Adjuvant combinations for immunization composition and vaccines

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...

Reexamination Certificate

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C424S184100, C424S234100, C424S278100, C424S279100, C424S280100, C424S282100, C424S283100, C435S822000, C530S300000, C530S350000

Reexamination Certificate

active

06649170

ABSTRACT:

The present invention relates to adjuvant combinations comprising two or more different adjuvants. In particular the invention relates to adjuvant compositions comprising the adjuvants in aqueous media for immunization and vaccines.
The invention also relates to vaccines and immunization combination kits comprising two or more adjuvants and an antigenic substance.
BACKGROUND OF THE INVENTION
Since the English doctor Edward Jenner in 1796 discovered that the infectious agency causing cowpox in cattle was able to produce immunity against smallpox in human beings without causing serious illness many efforts have been made in order to find other vaccines which can generate immunity against more or less severe diseases in animal and human beings without provoking the unpleasant, serious or fatal symptoms and reactions usually accompanying the ordinary diseases in question.
Thus, for example, tuberculosis in man has for many years been combated by vaccination with attenuated but living strains of
Mycobacterium bovis
(BCG vaccine). However, the efficacy of this procedure does not allways provide satisfactory resistance to human tuberculosis in every population.
Therefore, attempts have been made to isolate and use fragments or subfragments of strains of human
Mycobacterium tuberculosis
instead as immunogenic agent which when injected intradermally or subcutaneously in individuals would cause satisfactory immunity against infections with naturally occurring strains of human
Mycobacterium tuberculosis.
Thus, non-determined substances from culture filtrates as well as a few isolated molecules such as Ag85 and ESAT-6 of
Mycobacterium tuberculosis
have been shown to provide some degree of tuberculosis immunity.
In the future it would be desirable to have vaccines based on well-defined substances which would always create high immunity against tuberculosis and other diseases.
Unfortunately, many highly purified substances, e.g.. purified recombinant proteins, are not very immunogenic and do not generate an effective immune response protective against the real infectious disease. This fact has been recognized since the beginning of this century and it has been tried to counteract the low immunogenicity by combining the substance in question with immunogenic response potentiating agents, so-called adjuvants. A large number of such adjuvants and kind of adjuvants have been suggested but in general without any being ideal in all respects.
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 NR
1
R
2
R
3
R
4
-hal, wherein R
1
and R
2
independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R
3
and R
4
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 NR
1
R
2
R
3
R
4
-hal the R
1
and R
2
groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R
3
and R
4
may be dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl nonadecyl and eicocyl groups. However, also other C
12
-C
20
hydrocarbon groups are possible because even though the R
3
and R
4
groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g. methyl and ethyl side chains. R
3
and R
4
may also have a minor degree of unsaturation, e.g. containing 1-3 double bonds each, but preferably they are saturated alkyl groups. R
3
and R
4
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 NR
1
R
2
R
3
R
4
-hal, wherein R
1
and R
2
independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R
3
and R
4
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 NR
1
R
2
R
3
R
4
-hal, wherein R
1
and R
2
independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R
3
and R
4
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

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