Title:  Adjuvant combinations for immunization composition and vaccines

United States Patent:  6,649,170

Issued:  November 18, 2003

Inventors:  Lindblad; Erik B. (Frederiksberg, DK); Elhay; Martin J. (Hawthorn, AU); Andersen; Peter (Bronshoj, DK); Brandt; Lise Ostergaard (Copenhagen, DK)

Assignee:  Statens Serum Institut (Copenhagen, DK)

Appl. No.:  310551

Filed:  May 12, 1999

Abstract

A kit useful for immunization is described. The kit contains an antigenic substance from a Mycobacterium and an adjuvant combination of dimethyl dioctadecyl ammonium bromide and monophosphoryl lipid A.

DISCLOSURE OF THE INVENTION

The present inventors have now discovered that two particular classes of adjuvants possess the capability to elicit a strong and long persisting immune response when administered in combination with an antigenic substance, even though this substance may have only poor immunogenicity per se.

Thus, the present invention relates to an adjuvant combination comprising a first adjuvant component which is a quaternary hydrocarbon ammonium halogenide of the formula NR¹ R² R³ R⁴ -hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R³ and R⁴ independently each is a hydrocarbon group containing from 12 to 20 carbon atoms, preferably from 14 to 18 carbon atoms and hal is a halogen atom, and a hydrophobic second adjuvant component

In the formula NR¹ R² R³ R⁴ -hal the R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R³ and R⁴ may be dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl nonadecyl and eicocyl groups. However, also other C₁₂ -C₂₀ hydrocarbon groups are possible because even though the R³ and R⁴ groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g. methyl and ethyl side chains. R³ and R⁴ may also have a minor degree of unsaturation, e.g. containing 1-3 double bonds each, but preferably they are saturated alkyl groups. R³ and R⁴ are preferably saturated alkyl groups containing from 14 to 18 carbon atoms.

The halogen atom "hal" is preferably bromine or chlorine because the other halogens, fluorine and iodine, may have undesirable biochemical, physiological and injurious effects, but for some experimental purposes, where such effects can be accepted, they may also be selected.

Preferably the hydrophobic second adjuvant component is selected from the group comprising triterpenoid saponins and derivatives thereof, lipopolysaccharides (LPS) and derivatives thereof, Staphylococcus antigen A, carbohydrate coupled phospholipids, monophosphoryl lipid A (MPL-A), mineral oil , Neem oil, taxol, the squalane and squalene series of adjuvants, block co-polymer adjuvants, pleuronic bloc polymer adjuvants, and lipoglycanes.

Examples of block co-polymer adjuvants are described by e.g. Todd C. V. et al., Systematic development of a block copolymer adjuvant for trivalent influenza virus vaccine, Dev Biol Stand 1998; 92:341-51.

Amongst the hydrophobic second adjuvant components, lipophilic adjuvants, such as monophosphoryl lipids (MPL), are prefered.

The monophosphoryl lipids (MPL) are e.g. obtainable from microbial lipopolysaccharide (LPS) and are usually prepared from bacterial polysaccharides even though other microbial sources like viruses, moulds, fungi, yeasts and algae may be the source of origin for the phosphoryl lipid of choice. Suitable bacterial polysaccharides are e.g. described in "The Theory and Practical Applications of adjuvants"¹), chapter thirteen, pp. 287-313, Ed. by D. E. S. Stewart-Tull, 1995, John Wiley Sons Ltd., in "Methods in Microbiology"²), Vol. 25, pp. 471-502, Ed. Stefan A E Kaufmann and Dieter Kabelitz, 1998, Academic Press, San Diego, Calif., USA and London, UK, and in "Vaccine"³), vol. 15, No. 3, pp. 248-256, 1997, Elsevier Science Ltd., GB.

Also, the monophosphoryl lipids derivable from the microbial polysaccharides and suitable for use in the adjuvant combinations of the present invention are described in more details in the above referrences. The preferred monophosphoryl lipid is monophosphoryl lipid A (MPL-A) which is described in 1) on pp. 289-292. in 2) on pp. 483-484, and in 3) on page 252, column 2. The most preferred MPL-A is designated 3-O-deacylated monophosphoryl lipid A. However, also other derivatives of the MPL-A's may be applicable.

The adjuvant combination of the present invention may preferably be in the form of:

a) an aqueous composition comprising the quaternary hydrocarbon ammonium halogenide of the formula NR¹ R² R³ R⁴ -hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R³ and R⁴ independently each is a medium chain length hydrocarbon group containing 12 to 20 carbon atoms and hal is a halogen atom, and

b) an aqueous composition comprising the hydrophobic second adjuvant component.

The aqueous media in these aqueous compositions may be any suitable aqueous solvent. However, formation of useful possible micelle structures appears to be sensitive to anions, like phosphate and sulphate ions. Thus, it is preferred that the adjuvant compositions of the Inventions are formed in the absence or low levels of such ions.

The aqueous adjuvant compositions may be prepared by any suitable process or procedure, e.g. as described further on in the detailed part of this specification.

If expedient, the different adjuvant compositions may be combined into one single composition either as a stock composition or immediately before use.

The invention concerns also a kit for immunization, said kit comprising a first adjuvant component which is a quaternary hydrocarbon ammonium halogenide of the formula NR¹ R² R³ R⁴ -hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R³ and R⁴ independently each is a hydrocarbon group containing from 12 to 20 carbon atoms, preferably from 14 to 18 carbon atoms, and hal is a halogen atom, and a hydrophobic second adjuvant component and an antigenic substance.

Such kit may be presented in the form of individual containers or compartments containing the different adjuvants and the antigenic substance and any solvent necessary for effecting the immunization procedure as well as any necessary device for the performance thereof. If appropriate the adjuvants and the antigenic substance may also be combined and stocked in one single container. If the adjuvants and the antigenic substance each is contained in a separate container they may be mixed in any order before use. For some applications it may be advantageous, however, to mix the adjuvants and the antigenic substances in a particular order for obtaining optimum results.

In principle the antigenic substance may be any pure chemical species such as a protein or a fragment thereof or artificial mixtures prepared of such species. But it can also be any naturally occuring mixture of chemical species such as e.g. a cell homogenate or fractions thereof, a culture filtrate from microorganisms or cell tissues from multicellular organisms, e.g. higher animals.

Specifically the antigenic substance may be derived from a culture of metabolizing Mycobacterium tuberculosis, Mycobacterium bovis and other environmental Mycobacteria such as e.g. Mycobacterium avium. A particular interesting substance from the filtrate of such Mycobacteria is the ESAT-6 protein (Early Secretory Antigenic Target) which is a dominant target for cell mediated immunity in the early phase of tuberculosis in TB patients and in different animal models. It contains 95 amino acid residues and has a deduced molecular mass of approximately 10 kDa. Its immunogenicity per se is low, but in combination with the adjuvant combinations of the present invention it has turned out to be a potent candidate for provoking high and persisting immunity against tuberculosis as is demonstrated in the following detailed part of this specification.

ESAT-6 as well as many other antigens applicable in combination with the adjuvant combinations of the present invention, today can be produced artificially, e.g. synthetically or by genetic recombinant techniques.

In addition to provide immunity to diseases the adjuvant combinations of the present invention can also be used for producing antibodies against compounds which are poor immunogenic substances per se and such antibodies can be used for the detection and quantification of the compounds in question, e.g. in medicine and analytical chemistry.

Without being bound by theory it is believed that an adjuvant as DDA, which induces strong CMI (cell mediated immune) reponses, has the ability to form micelles in aqueous solutions. The lipid portion of this structure provides a matrix for the inclusion of other lipophilic compounds and the formation of composite micelles with increased adjuvant activity.

Preferred embodiments of the adjuvant combination and the immunization combination kit of the present invention are set forth in the dependent claims in the accompanying set of claims attached.

Claim 1 of 10 Claims

We claim:

1. An immunization combination kit comprising:

(a) an antigenic substance from a culture of metabolizing Mycobacterium tuberculosis; and

(b) an adjuvant combination consisting of dimethyl dioctadecyl ammonium bromide (DDA-Br) and monophosphoryl lipid A (MPL-A) in a ratio of from 30:1 to 4:1 by weight.

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