DESC:FIELD OF INVENTION
The invention relates to adjuvants. More particularly, invention relates to novel adjuvant formulations for vaccines.

BACKGROUND OF THE INVENTION

The most commonly used adjuvants in vaccine industry are alum and oil based emulsions. Adjuvants can modify immune response by activating T cells instead of antibody-secreting B cells or vice versa, or elicit a balanced immune response depending on the type of adjuvant as well as the antigen used.

Emulsions are available as water in oil emulsion or oil in water emulsion depending the excipients used in the formulation. The emulsions generally induce strong and long lasting immune response. But depending on the oil base used in emulsions, local reactions are common. The choice of excipients used in emulsions and indeed in any adjuvant-vaccine formulations depends on the type of antigen and the kind of immune response elicited by the combination.

The most established emulsion used as an adjuvant is MF59, which has been used commercially since 1997. MF59 has been used in flu vaccines. AS03 is an emulsion adjuvant which was approved for human use in 2009 to meet the challenges of the H1N1 pandemic. The oil component in both MF59 and ASO3 is squalene that is present in comparable quantities, but AS03 additionally contains an immune potentiator DL-a-tocopherol, in approximately a 1:1 (w/w) ratio. However, it has been also well recognized that squalene alone is not an effective adjuvant. Montanide is another notable emulsion adjuvant that contains an oil base emulsified with highly purified mannide monooleate. New categories of adjuvants that have emerged more recently, include liposomes, and micro and nano particle based adjuvants. Although squalene containing emulsions such as MF59 and ASO3 have been used in human vaccines, safety concerns have been raised on the use of squalene and mineral oil in adjuvants for human administration. More recent water in oil emulsions include the introduction of purified and metabolizable vegetable oils and emulsifiers. It is always desirable to make an emulsion based adjuvant composition which is safe and completely non-toxic in nature, naturally and capable of rapid absorption by the host without compromising the capability to boost the immune response.

It is the intention of the inventors of this patent application to make available new vaccine adjuvants and their formulation(s) which boosts immune response, safe for all age groups, using natural non-toxic compound(s) which can be used either alone as a vaccine adjuvant or in combination with other vaccine adjuvants to elicit robust immune response to diverse vaccine antigens either for prophylaxis or treatment in mammals.

The term vitamin D refers to a group of fat-soluble vitamins which are secosteroids that are involved in the intestinal absorption and regulation of mainly calcium and phosphate metabolism and mineralization of bone tissue. Major sources of vitamin D are diet and synthesis in skin. In skin, vitamin D synthesis starts with the formation of cholecalciferol which chemically is (3ß,5Z,7E)-9,10-secocholesta- 5,7,10(19)-trien-3-ol or vitamin D3) by the action of UV-B on 7-dehydrocholesterol. Both cholecalciferol formed in the skin and that ingested from dietary sources, as well as vitamin D2 (ergocalciferol) are hydroxylated in the liver via microsomal and mitochondrial enzymes to become 25-hydroxyvitamin D3 (25-(OH) D3) or 25-(OH) D2 respectively. 25-hydroxyvitamin D3 or 25-(OH) vitamin D3 is also known as 25-hydroxycholecalciferol or calcidiol. 25-(OH)D2 is also known as 25-hydroxyergocalciferol. Calcidiol (also known as calcifediol) or 25-hydroxycholecalciferol is the main systemically available form of vitamin D with a half-life of two to three weeks and its level in blood is indicator of vitamin D nutritional status. 25-hydroxycholecalciferol is further hydroxylated by 25-(OH) D-1-a-hydroxylase (mitochrondrial cytochrome P450 isoenzyme CYP27B1 and microsomal CYP2R1) in the kidneys to form 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) also known as calcitriol. 1,25-(OH)2D3, is a steroid hormone and is the metabolically most active form of vitamin D. The main vitamin D transport proteins are the vitamin D-binding protein (DBP) and albumin which has lower affinity than DBP. 1,25-(OH)2D3 mediates the transcription regulation and thereby the expression of several genes by binding to the nuclear vitamin D receptor (VDR). Receptors for vitamin D are present in a variety of cell types, and have biologic effects which extend far beyond the control of calcium homeostasis and mineral metabolism.

Calcitriol which is also known as 1,25-Dihydroxyvitamin D3 has the chemical formula C27H44O3. Calcitriol has a molecular mass of 416.64 g/mol. It is available under the trade names Rocaltrol®, Calcijex® and Decostriol® among others. Calcitriol has been tested as a vaccine adjuvant and there is a lot of published evidence for its immunomodulatory role in modulating the response to vaccines. Among those studies where calcitriol has been used as a vaccine adjuvant are: adjuvant for inactivated poliovirus vaccines (Ivanov et al. 2006); haemophilus influenza type b oligosaccharide conjugated to diphtheria toxoid (Enjoutina et al.,1999), influenza (Quintilio et al 2016) and hepatitis B vaccine (Daynes et al., 1996). It has also been observed that locally produced 1,25(OH)2VitD3 or calcitriol can stimulate antigen-specific T- and B-cells antibody responses induced by migration of dendritic cells from the site of vaccination to non-draining lymphoid organs, for generating immune response in diphtheria vaccination (Enjoutina et al., 2009). In contrast to the promising nature of adjuvant effect of calcitriol in animal models, however, it has been reported that calcitriol co-administered with influenza vaccine does not enhance humoral immunity in humans (Kriesel et al., 1999). Calcitriol has been found effective in prevention of autoimmune diseases (Lemire et al., 1992). It has been shown that human dendritic cells, through an induced ability to endogenously produce calcitriol are able to program activated T cell migration to inflamed skin (Sigmundsdottir et al., 2007).

However, there are several drawbacks to using calcitriol as vaccine adjuvant. Calcitriol is quite toxic and cannot be administered to all individuals as vaccination is primarily intended for mass administration. The normal permissible limit for calcitriol for therapeutic purpose is only 1 to 2 microgram maximum per dose even for adults. Further, due to the toxic nature of calcitriol, it is not recommended to give booster dose of the vaccine, thereby resulting in lesser period of immune response as well. Additionally, calcitriol has also been linked with calcinosis, which is a toxic calcification of soft tissues due to deposition of calcitriol. Over time, these deposits lead to calcinosis, where excess calcitriol accumulates in tendons, ligaments, cartilage, cardiovascular tissues, kidneys and skin. Chronic hypercalcemia can lead to generalized vascular (blood vessel) calcification, which is coronary artery disease or alternatively nephrocalcinosis which is calcification of the kidneys and osteoarthritis (available at http://www.westonaprice.org/health-topics/nightshades/). According to Medline, common side-effects of calcitriol injections include weakness, headache, somnolence, nausea, vomiting, dry mouth, constipation, muscle pain, bone pain and metallic taste.
Synthesis of calcitriol from its precursors such as cholecalciferol is self-regulated and tightly controlled under normal physiological conditions. Hence its toxicity under normal physiological conditions is not evident. However any extraneously administered calcitriol is a potentially safety issue as it is not feasible to ascertain the vitamin D status of all individuals for mass vaccination. Hence there are safety issues related to use of calcitriol as vaccine adjuvant.

In this present invention, the limitations of calcitriol for use as vaccine adjuvant has been overcome by disclosing novel adjuvant formulations with Cholecalciferol.

Cholecalciferol is the non-toxic precursor of calcitriol. Completely new adjuvant formulations has been made available herein which overcomes the issues faced with calcitriol as a vaccine adjuvant such as toxicity and efficacy. Cholecalciferol is chemically different from calcitriol. Cholecalciferol is activated 7-dehydrocholesterol and it’s IUPAC id is (3ß,5Z,7E)-9,10-secocholesta- 5,7,10(19)-trien-3-ol. It has a molecular mass of 384.64 g/mol. It’s chemical formula is C27H44O. Structurally and chemically, it is different from the metabolically the active form of vitamin D which is calcitriol. Unlike calcitriol, cholecalciferol is completely safe in humans at doses several times higher than calcitriol and is the only form of vitamin D recommended for use in infants and toddlers for vitamin D supplementation. A study of intramuscular injection of a mega dose of cholecalciferol up to 250 microgram/dose (or 10,000 IU) was found is a safe and effective therapy for treatment of vitamin D deficiency induced rickets in infants and toddlers (Soliman et al., 2009). In adults, doses up to 100000 IU or 2500 microgram /dose once every 3 months have been shown to be effective in maintaining 25-hydroxyvitamin D levels at 20ng/ml or higher with no adverse effects (Trivedi et al., 2003). Intramuscular mega-dose of vitamin D was used in two studies both in children and adults 300000 IU and 600000 IU, respectively with vitamin D deficiency and reported improvement of biochemical and radiological abnormalities with no adverse effects (Khaw et al., 1994; Wu et al., 2003).

OBJECTS OF THE INVENTION

One object of the invention is to provide new adjuvants for vaccine compositions which are non-toxic and metabolized easily by mammals.

Another object of the invention is to provide better and effective and safe adjuvant compositions for use in mammals.

Yet another object of the invention is to provide novel vaccine adjuvant Cholecalciferol which is a precursor of active form of Vitamin D in humans and is safer than calcitriol for use as vaccine adjuvant.

One more object of the invention is to provide different adjuvant formulations as emulsions based on Cholecalciferol.

Yet another object of the present invention is to provide novel Cholecalciferol based adjuvant
formulations which are compatible with all major vaccine antigen platform technologies and at the same time compatible with a wide range of specific vaccine antigens.

Yet another object of the present invention is to provide several vaccine formulations of Cholecalciferol either alone or emulsified with other excipients to obtain stable formulation

Yet another object of the present invention is to provide a combination of other adjuvant(s) with Cholecalciferol to provide a balanced and potent immune response when administered with vaccine antigens in mammals.

Yet another object of the present invention is to provide formulation(s) of Cholecalciferol with other organic and inorganic compounds that have immunopotentiating role, one such example being alpha tocopherol.

Yet another object of the present invention is to provide metabolites and analogues of Cholecalciferol as potential vaccine adjuvant.

Yet another object of the invention is to provide formulations and methods of preparation of Cholecalciferol containing adjuvants that are applicable to other secosteroid derivatives and all vitamin D metabolites and their analogues.

One objective of the invention provides Cholecalciferol as vaccine adjuvant in vaccine compositions which are non-toxic and metabolized easily by mammals.

One more objective of the invention provides different adjuvant formulations as emulsions based on Cholecalciferol.

Yet another objective of the invention provides novel vaccine adjuvant Cholecalciferol which is a precursor of the active form of Vitamin D in humans and is safer than calcitriol for use as vaccine adjuvant.

Yet another objective of the present invention provides novel Cholecalciferol based adjuvant
formulations which are compatible with all major vaccine antigen platform technologies and at the same time compatible with a wide range of specific vaccine antigens.

Yet another objective of the present invention provides formulation(s) of Cholecalciferol with other organic and inorganic compounds that have immunopotentiating role, one such example being alpha tocopherol.

Further, all the formulations with Cholecalciferol and/or in combination with other adjuvants were found to be stable and is suitable to be used as a vaccine adjuvant at a concentration of 10 µg to 250 µg per dose for any animal or human prophylactic and/or therapeutic vaccines.

SUMMARY OF THE INVENTION

According to one embodiments of the invention, novel adjuvant compositions with Cholecalciferol are disclosed.

According to one more embodiment of the invention, the new adjuvant compositions with Cholecalciferol are formulated with different vaccine antigens.

According to the embodiments for adjuvant compositions, disclosed are stable adjuvant formulations comprising

(a) cholecalciferol;
(b) an oil base;
(c) a lipid base;
(d) a fat soluble hydrophobic solvent;
(d) a surfectant; and
(e) a pharmaceutically acceptable buffer.

The adjuvant formulation wherein the fat soluble hydrophobic solvent is selected from phospholipids, cholesterol, chylomicrons, Vitamin E (alpha tocopherols), Vitamin B complex (methylcobalamin), and Vitamin C (ascorbic acid) or combinations thereof.

The adjuvant formulation wherein the lipid base is selected from any long, short or medium chain triglycerides, the said triglyceride selected from saturated and unsaturated fatty acids, or combinations thereof.

The adjuvant formulation wherein the saturated fatty acids are selected from palmitic acid, stearic acid, or caprylic acid or combinations thereof.

The adjuvant formulation wherein the unsaturated fatty acids are selected from oleic acid, linoleic, linolenic and arachidonic acids or combinations thereof.

The adjvant formulation wherein the lipid base is a medium chain triglyceride selected from glyceryl trioctanoate or glyceryl tricaprylate, omegavan or combinations thereof.

The adjuvant formulation wherein the oil base is selected from squalene or analogs thereof, vegetable oils, fish oils, animal oils, or synthetic oils or combinations thereof.

The adjuvant formulation wherein the fat soluble hydrophobic solvent is selected from phospholipids, cholesterol, chylomicrons, Vitamin E (alpha tocopherols), Vitamin B complex (methylcobalamin), and Vitamin C (ascorbic acid) or combinations thereof.

The adjuvant formulation wherein the surfactant is any organic or inorganic surfactants selected from polysorbates, sodium lauryl sulfate, sorbitan trioleates, mannide monooleates, lecithin, TEA (triethanolamine lauryl sulfate), ammonium and magnesium lauryl sulfates, any poloxamers said poloxamers being poloxamer 188 (Pluronic F68), polyoxyethylene9-10 nonylphenol (Triton N-101 octoxynol 9), any polyoxyethylated octyl phenol said polyoxyethylated octyl phenol being Triton-X100, polyvinyl alchohols, sodium deoxycholates, sodium decosates or combinations thereof..

The adjuvant formulation wherein the oil base is selected from omega 3 or omega 6 fatty acids.

The adjuvant formulation wherein the lipid base is selected from glyceryl trioctanoate also known as glyceryl tricaprylate.

The adjuvant formulation wherein the surfactant or the emulsifiers is suitable for any vaccine composition selected from polysorbate such as tween-80 of any grade or purity, either used alone or in combination thereof.

The adjuvant formulation wherein the emulsifier is sorbitan trioleate.

The adjuvant formulation in combination with one or more any other known adjuvants selected from MF59, ASO3, monanise, MPL (monophosphoryl lipid A), MDP (muramic dipeptides), resiquimoid or any of its analogs, PolyIC or any oligonucleotide such as for example CpG containing oligonucleotides, N-glycolyl dipeptide (GMDP), inulin compounds or any analogues thereof for use in vaccine compositions.

The adjuvant formulation for use as vaccine compositions with any given vaccine antigens, the said vaccine antigen being any live attenuated vaccine antigen, an inactivated vaccine antigen, a subunit vaccine antigen, a vector based vaccine antigen, mRNA or DNA based vaccine antigen or any other vaccine antigen.

The adjuvant formulation wherein the vaccine antigen is selected from as hepatitis B small antigen, Japanese encephalitis virus antigens or zika virus vaccine antigen, mayaro virus antigen, chikungunya vaccine antigen, rabies virus antigen, influenza virus antigen or a dengue virus antigen. It is to be noted that, the list of vaccine antigens disclosed herein is non-exhaustive in nature. Any vaccine antigen can be used with the novel Cholecalciferol based vaccine formulations of this invention.

The adjuvant composition wherein the Cholecalciferol is used at a concentration of 10 µg to 250 µg per dose, preferably from 50 µg to 100 µg per dose.

The adjuvant composition wherein the pharmaceutically acceptable buffer is phosphate buffer at a range of 1-100 mM at a pH of 6.5 to 7.5.

One more embodiment of the invention discloses stability of the vaccine formulations with different vaccine antigens and adjuvant compositions with Cholecalciferol.

Further embodiments of the invention includes testing of immunogenicity with different vaccine antigens with cholecalciferol based adjuvant compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. CCFL adjuvant formulation characterization
Particle size distribution (by Number) of the adjuvant composition (CCFL) comprising cholecalciferol with glyceryl trioctanoate as lipid base in combination with sorbitan trioleate and Tween 80 as emulsifiers prepared in phosphate buffer, pH 7.5. The particle size distribution of the emulsion was estimated using Malvern zetasizer.

Figures 2A to 2D: Immunogenicity of the vaccine formulations with CCFL
Antibody titers elicited by the vaccine compositions comprising either A) Hepatitis B small antigen (HbsAg) B) Japanese encephalitis (JE) vaccine antigen C) Zika virus (ZIKV) vaccine in combination with adjuvant emulsion CCFL comprising cholecalciferol, glyceryl trioctanoate, sorbitan trioleate and tween-80 in phosphate buffer, pH 7.5. D) ZIKV vaccine in combination with adjuvant emulsion CCFLT comprising cholecalciferol, glyceryl trioctanoate, sorbitan trioleate and tween-80 in phosphate buffer, pH 7.5 and alpha tocopherol. Vaccine specific antibody titers were measured in serum of mice immunized with two doses of the respective vaccine antigens. 50% plaque reduction neutralization titers (PRNT50) was used to measure neutralizing antibody responses to JE, and ZIKV vaccines as described in Example 3 and anti-HbsAg antibodies were measured by ELISA by the method disclosed in Example 4. The PRNT50 titers of control animals for JE and ZIKV were <10 and not depicted in the figure. The average (mean) control value for the HbsAg control animals was 1.02 and not depicted in the figure.

Figure 3. Stability of CCFL vaccine formulations
Comparative stability of the ZIKV vaccine antigen with and without CCFL adjuvant formulation. The vaccine antigen in the adjuvant emulsion had a higher stability as seen by higher immunogenicity than when stored without the adjuvant emulsion. Accelerated stability studies were carried out at 37 degree C by storing the vaccine antigen in aqueous phosphate buffered saline, pH 7.5 and as vaccine antigen formulated as an emulsion with CCFL. Immunogenicity testing was carried out in Balb/c mice by the methods described in Examples 2 & 3.

DETAILED DESCRIPTION OF THE INVENTION

The Cholecalciferol containing adjuvant formulations disclosed in this invention confer better stability, render the vaccine antigen more immunogenic, is safe and is used at a fractional dose than what is normally administered to humans for vitamin D supplementation.

Cholecalciferol is emulsified in a formulation that is safe and free from toxic effects. Cholecalciferol was emulsified in lipid emulsions that are safe for human administration and suitable for parenteral administration in infant for nutritional supplementation. Long, short or medium chain triglycerides can be used to emulsify Cholecalciferol. Triglycerides occur naturally in human body and hence are non-toxic and easily metabolized without any side effects. Triglycerides can contain saturated fatty acids selected from a list but not limited of palmitic acid, stearic acid, caprylic acid etc. or any unsaturated fatty acids selected from a list but not limited to oleic acid, linoleic, linolenic and arachidonic acids etc. Medium chain triglyceride glyceryl trioctonoate or glyceryl tricaprylate has been used in the current invention. Any of the above mentioned triglycerides can be used. In lieu of triglycerides any other oil base can be used selected from list but not limited to squalene or its analogs, any vegetable or plant derived oil, fish oils, animal source derived oils, oil from any source, or any synthetic oil substitute that is useful as a solvent for the hydrophobic compounds, phospholipids, cholesterol, chylomicrons, vitamins with antioxidant properties such as vitamin E (alpha tocopherols), methylcobalamin or any vitamins including B complex vitamins, and ascorbic acid i.e. Vitamin C that provide antioxidant properties can be part of the formulation and can be used with Cholecalciferol either singly or in combinations. The Cholecalciferol based adjuvant formulations of the present invention also contains surfactants or emulsifiers. Suitable surfactants (emulsifiers) for use as adjuvant formulations with Cholecalciferol were selected from any ionic or non-ionic list of compounds that include but are not limited to: polysorbates, sodium lauryl sulfate, sorbitan trioleates, mannide monooleates, lecithin, TEA (triethanolamine lauryl sulfate), ammonium and magnesium lauryl sulfates, any poloxamers such as poloxamer 188 (Pluronic F68), polyoxyethylene9-10 nonylphenol (Triton N-101 octoxynol 9), any polyoxyethylated octyl phenol such as Triton-X100, polyvinyl alchohols, sodium deoxycholates, sodium decosates etc. or any organic compound that is pharmaceutically acceptable and has surfactant property. A combination of the either of the above surfactants were found to be useful as well. The aqueous phase in the emulsion is water or any pharmaceutically acceptable buffer systems that provided a pH range from pH 6.0 to pH 8.5 to ensure stability of the vaccine antigen in the adjuvant emulsion, and depends on the nature of the vaccine antigen used in the emulsion.

Cholecalciferol can also combined with a variety of presently known adjuvants which have similar composition as MF59, ASO3 and Montanide either singly in any combination thereof. Further, use of Cholecalciferol as a vaccine adjuvant in a suitable formulation can also be used with wide variety of vaccine antigens. The adjuvant formulations of the present invention are highly immunogenic and the excipients are safe for animal and human use. It was observed that Cholecalciferol induced high antibody titers to vaccine antigens in mice. In order to augment cell mediated immunity in addition to B cell immunity, other adjuvants can be added to Cholecalciferol containing preparations. For example alum (any aluminum containing adjuvant) can be added wherein the vaccine antigen is adsorbed to alum and added as an insoluble particulate matter to Cholecalciferol containing emulsion. This can enhance the immunogenicity further. Other adjuvants that can be added along with Cholecalciferol can selected from the following list but is not limited to: MPL (monophosphoryl lipid A), MDP (muramic dipeptides), resiquimoid or any of its analogs, PolyIC or any oligonucleotide such as for example CpG containing oligonucleotides, N-glycolyl dipeptide (GMDP), inulin compounds or any analogues of the aforementioned class of adjuvants or for that matter any organic or inorganic compound that has immunopotentiating activity.

The adjuvants of the present invention are safe for animal and human use, as the adjuvant itself and all the excipients are being used for human administration in quantities much larger than that used as adjuvant in the present invention. The adjuvant of the present invention can be used for all human and animal viral, bacterial and parasitic vaccines and indeed vaccines for any pathogen. The animal vaccines derived from any platform technology could be used to formulate any zoonotic vaccines for dogs, cattle, sheep, camel, horses and indeed any animal species. The adjuvant disclosed in the invention can be used as an adjuvant to formulate any viral, bacterial, parasitic vaccines and indeed vaccines for any pathogens for human use, and is suitable for antigens derived from any platform technology as long as the vaccine antigen can be easily formulated with the Cholecalciferol adjuvant.

The adjuvants of the present invention are stable with vaccine antigens such as hepatitis B small antigen, inactivated Japanese encephalitis virus antigen, Zika virus and Chikungunya virus vaccines. It is also suitable to be used as adjuvant for live attenuated, inactivated (killed) and recombinant subunit, virus like particles or other subunit or chimeric vaccine antigens and nucleic acid vaccines such as DNA and RNA vaccines, polysaccharide and polysaccharide conjugate vaccines. Addition of vaccine adjuvant elicited significantly higher antibody titers to the vaccine antigens than when the vaccine antigen was administered alone in mice.
The findings of the current invention could potentially apply to all precursors, metabolites and analogues of Cholecalciferol and other secosteroid derivatives and indeed all vitamin D metabolites and their analogues.

The vaccine formulations were found to be more stable and immunogenic in the cholecalciferol adjuvant based emulsion than when used alone without the adjuvant emulsion.

EXAMPLES

Example 1: Preparation of Cholecalciferol adjuvant formulations
Cholecalciferol (catalog No. C9756, Sigma Aldrich) was used in the preparation of adjuvant formulations along with the other excipients. Different stock concentrations of Cholecalciferol were made ranging from 1 mg/mL to 50 mg /mL in 10 mM phosphate buffer, pH 7.5 containing glyceryl trioctanoate, tween 80 and sorbitan trioleate. The adjuvant formulations were emulsified using by homogenization until a uniform emulsion was formed. Various concentration of the excipients were tested to obtain a uniform emulsion. The final concentration of the adjuvant used for animal testing contained 100 µg of Cholecalciferol, 15.53 mg of glyceryl trioctanoate, 1.1 mg of tween-80 and 1.175 mg of sorbitan trioleate per vaccine dose of 100 µl used in the animal experiments. There was no significant difference in the levels of antibody titers elicited when 10 to 100 µg of Cholecalciferol was used. The particle size distribution of the Cholecalciferol emulsion and the background diffraction of the buffer was measured using a Malvern zetasizer (Fig.1a and 1b). The adjuvant formulation was designated as CCFL.

Other emulsions of Cholecalciferol were made by addition of alpha-tocopherol to CCFL (CCFLT) to the aforementioned excipients. Alpha tocopherol was used at a concentration of 11.86 mg /per vaccine dose. Omegavan (Fresenius Kabi) was added in lieu of glyceryl trioctanoate and contained other excipients as mentioned above. The formulation was designated as CC-OMG. All the adjuvant formulations were found to be stable for at least 12 months at 2-8 degree C. CCFL and CCFLT formulations were found to be more stable at 37 degree C than CC-OMG.

Example 2: Immunogenicity testing in mice
Female Balb/c mice, 18-22 g body weight were injected with either of the below vaccine formulations containing the adjuvant CCFL at the excipient concentrations mentioned in Example 1. The test group consisted of 6 nos of mice along with 6 nos of placebo control. The HbsAg vaccine formulation comprised 22 µg of antigen/dose, inactivated JE antigen 6 µg per dose and inactivated Zika virus antigen, 10 µg per dose. Each of the vaccine formulations were formulated with CCFL adjuvants and were administered in a volume of 100 µl to 150 µl/dose by intramuscular route based on the antigen concentration in the final formulation. Two doses of each vaccine was administered at day 0 and day 21 and the antibody titers were measured in serum samples 10 days after last vaccine dose administration by the methods described in Example 3 and 4. In another experiment, 10 µg per dose of ZIKV antigen was formulated with CCFLT adjuvant at the concentrations of the excipients / dose described in Example 1 and administered in mice by intramuscular route on days 0 and 21. Blood was drawn 10 days after the last vaccine dose and the PRNT50 titers were estimated by the method described in Example 3. No visible toxicity was observed in any of the animals in all the vaccine and placebo groups.

Example 3: 50% Plaque Neutralization Reduction Assay
Estimation of JE and ZIKV neutralizing antibodies was estimated by 50% Plaque Reduction Neutralization Test (PRNT50) by standardized procedures. Briefly, one day prior to the assay, 6-well plates were seeded with 2.5 x 103 Vero cells (ATCC CCL-81) per well and the plates were incubated at 37°C in a 5% CO2 incubator. To 4-fold dilutions of the sera samples in MEM, equal volume of the standardized Zika virus strain (105 pfu/mL) was added and incubated at 37°C with 5% CO2 for 90 min. The cells were washed twice with 1 x PBS pH 7.4 (10 mM phosphate with 150 mM NaCl) and 0.30 ml of each dilution of the serum-virus mixture was added to the corresponding well and incubated for 90 min at 37°C in a 5% CO2 incubator. Each assay was carried out in triplicates. The cells were overlaid with 2 ml of 0.85% methyl cellulose in MEM with 1% penicillin-streptomycin and 1% L-glutamine. The plates were incubated at 37°C in a 5% CO2 incubator for 4 days. At the end of incubation, the plaques were fixed with 10% formalin, washed with 1 x PBS, pH 7.4 and were visualized with 0.1% crystal violet. The highest dilution of serum causing 50% reduction in the number of plaques formed by the control virus sample was estimated as the PRNT50 titer. The vaccine antigens elicited high level of neutralizing antibodies as depicted in Figure 2.

Example 4: Estimation of HbsAg antibodies

Hepatitis B small antigen (HbsAg) antibodies were estimated using the ARCHITECT anti-HBs immunoassay (Abbot Diagnostics Core Laboratories) based on chemiluminescent microparticle technology. It is an automated process for estimating the concentration of antibody to Hepatitis B surface antigen (anti-HBs) in human serum/plasma by the ARCHITECT i System. It is a two-step process where Anti-HBs from human sera initially binds to recombinant HBsAg (rHBsAg) coated microparticles followed by a secondary binding to another acridinium-labeled rHBsAg conjugate. After subsequent washes, addition of 1.32% w/v H202 and 0.35N NaOH solutions result in a chemiluminescent reaction that is measured as relative light units (RLUs) by the ARCHITECT i System optics. The assay uses appropriate calibrators and control and the sera samples for estimation are diluted if the titers are high, and the dilution factor is factored in the final calculation. The result is generated in an automated manner via an ARCHITECT Anti-HBs calibration curve. Specimens with anti-HbsAg titers greater than or equal to 10 mIU/mL are only considered to be reactive for anti-HBsAg.

Example 5: Stability of the vaccine formulation with CCFL adjuvant:
Stability of ZIKV vaccine formulated with the CCFL emulsion of the composition described in Example 1 was tested in accelerated stability studies at 37 degree C for six weeks. The control sample had equivalent content of ZIKV antigen in 10 mM phosphate buffered saline, pH 7.5. Both the vaccine formulation and the control ZIKV antigen sample were stored at 37 degree C for six weeks. At the end of six weeks, the vaccine composition with adjuvant and the control antigen composition in aqueous buffer were tested for immunogenicity in Balb/c mice as per the methods described in Example 2. The PRNT50 titers were estimated in the serum samples of individual mice (6 nos) per group 10 days after the administration of the last vaccine dose. ZIKV vaccine formulated with CCFL in emulsion had higher stability as evidenced by higher immunogenicity when compared to ZIKV antigen alone without adjuvant. Thus CCFL adjuvant formulation conferred stability and higher immunogenicity to the antigen as seen in Figure 3.
,CLAIMS:We claim

1. A stable adjuvant formulation comprising:
(a) cholecalciferol;
(b) an oil base;
(c) a lipid base;
(d) a fat soluble hydrophobic solvent;
(d) a surfectant; and
(e) a pharmaceutically acceptable buffer.

2. The adjuvant formulation of claim 1, wherein the lipid base is selected from any long, short or medium chain triglycerides, the said triglyceride selected from saturated and unsaturated fatty acids, or combinations thereof.

3. The adjuvant formulation of claim 2, wherein the saturated fatty acids are selected from palmitic acid, stearic acid, or caprylic acid or combinations thereof.

4. The adjuvant formulation of claim 2, wherein the unsaturated fatty acids are selected from oleic acid, linoleic, linolenic and arachidonic acids or combinations thereof.

5. The adjvant formulation of claim 1, wherein the lipid base is a medium chain triglyceride selected from glyceryl trioctanoate or glyceryl tricaprylate, omegavan or combinations thereof.

6. The adjuvant formulation of claim 1, wherein the oil base is selected from squalene or analogs thereof, vegetable oils, fish oils, animal oils, or synthetic oils or combinations thereof.

7. The adjuvant formulation of claim 6, wherein the fat soluble hydrophobic solvent is selected from phospholipids, cholesterol, chylomicrons, Vitamin E (alpha tocopherols), Vitamin B complex (methylcobalamin), and Vitamin C (ascorbic acid) or combinations thereof.

8. The adjuvant formulation of claim 1, wherein the surfactant is any organic or inorganic surfactants selected from polysorbates, sodium lauryl sulfate, sorbitan trioleates, mannide monooleates, lecithin, TEA (triethanolamine lauryl sulfate), ammonium and magnesium lauryl sulfates, any poloxamers said poloxamers being poloxamer 188 (Pluronic F68), polyoxyethylene9-10 nonylphenol (Triton N-101 octoxynol 9), any polyoxyethylated octyl phenol said polyoxyethylated octyl phenol being Triton-X100, polyvinyl alchohols, sodium deoxycholates, sodium decosates or combinations thereof..

9. The adjuvant formulation of claim 1 wherein the oil base is selected from omega 3 or omega 6 fatty acids.

10. The adjuvant formulation of claim 1 wherein the lipid base is selected from glyceryl trioctanoate also known as glyceryl tricaprylate.

11. The adjuvant formulation of claim 1, wherein the surfactant or the emulsifiers is suitable for any vaccine composition selected from polysorbate such as tween-80 of any grade or purity, either used alone or in combination thereof.

12. The adjuvant formulation of claim 11, wherein the emulsifier is sorbitan trioleate.

13. The adjuvant formulation of claim 1, in combination with one or more any other known adjuvants selected from MF59, ASO3, monanise, MPL (monophosphoryl lipid A), MDP (muramic dipeptides), resiquimoid or any of its analogs, PolyIC or any oligonucleotide such as for example CpG containing oligonucleotides, N-glycolyl dipeptide (GMDP), inulin compounds or any analogues thereof for use in vaccine compositions.

14. The adjuvant formulation of claim 1 for use as vaccine compositions with any given vaccine antigens, the said vaccine antigen being any live attenuated vaccine antigen, an inactivated vaccine antigen, a subunit vaccine antigen, a vector based vaccine antigen, DNA vaccine, mRNA vaccine or any other vaccine antigen for human and animal administration

15. The adjuvant composition of claim 1, wherein the cholecalciferol is used at a concentration of 10 µg to 250 µg per dose, preferably from 50 µg to 100 µg per dose.

16. The adjuvant composition of claim 1 wherein the pharmaceutically acceptable buffer is phosphate buffer at a range of 1-100 mM at a pH of 6.5 to 7.5.

Dated this 7th day of August 2017