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Document AMI 
Appl.No. 09/848,616 



® 



J 



Europaisches Patentamt 
European Patent Office 
Office europ^en des brevets 



0 Publication number: 



0 283 505 B1 



Q. 

o 

O 

HI 
_J 

I 

CQ 



® 



EUROPEAN PATENT SPECIFICATION 



@ Date of publication of patent specification: 06.07.94 ® Int. C|5:A61K 39/02, A61K 39/12, 

A61 K 39/295, A61 K 39/385 

0 Application number: 87906496.2 

0 Date of filing: 19.08.87 

0 International application number: 
PCT/US87/02056 

0 International publication number: 
WO 88/01873 (24.03.88 88/07) 



0 VACCINE AND METHOD OF PREPARATION. 



0 Priority: 22.09.86 US 909964 


0 


Proprietor: EMORY UNIVERSITY 


16.07.87 US 75187 




1380 South Oxford Road 


0 Date of publication of application: 




Atlanta, GA 30322(US) 


© 




28.09.88 Bulletin 88/39 


Inventor: HUNTER, Robert, L 






3640 Churchwell Court 


0 Publication of the grant of the patent: 




Tucker, GA 30084(US) 


06.07.94 Bulletin 94/27 






0 Designated Contracting States: 


0 


Representative: Sternagel, Hans-GUnther, Dr. 


AT BE CH DE FR GB IT LI LU NL SE 




et al 






Patentanwalte Dr. Michael Hann 


0 References cited: 




Dr. H.-G. Sternagel 


US A- 2 674 619 US-A- 2 979 528 




Sander Aue 30 


US-A- 3 022 335 US-A- 3 036 1 1 8 




D-51465 Berglsch QIadbach (DE) 


US-A- 4 372 945 US-A- 4 400 376 






US-A- 4 478 823 US-A- 4 503 036 






US-A- 4 711 876 






CHEMICAL ABSTRACTS, vol. 105, no. 1, July 






7, 1986, Columbus. OH (US); N.Y. ALEK- 






SEEVA et al., p. 450, no. 4730x# 







OQ 

in 
o 
in 

CO 
00 
CM 



Q. 
Ill 



Note: Within nine months from the publication of the mention of the grant of the European patent, any person 
may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition 
shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee 
has been paid (Art. 99(1) European patent convention). 



Rank Xerox (UK) Business Sereices 

(3. 10/3.09/3.3.3) 



EP 0 283 505 B1 



JOURNAL OF EXPERIMENTAL MEDICINE, vol. 
134, 1971; FELDMAN. pp. 103-119# 



NEW ENGLAND JOURNAL OF MEDICINE, vol. 
315. no. 10, 04 September 1986; HOFFMAN, 
pp. 601-606# 

ARCH. OF BIOCHEMISTRY AND BIOPHYSICS, 
vol. 84, 1959; KOBAYASHI, pp. 342-362# 



2 



EP 0 283 505 B1 



Description 
Technical Field 

5 The present invention relates to a vaccine and more particularly to a vaccine comprising a bacterial 
flagella which, when conjugated with an antigen moiety, amplifies the immune response to the antigen. 

Background Art 

JO A vaccine is defined herein as a suspension of antigenic moieties, usually consisting of infectious 
agents, or some part of the infectious agents, that is injected into the body to produce active immunity. The 
antigenic moiety making up the vaccine can be either a natural product purified from a microorganism, a 
synthetic product or a genetically engineered protein peptide or similar product. An adjuvant is defined 
herein as any substance whose admixture with an injected immunogen increases the immune response. A 

15 hapten is defined herein as a substance that reacts selectively with appropriate antibodies but the hapten 
itself is usually not immunogenic. Most haptens are small molecules, 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 

20 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 epitopes that were previously impossible to produce. Current vaccine candidates, by way of 

25 example, include synthetic peptides that immunogenically mimic streptococcal, gonococcal, hoof and mouth 
disease, AIDS (HIV-1 virus) and malarial antigens. 

The work on the parasitic disease malaria is especially important. This disease affects in excess of 
200,000,000 people per year worldwide and is the most important disease in the world in terms of morbidity 
and loss of work. The techniques of genetic engineering have been used to identify, and now to produce in 

30 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. Antibodies against this 
particular peptide can kill the parasite immediately after it is injected. Unfortunately, this peptide, by 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 

35 administered with adjuvants. To date, however, the adjuvants used with the peptide 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 conven- 
tional 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. 

40 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 under- 
standing of the immune system. 

45 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 nonbacterial adjuvants, such as aluminum compounds. The first use of 
aluminum compounds as adjuvants was described in 1926. Since that time antigens precipitated with 
aluminum salts or antigens mixed with or adsorbed to performed aluminum compounds have been used 

60 extensively to augment immune responses in animals and humans. Aluminum compounds and 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 
follicular-dendritic cells. In addition, immunocompetent cells are attracted to the area of injection. Aluminum 
particles have been demonstrated in regional lymph nodes of rabbits seven days following immunization. 

55 and it may be that another significant function is to direct antigen to T cell-containing areas in the nodes 
themselves. Adjuvant potency has been shown to correlate with inflammation of the draining lymph nodes. 
While many studies have confirmed that antigens administered with aluminum salts led to increased 
humoral immunity, cell mediated immunity appears to be only slightly increased, as measured by delayed- 



3 



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type 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 exc llent record of safety, aluminum compounds are presently the most 
5 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 
10 vaccines cannot be lyophilized, thus necessitating refrigerated transport and storage with the resulting risk 
of contamination. 

Rnally. and most importantly, aluminum compounds are not always successful in inducing sustained 
protection from disease. Thus, while aluminum salts have been a sufficient adjuvant for strong immunogens 
that require antibody responses only to elicit protection, they are not effective when used with weak 

15 immunogenic-like synthetic peptides of malaria for introducing cell-mediated immune responses of the type 
required for many infections. 

Another large group of adjuvants are those of bacterial origin. Adjuvants with bacterial origins have 
recently been purified and synthesized (e.g. muramyl depeptides, lipid A) and host mediators have been 
cloned (Interleukin 1 and 2), providing chemically characterized products for study. The last decade has 

20 brought significant progress in the chemical purification of three adjuvants of active components of bacterial 
origin: Bordetella pertussis, lipopoly saccharide and Freund's complete adjuvant. 

B.pertussis is of interest due to its ability to modulate cell-mediated immunity through action on T- 
lymphocyte populations. For lipopoly saccharide and Freund's complete adjuvant, adjuvant-active moieties 
have been identified and synthesized, which permit study of structure-function relationships and the 

25 possibility of modifying the original adjuvant to create a more beneficial toxic-therapeutic ratio. 

Lipopoly saccharide 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 tow 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 

30 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 classifica- 
tion is necessarily somewhat arbitrary because most adjuvants appear to function by more than one 
mechanism. Adjuvants may act through antigen localization and delivery, or by direct effects on cells 
making up the immune system, such as macrophages and lymphocytes. Another mechanism by which 

35 adjuvants enhance the immune response is by creation of an antigen depot. This appears to contribute to 
the adjuvant activity of aluminum compounds, oil emulsions, liposomes, and synthetic polymers. The 
adjuvant activity of lipopolysaccharides and muramyl dipeptides appears to be mainly mediated through 
activation of the macrophage, whereas 8. pertussis affects both macrophages and lymphocytes. Recent 
and speculative approaches to immunopotentiation, such as the utilization of monokines and lymphokines, 

40 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 complexity of an 
antigen relative to immunogenicity reflects the availability of antigenic determinants on the molecule. This 

45 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 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 

50 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 

55 synthetic terpolymer 1-glutamic acid^°-L-alanine^°-L-tyrosine^° (GAT). While C57B1/6 mice respond to this 
polypeptide, DBA/1 mice will respond only if the GAT is coupled to methylated bovine serum albumin 
(MBSA). However, if the mice are injected with GAT prior to immunization with GAT-MSBA, a detectable 
antibody response to GAT does not occur. The explanation for these observations is that GAT stimulates 



4 



EP 0 283 505 B1 



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 MBS A. However, 
if primary immunization is with GAT-MBSA. activation of helper T celts by the carrier moi ty provides help 
that overrides the effect of any suppressor cells activated by GAT. 

5 Determinants associated with a native protein molecule have also been demonstrated to contribute 
differently to help and suppression. Conjugation of an immunogenic carrier to an antigen can change the 
isotype of antibodies produced in response to that antigen. Purified polysaccharides from a variety of 
encapsulated 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 

10 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 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 

15 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 IgfA antibody responses. (See Feldmann, M, ef a/., 
"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 

20 Recovery of the Monoclonal Response to Phosphorylcholine during Repeated Immunization" JJmmun., 
Vol.113, pp 1644-1646, 1974) This published data would lead one to believe that they have little potential as 
adjuvants or carriers for malaria peptides or other small antigens which require thymic-dependent IgG 
antibody responses. 

Feldman et al discloses in J. Exp. Med. (1971) 134 pp 103-1 19 that monomeric flageilin conjugated with 
25 donkey red cells produces a thymus-dependent antibody response. However, this reference also concludes 
that polymerized flageilin produces a thymus independent response which is related to the quaternary 
structure of the polymerized flageilin. 

In Chem. Abs. (1986) vol. 105 abs No. 4730x it is described that the addition of antigenic flageilin 
determinants enhances the IgG response and protects mice from infection by Salmonella typhimurium. 
30 There probably is no precise point of transition that distinguishes a carrier from an adjuvant. Obviously, 
the carrier moiety is contributory to a property of antigens that has been termed intrinsic adjuvanticity. The 
capacity of certain materials to convert a 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 immunogenic or inert carriers. Thus materials, such as aluminum hydroxide, latex 
35 particles, bentonite, or liposomes that adsorb antigen and enhance the immune response, are termed 
adjuvants. However, this observed effect of aggregation of antigen represents 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 
40 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. 

Accordingly it is an object of the present invention to provide a vaccine that is particularly effective in 
45 providing a prolonged and potent immune response to small immunogenic determinants. 

This object has been attained by a vaccine comprising 5ug-500ug per dose of a polymerized bacterial 
flageilin protein conjugated to a non-Salmonella antigen, wherein said antigen is chosen from the group 
consisting of low molecular weight peptides, polysaccharides, glycopeptides, drugs and haptens. 
Additionally a method of producing this improved vaccine comprising the steps of: 
50 a) isolating polymerized flageilin protein from a bacteria and 
b) conjugating said flagella to a non-Salmonella antigen 
wherein said antigen is chosen from the group consisting of low molecular weight peptides, polysac- 
charides, glycopeptides, drugs and haptens, 
is provided. 

55 This polymerized bacterial flageilin protein conjugated to a non-Salmonella antigen can be used for the 
production of a vaccine against the antigen for the immunization of a human or animal. 



5 



EP 0 283 505 B1 



Summary of the Invention 

In accordance with the present invention, a vaccine that is especially effective for vaccinating a human 
or animal against low molecular weight peptides, polysaccharides, glycopeptides. drugs or haptens is 
5 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 determinants such as 
low molecular weight peptides, polysaccharides, glycopeptides, drugs or haptens The bacterial protein that 
is used In the present invention is bacterial flagella. The flagella may be derived from any flagellated 

70 mocroorganisms; however, those from Salmonella species are preferred. However, it is to be understood 
that the preferred bacterial species from 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. 
The bacterial flagella can be in the native polymerized form or can be repolymerized flagellln. 

The present invention also Includes combination of the conjugated flagella and antigen with an adjuvant, 

75 such as a block copolymer. The preferred adjuvant 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 block of 
hydrophilic polyoxyethylene (POE). This results in an octablock copolymer with the following general 
formula: 



20 



25 



Hy droohobe-l Hydrophi 1 e ■ 



rHydrophobe 

POP POE POE POP 



wherein: 

35 a is a number such that the hydrophile portion represented by polyoxyethylene (C2H*0)a (POE) 
constitutes between 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 (C3H6 0)b (POP) is between approximately 4000 and 9000 daltons; and 

b is a number such that the polyoxypropylene (C3H6 0)b (POP) portion of the total molecular weight of 
40 the octablock copolymer constitutes between approximately 60% and 90% of the compound. 

Flagella can be used as a very effective adjuvant and carrier for inducing antibody responses which are 
long-lasting, high titer, and of high avidity against small antigenic determinants, such as low molecular 
weight peptides, polysaccharides, glycopeptides, drugs and haptens. The low molecular weight peptides 
can be either synthetic or genetically engineered. Examples of a genetically engineered peptides are those 
45 currently available for malaria. The improved vaccine comprising a conjugate of a small antigenic 
determinant and flagella can be used to induce strong and prolonged thymic-dependent IgG antibody 
responses. 

An advantage of the present invention is that an effective vaccine that can utilize a synthetic peptide, 
such as malaria, to produce a sustained immune response capable of protecting an individual from infection 
50 by the malaria parasite, is provided. 

An application of the present invention might lead to an effective vaccine that can utilize a synthetic 
peptide of the AIDS virus to produce an immune response that is effective in preventing the disease. 

Yet another advantage of the present invention is that 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 
55 immunogenic determinant, such as a peptide or hapten, is provided 

Another advantage of the present invention is that the vaccine has very low toxicity for humans or 
animals. 



6 



EP 0 283 505 B1 



Yet another advantage of the present invention is that a vaccine which cases little or no local allergic 
reaction, is provided. 

A further advantage of the present invention is that a vaccine which can be lyophilized. is provided. 
Another advantage of the pres nt invention is that an adjuvant that can be used with a vaccine 
5 preparation, is provided 

Brief Description of the Drawings 

Fig. 1 is a graph illustrating the antibody titer in a mouse immunized with trinitrophenol (TNP) 
70 conjugated to flagella protein from Salmonella. 

Fig. 2 is a graph illustrating the dose response of a mouse Immunized 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) and TNP conjugated to flagella protein from Salmonella, The graph also compares using 
75 the two compounds with and without the adjuvant Polyphore 32:5 (CytRx Corporation, Atlanta, Georgia). 

Detalied Description 

The present invention comprises a vaccine that is especially useful for immunizing an animal or human 

20 against low molecular weight peptides, polysaccharides, glycopeptides, drugs or haptens. According to the 
present invention, the low molecular weight peptides polysaccarides, glycopeptides, drugs or haptens are 
conjugated to flagella that is derived from a microorganism. The flagella may be derived from an flagellated 
microorganism; however, those from Salmonella species are preferred. It is to be understood that the 
preferred bacterial species from which the flagella are derived for any particular application is dependent 

25 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 tuft of flagella 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 um 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 nm. 

30 When bacterial flagella, which are protein in nature, are acidified to pH3, they dissociate into identical 
monomeric subunits called flagellin. which has a molecular weight of approximately 40,000 in most species. 
Under appropriate conditions of pH and salt concentration, flagellin monomers will spontaneously reagg- 
regate to form structures that appear to be identical with intact flagella possessing periodic curls of the 
same wavelength as the native flagella. 

35 Intact bacterial flagella in the native form or fixed with a number of fixative agents can be used in 
practicing the present invention. Additionally, repolymerized flagellin is satisfactory in practicing the present 
invention. It is believed that an essential component of the present invention Is that 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. 

40 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 low 
molecular weight peptides, polysaccharides, glycopeptides, drugs and haptens 

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 

45 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 been developed and are 
well-known to those of ordinary skill in the art. The preferred procedure is a modification of the procedure of 

50 Kobayashi, Rinker. and Koffler Arch. Biochem. Biophys. 84. 342-362 (1959) as described herein. 

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 incubated at 37 'C for approximately 30 hours until the end of the log phase of 
growth. The organisins may be killed at this time by the addition of formaldehyde to produce a 0.3% 

55 suspension. The organisms are preferably collected by centrifugation; however, care should be taken to 
avoid production of excessive shear force. The flagella are then removed from th organisms by shaking 
vigorously for 20 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. 



7 



EP 0 283 505 B1 



The flagella are then separated from the cell bodies by differential centrifugation. The cell bodies are 
removed by 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 ultracentrifug . and soluble contaminating materials are pour d off. Large contaminating materials will 
5 form a black spot at the bottom of the transparent flagella pellet. This material is physically removed and 
discarded. The end product derived from 20 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 
70 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 proteolytic 
cleavage fragments of flagellin protein are very much less effective. 

The antigen moieties can be chemically conjugated to the flagella by any one of the standard means 
15 well-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 antigen 
to the flagella and further fixes the flagella preparation. Other chemical cross-linking reagents or chemical 
antigen derivatives, such as dinitrofluorobenzene, are effective. 

The amounts of antigen attached to the flagella varies with the particular application and is not a critical 
20 component of this invention. Preferably, between 2 and 10 peptide or hapten units per flagellin monomer In 
the flagella preparation is sufficient. 

The conjugated flagella preparation Is purified by dialysis, centrifugation, or any other standard method. 
The material is then resuspended In saline at a concentration approximating 100 ug/ml. This preparation is 
effective in low doses between 1 and 100 ug per injection. A dose of 10 ug produces a satisfactory 
25 response in many situations. The material can be injected by any convenient route, intravenous, subcutane- 
ous, intramuscular, or Intraperitoneal. The subcutaneous or Intramuscular route is usually the most 
convenient for many vaccine purposes. 

As an example, injections of 20 ug 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 
30 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. 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 
35 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 sharply with oil 
emulsions or mineral salts, such as aluminum. Very small doses of antigen are required to produce 
prolonged immmune responses. A significant portion of the antibody Is complement-fixing IgG which Is the 
40 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 
45 preparation consists only of polymerized protein with traces of polysaccharide. 

The dose of a vaccine prepared according to the present invention Is between 5ug ad SOOug. 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 
50 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 hydrophillc polyoxyethylene (POE) built on a ethylene diamine initiator. Polymers of 
hydrophobic polyoxypropylene (POP) are then added to a block of hydrophillc polyoxyethylene (POE). This 
results In an octablock copolymer with the following general formula: 

55 



8 



EP 0 283 505 B1 




Hydrophlle 



/ 
\ 



I-Hydrophobe 

(C2H^0).(C5H^0), 



(C3H^0VC2H^0)^ 
POP POE 



^ 



N-HjC-CHjN 



POE POP 



wherein: 

a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 0)a (POE) 
constitutes between 1 0% 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 (C3HsO)b (POP) is between 4000 and 9000 daltons; and 

b is a number such that the polyoxypropylene (C3H6 0)b (POP) portion of the total molecular weight of 
the octablock copolymer constitutes between 60% and 90% of the compound. 

The preferred adjuvant has the following formula: 



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: 

H0(C2 H4 0)a(C3 Hg 0)b(C2 0)aH 

wherein the molecular weight of the hydrophobe (C3H5O) is between 2000 to 5000 and the total molecular 
weight of the compound is between 2300 and 6000 (CytRx Corporation, Atlanta, Georgia). 
The preferred adjuvant has the following formula: 

HO(C2H4 0)o(C3HGO)b (C2H4 0)aH 

wherein the molecular weight of the hydrophobe (CsHeO) is approximately 4300 and the percentage of 
hydrophile (C2H4 0)a is approximately 10% by weight. (CytRx Corporation, Atlanta, Georgia). 

The polymer blocks are formed by condensation of ethylene oxide and propylene oxide onto a 
tetrafunctlonal ethylene diamine 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-A-2,674,619 and U S-A-2,979,528 which are incorporated herein by reference. (Also see "A 
Review of Block Polymer Surfactants", Schmolka. I.R., J. Am Oil Chemists' Soc.» 54:110-116 (1977) and 
B/oc/r and Graft Copolymerization, Volume 2 edited by R.J. Ceresa, John Wiley & Sons, New York (1976) 
which are incorporated herein by reference.) 





Hydrophile 



I-Hydrophobe 

(C2H,0),(C3H,0)^ 



POP POE 



POE POP 



9 



EP 0 283 505 B1 



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 0.1 mg and 5.0 mg with the most preferred amount between 0.5 mg and 2 
mg. 

5 The following specific examples will illustrate the invention as it applies to enhancing the immune 
response of an organism to small haptens. 

Example I 

10 Salmonella typhi organisms of strain of TY2 are grown in motility agar. Organisms are then inoculated 
in 20 liters of trypticase soy broth and incubated at 37* for 30 hours until the end of the log phase of 
growth. The organisms are killed at this time by the addition of formaldehyde to produce a 0.3% 
suspension. The organisms are collected by centrifugation. Care should be taken to avoid production of 
excessive shear force. The flagella are then removed from the organisms by shaking vigorously for 20 

/5 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. 

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 ultracentrifugation, the flagella are resuspended and recen- 

20 trifuged 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 20 liters of bacterial culture is approximately 100 
mg or purified flagella. 

25 Example II 

Flagellln 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 flagellln which have a molecular 
weight of approximately 30.000. The monomers reassemble into the polymerized flagella when allowed to 
30 stand at neutral pH for a period of at least 24 hours. 

Example III 

Gluteraldehyde is a divalent cross-linking compound which covalently attaches the peptide to the 
35 flagella and further fixes the flagella preparation. These methods of conjugating a functional group to a 
protein are well-known to one of ordinary skill in the art. Other chemical cross-linking reagents or chemical 
antigen derivatives, such as dinltrofluorobenzene are effective. 

Example IV 

40 

The conjugated flagella preparation Is purified by dialysis, centrifugation, or any other standard method. 
The material is then resuspended in saline at a concentration approximating 100 ug/ml. This preparation Is 
effective In low closes between 1 and 100 ug per injection. A dose of 10 ug produces a satisfactory 
response In many situations. The material can be Injected by any convenient route, Intravenous, subcutane- 
45 ous, Intramuscular, or Intraperitoneal. The subcutaneous or Intramuscular route Is usually the most 
convenient for many vaccine purposes. 

Example V 

50 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 Clinical Laboratory Alan R. 
Llss. New York, pp. 111-112. 1979). 

The assay uses a protein, bovine serum albumin, hydrogel to reduce denaturatlon of proteins adherent 

55 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 g6-well microliter plates. Unbound glutaraldehyde Is washed off. Antigen added to the plates 
attaches to the plate covalently via the free aldehyde groups of gluteraldehyde. 



10 



EP 0 283 505 B1 



Remaining aldehyde groups are blocked with lysine and the plate Is ready to use. The plates are 
incubated with various dilutions of antiserum, washed and then a second antibody such as peroxidase- 
conjugated goat anti-mouse IgG or one of the subclasses. The plates are washed and substrate (e.g., 
orthophenylene diamine with peroxid ) is added. The resulting absorbanc at 492 nm is read by a Titertek 
5 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 estimated by analysis of the slope of the curve of O.D. 
versus serum dilution. 

10 

Example VI 

In the following experiment, 25 ug 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 

75 volume 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 90 days. 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 

20 frequently do not respond at all with detectable antibody to a hapten on a soluble protein carrier after a 
single injection. 

Example VII 

25 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 (molecular weight approxi- 
mately 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. The following concentrations of TNP-conjugated flagella are administered to 
mice: 4 ug, lOug, 25jLLg and 50ug. The antibody produced in response to the TNP-conjugated flagella is 

30 measured 8 days and 19 days after administration of the TNP-conjugated flagella. The results of this 
experiment Is shown in Fig. 2. 

Example VIII 

35 A comparison of the immunologic response of mice to TNP conjugated to hen egg albumin (hEA) and 
TNP conjugated to bacteria flagella protein Is shown in Fig. 3. In this experiment. TNP is conjugated to hEA 
using the reactive derivative trinitrobenzene sulfonic acid (TNBS) in the same fashion as flagella. 100 ug of 
the TNP-conjugated hEA or 25 ug 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 

40 Example V. As shown in Fig. 3, the TNP-conjugated flagella 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 
(lOOug of TNP-hEA vs 25ug of TNP-conjugated flagella). 

45 Example IX 

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). 100 ug of the TNP-conjugated hEA or 25 ug 
of TNP-conjugated flagella are administered to mice via a hind footpad. Ten days after administration of the 
60 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 response to both 
the TNP-conjugated hEA and the TNP-conjugated flagella. However, the TNP-conjugated flagella induced a 
significantly greater immune response than did the TNP-conjugated hEA. 

55 Claims 

1. A vaccine comprising 5ug-500ug per dose of a polymerized bacterial flagellin protein conjugated to a 
non-Salmonella antigen, wherein said antigen is chosen from the group consisting of low molecular 



11 



EP 0 283 505 B1 



weight peptides, polysaccharides, glycopeptides. drugs and haptens. 
2. The vaccine of claim 1 wherein the bacterial flagella is isolated from Salmonella species. 
5 3. The vaccine of claim 2 wherein the Salmonella species is Salmonella typhi. 

4. The vaccine of claim 1 wherein the vaccine is combined with an adjuvant. 

5. The vaccine of claim 4 wherein the adjuvant has the following formula 



10 



15 



25 



30 



35 



C H 0 C H 0 AC HO) C H 0) 

3 6 b 2 4 r\ ^ 2 4 a 3 6 b 
^N-H C-CH 

(C HO) (C H 0)^ ^ ^ ^(C H 0) (C H 0) 

36 b 24 a 24 a36b 



wherein 

a is a number such that the hydrophile portion represented by pofyoxyethylene (C2H4 0)a (POE) 
20 constitutes between 10% and 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 (CsHsOb (POP) is between 4000 and 9000 daltons, and 

b is a number such that the polyoxypropylene (CsHgO^ (POP) portion of the total molecular weight of 
the octablock copolymer constitutes between 60% and 90% of the compound. 



6. The vaccine of claim 4, wherein the adjuvant has the following formula 



C H 0) (C H 0).. .(C H 0) (C H 0) 

3 6 b 2 4 a\ . ^ 2 4 a 3 6 b 

^ N-H C-CH -N 



(C H 0) (C H 0)^ \(C HO) (C H 0) 

36b24a 24a36b 



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

7. The vaccine of claim 4, wherein the adjuvant has the following formula 

40 H0(C2 Ha 0)a (Ca Hs 0)b (C2 H4 0)aH 

wherein the molecular weight of the hydrophobe (CsHgO) is between 2000 to 5000 and the total 
molecular weight of the compound is between 2300 and 6000. 

45 8. The vaccine of claim 4. wherein the adjuvant has the following formula 

HO(C2H40)a (C3H60)b (C2H40)aH 

wherein the molecular weight of the hydrophobe (CsHeO) is approximately 4300 and the percentage of 
50 hydrophile (C2H40)a is approximately 10% by weight. 

9. A method of producing an improved vaccine comprising 

(a) isolating polymerized flagellin protein from a bacteria, and 

(b) conjugating said flagella to a non-Salmonella antigen, wherein said antigen is chosen from the 
55 group consisting of low molecular weight peptides, polysaccharides, glycopeptides, drugs and 

haptens. 

10. The method of claim 9, wherein the vaccine is combined with an adjuvant. 



12 



EP 0 283 505 B1 



10 



20 



30 



11. The method of claim 10, wherein the adjuvant has the following formula 



(C HO) (C H 0)^ AC HO) (C H 0) 

2 ' \| 



(C HO) (C H 0)^ ^(C HO) (C H 0) 

3 6 b 2 4 a 2 4 a 3 6 b 



wherein 

a is a number such that the hydrophile portion represented by polyoxyethylene {C2H4 0)a (POE) 
constitutes between 10% and 40% of the total molecular weight of the compound, 
the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting 
75 of polyoxypropylene (C3H5 0)b (POP) is between 4000 and 9000 daltons. and 

b is a number such that the polyoxypropylene (C3HeO)b (POP) portion of the total molecular weight of 
the octablock copolymer constitutes between 60% and 90% of the compound. 



12. The method of claim 10, wherein the adjuvant has the following formula 



(C HO (C H 0)>. / C H 0) C H 0) 

3 6 b 2 4 a\ 2 4 a 3 6 b 

^N-H C-CH -N^ 
(C HO) (C H 0)^ ^ ^ ^{C H 0) (C H 0) 

3 6 b 2 4 a 2 4 a 3 6 b 



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

13. The method of claim 10, wherein the adjuvant has the following formula 

H0(C2 0)a(C3 Hg 0)b(C2 Ha 0)aH 

35 wherein the molecular weight of the hydrophobe (CsHgO) is between 2000 to 5000 and the total 
molecular weight of the compound is between 2300 and 6000. 

14. The method of claim 10, wherein the adjuvant has the following formula 

40 H0(C2 H* 0)a (03 He 0)b (O2 0)aH 

wherein the molecular weight of the hydrophobe (CsHbO) is approximately 4300 and the percentage of 
hydrophile (C2H4 0)a is approximately 10% by weight. 

45 15. Use of a polymerized bacterial flagellin protein conjugated to a non-Salmonella antigen for the 
production of a vaccine against the antigen for the immunization of a human or animal, wherein said 
antigen is chosen from the group consisting of low molecular weight peptides, polysaccharides, 
glycopeptides, drugs and haptens. 

50 16. The use of claim 15, wherein the vaccine is combined with an adjuvant. 



55 



13 



EP 0 283 505 B1 



17. The use of claim 16, wherein the adjuvant has the following formula 

(C HO) (C H 0)^ fC H 0) (C H 0) 

3 6 b 2 4 ^ 2 4 a 3 6 b 

5 ^N-H C-CH -N 

(C HO) (C H 0)-^ ^ ^ ^(C HO) (C H 0) 
36 b 24 a 24 a 36b 

70 

wherein 

a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 0)a (POE) 
constitutes between 10% and 40% of the total molecular weight of the compound, 
the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting 
75 of polyoxypropylene (C3H6 0)b (POP) is between 4000 and 9000 daltons, and 

b is a number such that the polyoxypropylene (C3HsO)b (POP) portion of the total molecular weight of 
the octablock copolymer constitutes between 60% and 90% of the compound. 



20 



25 



30 



ia The use of claim 16, wherein the adjuvant has the following formula 



(C HO) (C H 0)^ ^(C HO) (C H 0) 

3 6 b 2 4 ^ 2 4 a 3 6 b 

^ N-H C-CH -N^ 

2 ^ \- 



(C HO) (C H 0)^ ^(C HO) (C H 0) 

3 6 b 2 4 a 2 4 a 3 6 b 



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

19. The use of claim 16, wherein the adjuvant has the following formula 

H0(C2 H4 0)a(C3 He 0)b(C2 H* 0)aH 

35 wherein the molecular weight of the hydrophobe (CsHsO) is between 2000 to 5000 and the total 
molecular weight of the compound is between 2300 and 6000. 

20. The use of claim 16, wherein the adjuvant has the following formula 

40 H0(C2 H4 0)a(C3 Hg 0)b(C2 H, 0)aH 

wherein the molecular weight of the hydrophobe (CaHeO) is approximately 4300 and the percentage of 
hydrophile (C2H4 0)a is approximately 10% by weight 

45 PatentansprUche 

1. Vakzine, enthaltend 5ug-500ng pro Dosis eines polymerisierten Proteins von bacteria flagella, konju- 
giert mit einem Nicht-Salmonella-Antigen, wobei das Antigen aus der aus Peptiden mit niedrigem 
Molekulargewicht, Polysacchariden, Glycopeptiden, Arzneimittein und Haptenen bestehenden Gruppe 

50 ausgew^hlt ist. 

2. Vakzine nach Anspruch 1 , 
dadurch gekennzelchnet, 

daB die bacteria flagella aus Salmonellenspezies isoliert sind. 



55 



3. Vakzine nach Anspruch 2, 
dadurch gekennzeichnet, 

daB die Salmonellenspezies Salmonella typhi sind. 



14 



EP 0 283 505 B1 



Vakzine nach Anspruch 1 . 
dadurch gek nnzeichnet, 

daS der Impfstoff mit einem Hilfsstoff kombiniert ist. 

Vakzine nach Anspruch 4, 
dadurch gekennzelchnet, 

daB der Hilfsstoff der folgenden Formel genUgt 



(C H 0) (C H 0)^ (C H 0) (C H 0) 

3 6 b 2 4 a\ / 24 a 36 b 

^ N-H C-CH -M^ 4 a 4 0 i> 

(C H 0) (C H 0)^ ^ ^ ^(C H 0) (C H 0) 
36b248 24 a 36 b 



in der a eine solche Zahl ist, dafi der hydrophile Teil, wiedergegeben durch Polyoxyethylen (C2H4 0)a - 
(POE). zwischen 10% and 40% des gesamten Molekulargewichtes der Verblndung ausmacht und das 
mittlere Aggregatmolekulargewicht des hydrophoben Teils des Octablockcopolymeren, bestehend aus 
Polyoxypropylen (C3H6 0)b (POP), zwischen 4000 and 9000 Daltons betragt und b eine solche Zahl ist, 
daB der Polyoxypropylenanteil (C3H6 0)b (POP) des gesamten Molekulargewichts des Octablockcopoly- 
meren zwischen 60% and 90% der Verbindung ausmacht. 

Vakzlne nach Anspruch 4, 
dadurch gekennzelchnet, 

daB der Hilfsstoff der folgenden Formel genugt 



(C HO) (C H 0). AC HO) (C H 0) 

3 6 b 2 4 a\ y 2 4 a 3 6 b 

N-H C-CH 'H(^ 

(C HO) (C H 0)^ ^ ^ ^(C HO) (C H 0) 
3 6 b 2 4 a ' 2 4 a 3 6 b 



in der a gleich 5 und b gleich 32 ist. 

Vakzine nach Anspruch 4, 
dadurch gekennzelchnet, 

daB der Hilfsstoff der folgenden Formel genugt 

H0(C2 H* 0)a(C3 Ms 0)b(C2 H4 0)aH 

in der das Molekulargewicht des hydrophoben Teils (CaHsO) zwischen 2000 und 5000 betrSgt und das 
Gesamtmolekulargewicht der Verblndung zwischen 2300 und 6000 liegt. 

Vakzine nach Anspruch 4, 
dadurch gekennzelchnet, 

daB der Hilfsstoff der folgenden Formel genugt 

H0(C2 H4 0)a(C3 He 0)b(C2 H, 0)aH 

In der das Molekulargewicht des hydrophoben Teils (CsHeO) etwa 4300 betragt und der Prozentsatz 
des hydrophilen Teils (C2H4 0)a etwa 10 Gew.-% betragt. 

Verfahren zum Herstellen einer verbesserten Vakzine durch 

(a) Isolieren des polymerisierten Proteins von bacteria flagella aus Bakterlen und 



15 



EP 0 283 505 B1 



(b) Konjugieren der Flagella mit einem Nicht-Salmonellen- Antigen, wobei das Antigen aus der aus 
Peptiden mit niedrlgem Molekulargewicht, Polysacchariden, Glycopeptiden, Arzneimlttein und Hapte- 
nen bestehenden Gruppe ausgewahit 1st. 

10. Verfahren nach Anspruch 9, 
dadurch gekennzeichnet, 

daB der Impfstoff mit einem Hilfsstoff kombiniert ist. 

11. Verfahren nach Anspruch 10, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genugt 



(C HO) (C H 0)>^ AC HO) (C H 0) 

3 6 6 2 4 ^ ^ 2 4 a 3 6 b 

^M-H C-CH -N 

(C HO) (C H 0)^ ^ ^ ^(C HO) (C H 0) 
36b24 a 24 a 36 b 



in der a eine solche Zahl ist, daB der hydrophile Teil, wiedergegeben durch Polyoxyethylen (CaH^Oja - 
(POE), zwischen 10% and 40% des gesamten Molekulargewichtes der Verbindung ausmacht und das 
mittlere Aggregatmolekulargewicht des hydrophoben Tails des Octablockcopolymeren, bestehend aus 
Polyoxypropylen (C3HG0)b (POP), zwischen 4000 and 9000 Daltons betragt und b eine solche Zahl ist, 
daB der Polyoxypropylenanteil (C3H6 0)b (POP) des gesamten Molekulargewichts des Octablockcopoly- 
meren zwischen 60% and 90% der Verbindung ausmacht. 

12. Verfahren nach Anspruch 10, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genOgt 



(C HO) (C H 0)^ (Z HO) (C H 0) 

3 6 b 2 4 a\^ ^ 2 4 a 3 6b 

^ N-H C-CH -N^ 

(C HO) (C H 0)^ ^ ^ \(C H 0) (C H 0) 
3 6 b ' 2 4 a 2 4 a 3 6 \ 



in der a gleich 5 und b gleich 32 ist. 

13. Verfahren nach Anspruch 1 0, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genUgt 

HO(C2H40)a(C3H6 0) b(C2H4 0)aH 

in der das Molekulargewicht des hydrophoben Teils (CaHeO) zwischen 2000 und 5000 betragt und das 
Gesamtmolekulargewicht der Verbindung zwischen 2300 und 6000 liegt. 

14. Verfahren nach Anspruch 10. 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genOgt 

H0(C2 H4 0)a(C3 Hs 0)b(C2 H* 0)aH 

in der das Molekulargewicht des hydrophoben Teils (CaHtO) etwa 4300 betragt und der Prozentsatz 
des hydrophilen Teils (C2H4 0)a etwa 10 Gew.-% betragt. 



16 



EP 0 283 505 B1 



15. Verwendung eines polymerisierten Proteins von bacteria flagella, konjugiert nait einem Nicht-Salnnonel- 
la-Antigen zur Herstellung eines Impfstoffes gegen das Antigen zur Immunisierung eines Menschen 
Oder Tieres, wobel das Antigen aus der aus Peptiden mit niedrlgenn Molekulargewicht, Polysacchariden. 
Glycopeptiden, Arzneimittetn und Haptenen bestehenden Gruppe ausgewahit ist 

16. Verwendung nach Anspruch 15, 
dadurch gekennzeichnet, 

daB der Impfstoff nnit einem Hilfsstoff korDbiniert ist. 

17. Verwendung nach Anspruch 16, 
dadurch gekennzeichnet, 

daS der Hilfsstoff der folgenden Forme! genugt 

^ N-H C-CH -N^ 
(C H 0) (C H 0)^ ^ ^ ^{C H 0) (C H 0) 

3 6 b 2 4 a 2 i a 3 6 b 



in der a eine solche Zahl ist, daB der hydrophile Tell, wiedergegeben durch Polyoxyethylen (C2H4 0)a - 
(POE), zwischen 10% and 40% des gesamten Molekulargewichtes der Verblndung ausmacht und das 
mittlere Aggregatmolekulargewicht des hydrophoben Teils des Octablockcopolymeren, bestehend aus 
Polyoxypropylen (C3H6 0)b (POP), zwischen 4000 and 9000 Daltons betragt und b eine solche Zahl ist, 
daB der Polyoxypropylenanteil (C3H6 0)b (POP) des gesamten Molekulargewichts des Octablockcopoly- 
meren zwischen 60% and 90% der Verbindung ausmacht. 

18. Verwendung nach Anspruch 16, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genugt 



(C H 0) (C H 0). ^{C H 0) (C H 0) 

3 6 b 2 4 ^ ^ 2 4 a 3 6 b 

^ N-H C-CH -N^ 

(C HO) (C H 0)^ ^ ^ \(C H 0) (C H 0) 

36b24a 24a36b 



in der a gleich 5 und b gleich 32 ist. 

19. Verwendung nach Anspruch 16, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genligt 

H0(C2 H* 0)a(C3 Hg 0)b(C2 H4 0)aH 

in der das Molekulargewicht des hydrophoben Teils (CaHgO) zwischen 2000 und 5000 betragt und das 
Gesamtmolekulargewicht der Verbindung zwischen 2300 und 6000 liegt. 

20. Verwendung nach Anspruch 16, 
dadurch gekennzeichnet, 

daB der Hilfsstoff der folgenden Formel genUgt 

H0(C2 H* 0)a(C3 He 0)b(C2 H^ 0)aH 

in der das Molekulargewicht des hydrophoben Teils (C3H6O) etwa 4300 betragt und der Prozentsatz 



17 



EP 0 283 505 B1 



des hydrophilen Teils (C2H4 0)a etwa 10 Gew.-% betragt. 
Revendications 

5 1. Vaccin comprenant 5 jxg ^ 500 ug par dose d'une prot^ine de flagelline bact^rienne polym^ris^e 
conjugu^e h un antig^ne non salmonella, ou I'antigfene est choisi parmi le groupe consistant en des 
peptides, des polysaccharides, des glycopeptldes. des medicaments et des heptanes de poids 
mot^culaire faible. 

10 2. Vaccin suivant la revendication 1 , ou les flagelles bact§riens sent isol§s d'especes de salmonella. 

3. Vaccin suivant la revendication 2, ou I'espece de salmonella est Salmonella typhi . 

4. Vaccin suivant la revendication 1 , ou le vaccin est combing h un adjuvant. 

75 

5. Vaccin suivant la revendication 4, ou Tadjuvant a la formule suivante : 



20 



25 



(CjH^O)^ (CjH^O), ^ ^ (CgH^O), (CjH^O)^ 



N - CHg - CHg - N 



/ \ 



OU : 

a est un nombre tel que la partie hydrophile representee par le polyoxyethyldne (C2H^O)q (POE) 
constitue entre 10% et 40% du poids mol6culaire total du compost; 
30 le poids mol^culaire de Tagregat moyen de la partie hydrophobe du copotymfere octas^quence 

consistant en du poly oxy propylene (C3HG0)b (POP) se situe entre 4000 et 9000 daltons, et 

b est un nombre tel que la partie polyoxypropylene (CsHsO^ (POP) du compose constitue entre 
60% et 90% du poids moleculaire total du copolymere octasequence. 

35 6. Vaccin suivant la revendication 4. ou i'adjuvant a la formule suivante : 



^ N - CHp - CH;, - N 

/ \ 

45 

OU, a est 6ga\ ^ 5 et b est ^gal k 32. 
7. Vaccin suivant la revendication 4, ou I'adjuvant a la formule suivante : 

50 H0(C2 H4 0)a (C3 Hs 0)b (C2 H4 0)a H 

ou le poids moleculaire du (CaHeO) hydrophobe se situe entre 2000 et 5000 et le poids moleculaire 
total du compose se situe entre 2300 et 6000. 

55 8. Vaccin suivant la revendication 4, ou I'adjuvant a la formule suivante : 

H0(C2H4 0)a (CsHsOb (C2H4 0)a H 



18 



EP 0 283 505 B1 



ou, le poids mol4culaire du (CsHsO) hydrophobe se situe h environ 4300 et le pourcentage de 
(C2H4 0)a hydrophile est d'environ 10% en poids. 

9. Precede de production d'un vaccin am^liore comprenant : 
5 (a) risolement de la prot^ine de flagelline polym^ris^e d*une bact^rie, et 

(b) la conjugaison des flagelles h un antig^ne non salmonella, ou Tantigdne est choisi parmi le 
groupe consistant en des peptides, des polysaccharides, des glycopeptides, des medicaments et 
des hapt^nes de poids moleculaire faible. 

10 10, Precede suivant la revendication 9, ou le vaccin est combine k un adjuvant. 

11. Precede suivant la revendication 10, ou I'adjuvant a la formule suivante : 



;5 



20 



N - CH, - CHj - N 



/ \ 



ou : 

a est un nombre tel que la partie hydrophile representee par le polyoxyethylene (C2H4 0)a (POE) 
constitue entre 10% et 40% du poids moleculaire total du compose; 
25 le poids moleculaire de I'agregat moyen de la partie hydrophobe du copolymere octasequence 

consistant en du polyoxypropylene (C3H6 0)b (POP) se situe entre 4000 et 9000 daltons, et 

b est un nombre tel que la partie polyoxypropylene (CsHBOjb (POP) du compose constitue entre 
60% et 90% du poids moleculaire total du copolymere octasequence. 

30 12. Precede suivant la revendication 10, ou I'adjuvant a la formule suivante : 

(C3H,0), (CjH^O)^ ^ ^ (C2H4O), (CjA^O), 

35 N - CH2 - CH2 - N ^ 

(CjH^O)^, (CjH^O), (CjH^O^ (CjHjO)^ 

40 OU. a est egal ^ 5 et b est egal h 32. 

13. Precede suivant la revendication 10, ou Tadjuvant a la formule suivante : 

HO(C2H4 0)a (C3H6 0)b (C2H4 0)a H 



45 



50 



55 



ou, le poids moleculaire du (CsHsO) hydrophobe se situe entre 2000 et 5000 et le poids moleculaire 
total du compose se situe entre 2300 et 6000. 

14. Precede suivant la revendication 10, ou Tadjuvant a la formule suivante : 
H0(C2 H4 0)a (Cs He 0)b (C2 H4 0)a H 

ou, le poids moleculaire du (CaHeO) hydrophobe est d'environ 4300 et le pourcentage du (C2H40)a 
hydrophile est d'environ 10% en poids. 

15. Utilisation d'une proteine de flagelline bacterienne polymerisee conjuguee h un antigfene non salmonel- 
la pour la production d'un vaccin centre rantigfene pour I'immunisation d'un humain ou animal, ou 
I'antigene est choisi parmi le groupe consistant en des peptides, des polysaccharides, des glycopepti- 



19 



EP 0 283 505 B1 

des, des medicaments et des haptenes de poids mol^cularre faible. 
16. Utilisation suivant la revendication 15, ou le vaccin est combing h un adjuvant. 
5 17. Utilisation suivant la revendication 16, ou I'adjuvant a la formule suivante : 



(CjH^O)^ (CgH^O)^ ^ ^ (CzH^O)^ (CjH^O)^ 



70 



15 



N - CHg - CH2 - N 



/ \ 



ou : 

a est un nombre tel que la partie hydrophile representee par le polyoxyethylene (C2H4 0)a (POE) 
constitue entre 10% et 40% du poids moleculaire total du compose; 

le poids moleculaire de Tagregat moyen de la partie hydrophobe du copolymere octas^quence 
20 consistant en du polyoxypropylene (CsHeOb (POP) se situe entre 4000 et 9000 daltons, et 

b est un nombre tel que la partie polyoxypropylene (C3H6 0)b (POP) du compost constitue entre 
60% et 90% du poids moleculaire total du copolymfere octasequenc§. 

1& Utilisation suivant la revendication 16, ou {'adjuvant a la formule suivante : 

25 

{CjH^O)^, (C2H^O)3 ^ ^ (C2H^0)^ (CsH^O)^ 

N - CHp - CHp - N 
/ \ 
(CjH^Ot, (C2H,0)3 (C2H,0)3 (CjH^O), 



35 ou, a est §gal 5 et b est ^gal k 32. 

19. Utilisation suivant la revendication 16, ou Tadjuvant a la formule suivante : 

HO(C2H4 0)a (C3H6 0)b (C2H4 0)a H 

40 

Oil. le poids moleculaire du (CaHsO) hydrophobe se situe entre 2000 et 5000 et le poids moleculaire 
total du compose se situe entre 2300 et 6000. 

20. Utilisation suivant la revendication 16, ou Tadjuvant a la formule suivante : 

45 

H0(C2H4 0)a (C3HG0)b (C2H4 0)o H 

ou, le poids moleculaire du (CaHgO) hydrophobe se situe k environ 4300 et le pourcentage du 
(C2H4 0)a hydrophile est d'environ 10% en poids. 

50 



55 



20 



EP 0 283 505 B1 



Immunization with 25^g TNP - Flagella 




Days after immunization 

Fig.1 



21 



EP 0 283 505 B1 




22 



EP 0 283 505 B1 




0 2000 4000 6000 8000 



Dilution 



Fig. 3 



23