporozoites, and other important infections. The product is stable especially when prepared with fixatives, such as gluteraldehyde. It can be lyophilized and stored at room temperature indefinitely, When reconstituted with saline, it is stable for several weeks with refrigeration and several days at room temperature. Unlike live attenuated vaccines which may produce infections in susceptible hosts, this vaccine preparation consists only of polymerized protein with traces of polysaccharide. The preferred dose of a vaccine prepared according to the present invention is between 5gg and 500gg. The optimal dose for any vaccine will depend upon the antigen that is conjugated with the flagella protein and the immunological condition of the animal or human that is being vaccinated. The vaccine of the present invention also includes the administration of the vaccine with an adjuvant to further enhance the immune response. The preferred adjuvant (Polyphore" 32:5,

CytRx Corporation, Atlanta, Georgia) that can be used with the vaccine of the present invention is a block copolymer that comprises a polymer of hydrophilic polyoxyethylene (POE) built on an ethylene diamine initiator. Polymers of hydrophobic polyoxypropylene (POP) are then added to a block of hydrophilic polyoxyethylene (POE). This results in an octablock copolymer with the following general formula:

Hydrophob-e Hydrophile -Hydrophobe ('C3 H6 )b(Ci2 H )a (C2 H 0 )(C3 H 0)b N-HC-CH N H )b(C2H4 0 (C2 H 0 4 (C3 H 0)b POP POE POE POP

wherein: a is a number such that the hydrophile poition represented by polyoxyethylene (C2H40)a (POE) constitutes between approximately 10% to 40% of the total molecular weight of the compound; the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene (C3H60)b (POP) is between approximately 4000 and 9000 daltons; and b is a number such that the polyoxypropylene (C3H60)b (POP) portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound. The preferred adjuvant has the following formula:

Hydrophobe Hydrophi le -IHydrophobe (C3 H6 0)b(CH 4 (C H4 0) (C3 H6 0 )b

N-H2 C-CHiN

(CH 0)b(C2 I(C2 H 0 )(CH 0 POP POE POE POP

wherein a is equal to approximately 5 and b is equal to approximately 32. Another copolymer that can be used with the vaccine comprising the present invention has the following formula: HO(C H 0) (C H O) (C H 0) H 2 4 3 6 6 2 4 0 b0S900 g wherein the molecular weight of the hydrophobe (C3H60) is 10 between approximately 2000 to 5000 and the total molecular wveight of the compound is between approximately 2300 and )0 4 (CytRx Corporation, Atlanta, Georgia). The preferred adjuvant has the following formula: i*

HO(C H O) (C H (C 0) H

6 wherein the molecular weight of the hydrophobe (C3H60) is approximately 4300 and the percentage of hydrophile (C2H40)a is approximately 10% by weight. (CytRx Corporation, Atlanta, Georgia). The polymer blocks are formed by condensation of ethylene ~0 0

S S. Sb oxide and propylene oxide onto a tetrafunctional ethylene diamine ba 4

4 0S 15 initiator at elevated temperature and pressure in the presence of a basic catalyst. There is some statistical variation in the number of monomer units which combine to form a polymer chain in each copolymer. The molecular weights given are approximations of the average weight of copolymer molecule in each preparation. A further description of the preparation of these block copolymers is found in U.S. Patent No. 2,674,619 and U.S. Patent No. 2,979,528

1A J04 ji. T il which are incorporated herein by reference. (Also see "AReview of Block Polymer Surfactants", Schmollca, I. Am. Oil Chemists' Soc., 54:110-116 (1977) and Block and Graft Copolymerization, Volume 2 edited by RJ. Ceresa, John Wiley Sons, New York, (1976) which are incorporated herein by reference.) The vaccine which comprises the present invention is mixed with the octablock copolymer and administered to the human or animal. The preferred amount of adjuvant administered with the vaccine of the present invention is between approximately 0.1 mg and 5.0 mg with the most preferred amount between approximately mg and 2 mg. 5, 0

0 The following specific examples will illustrate the invention

SO S as it applies to enhancing the immune response of an organism to F I small baptens. It will be appreciated that other examples will be 6

S apparent to those of ordinary skill in the art and that the invention is 0 not limited to these specific illustrative examples. OS 0 See

Example I Salmonella ryphi organisms of strain of TY2 are grown in 0 motility agar. Organisms are then inoculated in 20 liters of 0 trypticase soy broth and incubated at 370 for 30 hours until the end 5005

0@ 55 S of the log phase of growth. The organisms are killed at thfs time by the addition of formaldehyde to produce a 0.3% suspension. TIhe organisms are collected by centrifugation. Care should be taken to avoid production of excessive shear force. The flagella are then @5 0 removed from the organisms by shaking vigorously for 20 minutes 55

S. 00 S in a shaker. Otlier mixes and devices which produce a shear force S. 50 to break off the flagella without disrupting the organism are equally satisfactory. The flagella are then separated from the cell bodies by differential centrifugation. The cell bodies are removed by centrifuging at 2000 rpm in a standard laboratory centrifuge. The flagella are then collected by ultracentrifugation at 30,000 rpm. After the ultracentrifuga-don, the flagella are resuspended and recenrifuged in an ultracentrifuge, and soluble contaminating materials are poured off. Large contaminating materials form a black spot at the bottom of the transparent flagella pellet. This material is physically removed and discarded. The end product derived from 20liters of bacterial culture is approximately 100 mg of purified flagella.

Example II Flagllin is produced by acidifying the flagella at a pH of approximately 2 for 12 hours. This treatment disassociates the flagellar proteins to produce the three monomers of flagellin which have/a molecular weight of approximately 30,000. The monomers ressniemble into the polymerized flagella when allowed to stand at neutral pH for a period of at least 24 hours.

Example III Gluteraidehyde is a divalent cross-linking compound which covalently attaches the peptide to the flagella and further fixes the flagella preparation. These methods of conjugating a functional group to a protein are wel-known to one of o_ -ary skill in the art. Other chemical cross,-inking reagents or chemical antigen derivatives, such as dinitrofluorobenzene ar effective.

Example IV The conjugated flagella preparation is purified by dialysis, centrifugation, or any other standard method. The material is then Sresuspended in saline at a concentration approximating 100 pg/ml. This preparation is effective in low doses between 1 and 100 tg per injection. A dose of 10 ug produces a satisfactory response in many situations. The material can be injected by any convenient route, intravenous, subcutaneous, intramuscular, or intraperitoneal. The subcutaneous or intramuscular route is usually the most convenient S for many vaccine purposes.

Example V

An ELISA assay is used for the determination of antibody directed against the trinitrophenol hapten. It is a modification of the method originally published by Saunders (See Saunders, G.C., "The art of solid phase enzyme immunoassay including selected protocols". in: Immunassays in the Cinical Laboratory, Ala R. Liss, New York, pp. 111-112, 1979). The assay uses a protein, bovine serum albumin, hydrogel to reduce denaturation of proteins adherent to the plastic support and the use of proteins and surfactants to reduce non-specific adsorption of proteins which tend to increase background and reduce sensitivity. Glutaraldehyde is used to attach antigen to BSA- coated 96-well microtiter plates. Unbound glutaraldehyde is washed off. Antigen added to the plates attaches to the plate covalently via the free aldehyde groups of gluteraldehyde. i Remaining aldehyde groups are blocked with lysine and the 10 plate is ready to use. The plates are incubated with various dilutions of antiserum, washed and then second antibody such as peroxidase-conjugated goat anti-mouse IgG or one of the subclasses. The plates are washed and substrate orthophenylene diamine with peroxide) is added. The resulting 15 absorbance at 492 nm is read by a Titertek Multiscan photometer. The titer of antibody is calculated as the dilution of antiserum required to produce a 1/3 to 1/2 maximal O.D. of the background. This is normalized by comparison to a reference antiserum simultaneously with the sample. This facilitates comparison of titers run on different days. The relative avidity of antibodies in relation to one another is estimat-d by analysis of the slope of the curve of O.D. versus serum dilution.

Example VII In the Ifollowing experiment, 25 tg of flagella conjugated with an average of 4 TNP molecules per flagella is administered to mice via a hind footpad. The TNP-conjugated flagella was administered in a volme of 0.5 ml of saline. Antibody specific for TNP is measured at the following times after administration of the

TNP-conjugated flagella: 8 days, 19 days, 30 days, 50 days and Sdays. The results of this experiment are shown in Fig 1.As can be seen, the immune response to the TNP-conjugated flagella is still significantly high even after 90 days. The response to conventional TNP conjugates, such as TNP-conjugated hen egg albumin is much shorter in duration and the antibody titers are much lower. Animals frequently do not respond at all with detectable antibody to a hapten on a soluble protein carrier after a single injection.

Example VIm The dose response of a mouse is measured by administering varying doses of TNP-conjugated flagella. Flagella conjugated with an average of 4 TNP molecules per flagellin molecule S (molecular weight approximately 40,000) is administered to mice via a hind footpad. The TNP-conjugated flagella is administered in a volume of 0.5 ml of saline. Thefollowing concentations of TNP-conjugated flagela are administered to mice: 4 and 50gg. The antibody produced i response to the TNP- conjugated flagella is measured days and 19 days after administration of the TNP-cojugatd fageih:r "The results of this experiment is shown in Fig. 2. O: 0

0000

Example IX OS S A comparison of the immunologic response of mice to TNP O 0 conjugated to hen egg albumin (hEA) and TNP conjugated to bacteria flagella protein is shown in Fig. 3. In this experiment, O SO TNP is conjugated to hEA using the reactive derivative S trinitro.-nzene sulfonic acid (TNBS) in the same fashion as 65 O o0 flagella. 100 gg of the TNP-conjugated hEA or 25 gLg of TNP- conjugated flagella are administered to mice via a hind footpad. Ten days after administration of the TNP-conjugated proteins, antibody titer is measured according to Example V. As shown in Fig. 3, the TNP-conjugated flag 1la induced a significantly greater immune response, as measured by antibody titer, than did the TNP- conjugated hEA. It should be noted that the amount of TNP-hEA administered in this experiment was four times the amount of TNP- conjugated flagella (100gg of TNP-hEA vs 25.g of TNP- conjugated flagella).

Example X The same preparations used in Example IX are administered to mice with the addition of 1.0 mg of Polyphore" 32:5 adjuvant (CytRx Corporation, Atlanta, GA). i00 pg of the TNP-conjugated hEA or 25 ,g of TNP-conjugated flagella are administered to mice via a hind footpad. Ten days after administration of the TNP- conjugated proteins with the adjuvant, antibody titer is measured according to Example V. The results of these experiments are summarized in Fig. 3. As shown, the adjuvant raised the immune respose to both the TNP-conjugated hEA and the TNP- 15 conjugated flagella. However, the TNP-conjugated flagella S. induced a significantly greater immune response than did the TNP- conjugated hEA. It should be understood that the foregoing relates only to a preferred embodiment of the present invention and that numerous modificati~rpsor alterations may be made without departing from the spirit and scope of the invention as set forth in the appended claims. 01

9* administered in this experiment was four times the amount of TNP- conjugated flagella (lO0pgg of TNP-hLEA vs 25g.g of TN?- conjugated flagella).

ExaMple .1 T"he same prepara~.ons used in Example IXare administered to mice with the addition of 1.0 mg of PolyphoreN1 32:5 adjuvant (CytRx Corporation, Atlanta, GA). 100 jpg of the TNP-conjugated bRA or 25 gg of TINP-conjugated flagella, are administered to mice 1') via a hind footpad. Ten days after administration of the TNP- conjugated proteins with the adjuvant, antibody titer is measured according to Example V. The results of these experiments are summarized in Fig. 3. As shown, t& adjuvant raised the immn 0 responseL to both the TNP-conjugated hEA and the TNP- 06 SO conjugated flagella. However, die TNP-ronjugated flagella S induced a significantly greater immune response than did the TN?- S S conjugated hEA. OSS..S 5 It should be understood that the foregofing relates only to a 5 .0B preferred embodiment of the present invention and that numerous modifications or alterations may be made without departing from 1 the spirit and scope of the invention as set forth, in the appended ciaims. 5

*00e OS 5O

5. S. S

05 5O 00 S. S 55 S.

Claims (2)

1. A

   23

   THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: A copolymer with the following formula:

   (C3H60)b(C2H4O)a (C2H40)a(C3H60 )b

   /N-H 2 C -CH N\

   (C3H60)b(C2H40)a- (C2H40)a(C3H60)b

   wherein: a is a number such that the hydrophile portion represented by polyoxyethylene (C2H40)a (POE) constitutes between approximately 10% to 40% of the total /0molecula,' weight of the compound; the mean aggregate molec,,iJ.ar weigh-t of the hydrophobe portion of the-- octablock copolymer consisting of polyoxyprapy ,,(C3H6O)b (POP) is between :approximatc-lv'56:0 and 7000 daltons; and b b is a number such that the polyoxypropylene (CH) (C3H6)b 0'(OP) portion of the total molecular weight of the octablock copolymer constitutes between Approximately 60% and 90% of the compound. When used a8 :a v.accine adjuvanit. 2. The vaccine adjuvant of claim 2 wherein the adj)uvant has the following formula:

   (C3H60)1b(C2H40)a (C2 H40) a(C3 H60) b

   N-H 2 C-CH

   (C3 H60)bC2HO)a' (C2Nl4)a(C3H6O)b

   wherein a is equal to 5 and b is equal to 32.

   DATED this 30 day of June 199 2

   EMORY UNIVERSITY Patent Attorneys for the Applicant:

   F.B. RICE CO,

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