FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10; rule 13)


AN ADJUVANT COMPOSITION FOR VACCINES
SERUM INSTITUTE OF INDIA LTD.,
an Indian Company
of Off Soli Poonawala Road, 212/2 Hadapsar, Pune 411 028,
Maharashtra, India;

THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION


Field of invention
The present invention relates to an adjuvant compostion, for vaccines. The invention also relates to a process for preparing the composition and to the vaccines prepared thereof.
Background
Vaccines based on polysaccharide antigens are Well known. Polysaccharide antigens are, T-lymphocyte independent antigens and therefore, IgG response elicited by the such antigens is very limited. Moreover, the immune response elicited by polysachcharide antigens is characterized by low immunological memory and therefore the immunity induced by the use of such antigens is short lived. Further, young children respond poorly to polysachcharide antigens and therefore vaccines containing the antigens have not been proven to be effective in young children. To enhance the immune response elicited by polysachcharide antigens, aluminium salts have been known to be used as adjuvants in vaccines containing these antigens. However, aluminium containing vaccines, when injected, have been reported to cause undesirable local reactions.
US 6645495 discloses adjuvant saponins extracted from the bark of the tree
Quillaja Saponaria Molina, for polysachcharide vaccines. Quillaja saponins
have, however, been found to have strong haemolytical activity and to cause
undesirable local reactions. The Indian patent application No.
1253/MUM/2003 published on 20th January 2006 discloses the use of Withania somnifera extract as an adjuvant in vaccines. The crude extract as disclosed here is known to contain a large proportion of polysaccharides that could adversely effect the bioavailability of the active compounds present therein.


Moreover, to attain the desired adjuvanticity, large quantity of the extract is required to be used in the vaccine.
There is a need for an adjuvant composition, for vaccines, that is devoid of the above-mentioned drawbacks.
Description
Accordingly, the invention provides an improved adjuvant composition, for vaccines.
In one embodiment, the invention provides an adjuvant composition, for vaccines, comprising a fraction of plant extract(s) selected from a group consisting of withania somnifera extract, asparagus racemosus extract, emblica officinalis extract, tribulus terrestris extract and tinospora cardifolia extract.
In another embodiment, the invention provides an adjuvant composition, for polysaccharide vaccines, comprising a fraction of withania somnifera extract, the fraction comprising around 4% by weight of total withanolides of the extract.
In another embodiment, the invention provides an adjuvant composition, for polysaccharide vaccines, comprising Withanolide A, withaferin A, Withanolide B, withanoside IV, Withanoside V and 12-deoxy withastramonolide.


In another embodiment, the invention provides an adjuvant composition, for polysaccharide vaccines, comprising at least 0.5%) by weight of Withanolide A, at least 0.1%) by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by weight of Withanoside IV, at least 0.5% by weight of Withanoside V and at least 0.8% by weight of 12-deoxy withastramonolide.
In another embodiment, the invention provides an adjuvant composition, for polysaccharide vaccines, comprising 0.5 to 1 % by weight of Withanolide A, 0.1 to 0.6 % by weight of Withaferin A, 0.01 to 0.1% by weight of Withanolide B, 0.8 to 1.2 % by weight of Withanoside IV, 0.5 to 0.8 % by weight of Withanoside V and 0.8 to 1.2% by weight of 12-deoxy withastramonolide.
In another embodiment, the invention provides an adjuvant composition, for polysaccharide vaccines, comprising 0.5 to 1 % by weight of Withanolide A, 0.1 to 0.6 % by weight of Withaferin A, 0.01 to 0.1% by weight of Withanolide B, 0.8 to 1.2 % by weight of Withanoside IV, 0.5 to 0.8 % by weight of Withanoside V and 0.8 to 1.2% by weight of 12-deoxy withastramonolide wherein ratio of Withaferin A and Withanolide A is in the range of 1:2 to 1:5.
In another embodiment, the invention provides a delivery system comprising an adjuvant composition, for polysaccharide vaccines.
In another embodiment, the invention provides a delivery system comprising an adjuvant composition, for polysaccharide vaccines, the delivery system being selected from liposomes, microspheres, emulsions.


In another embodiment, the invention provides a vaccine comprising a polysaccharide antigen and an adjuvant composition, the composition comprising at least 0.5% by weight of Withanolide A, at least 0.1% by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by weight of Withanoside IV, at ieast 0.5% by weight of Withanoside V and at least 0.8%) by weight of 12-deoxy withastramonolide.
In another embodiment, the invention provides a vaccine comprising a polysaccharide antigen and an adjuvant composition, the composition comprising at least 0.5%> by weight of Withanolide A, at least 0.1%) by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by weight of Withanoside IV, at least 0.5%> by weight of Withanoside V and at least 0.8% by weight of 12-deoxy withastramonolide, the vaccine additionally comprising other adjuvants selected from alum, QS-21, CpG, MPL, MF-59.
In another embodiment, the invention provides a vaccine comprising a polysaccharide antigen and an adjuvant composition, the composition comprising at least 0.5% by weight of Withanolide A, at least O.P/o by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by weight of Withanoside IV, at least 0.5%> by weight of Withanoside V and at least 0.8% by weight of 12-deoxy withastramonolide wherein the antigen is a capsular polysaccharide of a gram-positive or a gram negative bacteria
In another embodiment, the invention provides a vaccine comprising a polysaccharide antigen and an adjuvant composition, the composition comprising at least 0.5% by weight of Withanolide A, at least 0.1%) by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by


weight of Withanoside IV, at least 0.5% by weight of Withanoside V and at least 0.8% by weight of 12-deoxy withastramonolide wherein the antigen is a capsular polysaccharide of N. menigitidis types A, C, Y or W135.
In another embodiment, the invention provides a process for preparing an adjuvant composition, for polysaccharide vaccines, the process comprising heating a withania somnifera root material with water under reflux to form an aqueous fraction of withania somnifera extract, filtering the aqueous fraction, concentrating the extract under vacuum, treating the concentrated extract successively with a non polar organic solvent and a polar organic solvent to form an organic fraction of the extract and drying the organic fraction at a temperature not greater than 70°C.
In another embodiment, the invention provides a process for preparing an adjuvant composition, for polysaccharide vaccines, the process comprising heating a withania somnifera root material with water under reflux to form an aqueous fraction of withania somnifera extract, filtering the aqueous fraction, concentrating the extract under vacuum, treating the concentrated extract successively with a non polar organic solvent and a polar organic solvent to form an organic fraction of the extract and drying the organic fraction at a temperature not greater than 70 C wherein the non polar organic solvent is selected from a group of n-hexane, toluene and Benzene and the polar organic solvent is selected from a group consisting of butanol, dichloromethane, dichloroethane and chloroform.
In another embodiment of the invention, there is provided a process for preventing a disease caused by a polysaccharide antigen, the method

comprising administering a vaccine comprising a polysaccharide antigen and an adjuvant comprising at least 0.5% by weight of Withanolide A, at least 0.1% by weight of Withaferin A, at least 0.01% by weight of Withanolide B, at least 0.8% by weight of Withanoside IV, at least 0.5% by weight of Withanoside V and at least 0.8% by weight of 12-deoxy withastramonolide.
In a further embodiment of the invention, there is provided a process for preventing a disease caused by a polysachcharide antigen and one or more of other antigen(s) selected from polysaccharides, toxoids, proteins, the method comprising administering a vaccine comprising a polysaccharide antigen and an adjuvant comprising 0.5 to 1 % by weight of Withanolide A, 0.1 to 0.6 % by weight of Withaferin A, 0.01 to 0.1% by weight of Withanolide B, 0.8 to 1.2 % by weight of Withanoside IV, 0.5 to 0.8 % by weight of Withanoside V and 0.8 to 1.2% by weight of 12-deoxy withastramonolide.
The adjuvant composition of the invention can be present in the vaccine in amounts ranging from 0.5mg/vaccine dose to 80 mg/vaccine dose. Further, the adjuvant composition of the invention can have immunologic activity on simple formulation with antigen either prior to injection or overnight storage with antigen.
While considerable emphasis has been placed herein on the compositions of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the invention. These and other changes in the preferred embodiment as well as other embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is


to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

MOHAN DEWAN
OF R. K. DEWAN & COMPANY
APPLICANTS' PATENT ATTORNEY