FIELD OF INVENTION
The invention belongs to the technical field of adenovirus compositions, and particularly relates to a novel improved stable recombinant adenovirus compositions comprising recombinant adenovirus vector/s and its process of manufacture. The novel stable recombinant adenovirus compositions of the invention can be widely used for preventing infection of novel coronavirus (Covid-19). Other aspects of the invention are described in or are evident from the following disclosure, and are within the ambit of the invention
BACKGROUND OF THE INVENTION
The burst of the novel coronavirus (COVID-19) brings huge crisis to social public health, the infectivity is very strong, and the novel coronavirus brings huge social health and economic influences to human beings and is highly valued by people. There is currently no effective therapeutic and prophylactic vaccine. For the health of people's public, the safety of the country and society, there is an urgent need to develop vaccine products that can prevent the infection of novel coronavirus.
Specific antiviral drugs and preventive vaccines which are clearly verified at home and abroad currently have not been provided aiming at the novel coronavirus. Therefore, the prevention is well done, and the blocking of the spread of the virus is the key to controlling the epidemic situation. The use of vaccines has played an irreplaceable role in the elimination of a wide variety of infectious diseases. Published results of alignment of the genomes of a dozen SARS-CoV-2 viruses show that the difference between viruses is very small and no variation has been found at present. Therefore, if the SARS-CoV-2 vaccine is successfully developed, the outbreak of new epidemic situation can be inhibited to a great extent.
Adenoviruses are commonly used vectors in vaccine development and gene therapy, and are widely used in the biomedical field. Adenovirus is less toxic than other vectors (e.g., bacteria or other viruses), and infection with adenovirus causes only mild cold symptoms. The gene therapy product using adenovirus vectors has proved its safety in a plurality of clinical trials.

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The commonly used development methods of viral vaccines include inactivated viral vaccines, subunit vaccines, VLP (virus like particle) vaccines and RNA vaccines. However, the recombinant viral vector vaccines, the high-tech recombinant viral vector gene vaccine is the latest technology for vaccine development. The vaccine needs to be safe and effective, short development time, and the improvement of the stability of the vaccine composition is also an important aspect of research.
Granted patent CNI 11218459 discloses a novel coronavirus vaccine using human type 5 replication defective adenovirus as a carrier. The vaccine uses the replication-deficient human type 5 adenovirus with combined deletion of El and E3 as a carrier, and uses HEK293 cells integrating the adenovirus El gene as a packaging cell line, and the protective antigen gene carried is an optimized design of 2019 new coronavirus.
PCT application WO20I3135615 describes a vaccine composition comprising a plurality of recombinant human adenovirus of serotype 5, 26, 34, 35, 48, 49 or 50, characterized in that the genomes of essentially all adenovirus particles in said composition comprise as the 5' terminal nucleotides the nucleotide sequence.
Chinese patent application CNI 11494615 claims a recombinant adenovirus gene vaccine for preventing new coronavirus and its method for preparing the recombinant adenovirus gene vaccine using lyophilization or freeze drying technique.
All these cited prior arts indicate vaccine compositions with recombinant adenovirus for treating or preventing novel coronavirus. None of the cited prior art discloses stabilized/ thermo-stable adenovirus-based vaccine against new coronavirus (Covid-19) that meet all of the desired stability and compatibility. Also none of the prior art attempts ongoing challenges in the field of adenoviral vectors based vaccine research is to develop thermo-stable formulation(s) which are stable for longer periods of time within a useful temperature range (about 2-8°C). Poor storage stability of adenovirus at 2-8°C has limited its potential by restricting storage conditions to the frozen state (-18° or below), or by necessitating the use of lyophilization to enhance stability. The frozen and lyophilized vaccine have their own limitation and the frozen vaccine have limited shelf life and lyophilized vaccine using have higher processing time and need for reconstitution of vaccine.

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Therefore, there remains a need to discover novel improved stable recombinant adenovirus vaccine compositions comprising recombinant adenovirus and methods for enhanced protective against novel coronavirus (Covid-19).
To overcome above mentioned challenges, the present invention aims to develop an improved novel stable recombinant adenovirus vaccine compositions comprising recombinant adenovirus vector/s and its process of manufacture, which can be widely used for preventing infection of novel coronavirus (Covid-19).
It was surprisingly found by the present inventors that recombinant adenovirus vaccine compositions of the present invention are thermo-stable for longer periods and very effective vaccine against infection of novel coronavirus (Covid-19) with an improved efficacy.
SUMMARY OF THE INVENTION
The present invention relates to a novel improved stable recombinant adenovirus vaccine compositions comprising recombinant adenovirus vectors: recombinant human adenovirus of the 5th serotype (Ad5) or recombinant human adenovirus of the 26th serotype (Ad26).
The present invention relates adenoviral vectors based vaccine research to develop thermo-stable formulations which are stable for longer periods of time within a useful temperature range (about 2-8°C).
The invention provides the process of manufacturing stable recombinant adenovirus vaccine compositions using different stabilizers.
The invention also provides application of the recombinant adenovirus in preparation of a vaccine for preventing novel coronavirus (Covid-19).
The invention also provides a kit comprising all the antigenic components in a single vial/ prefilled syringe or a kit.
A composition according to the invention may be administered by any conventional route which is used in the field of vaccines, in particular by the systemic, i.e. parenteral route, e.g. by the

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subcutaneous, intramuscular, intradermal route for sequential or simultaneous administration of immunogenic compositions.
In a preferred embodiment, in the above application, the recombinant adenovirus vaccine composition is prepared as an injection, nasal drops or spray.
In a more preferred embodiment, the recombinant adenovirus vaccine composition is prepared as an intramuscular injection.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Effect of stabilizer on Virus particle in the Composition
FIG.2: Effect of stabilizer on pH of Composition
FIG.3: Effect of stabilizer on Immunogenicity of Composition for Intramuscular delivery
FIG.4: Effect of stabilizer on Immunogenicity of Composition for Intranasal administration
FIG.5: Virus particle present in the stabilized Composition
FIG.6: Immunogenicity effect of stabilized Composition for Intramuscular delivery
FIG.7: Immunogenicity effect of stabilized Composition for Intranasal administration
DESCRIPTION OF THE INVENTION
The first aspect of the present invention is to provide a novel improved stable recombinant adenovirus vaccine compositions comprising recombinant adenovirus vector/s and its process of manufacture, which can be widely used for preventing infection and infection of novel coronavirus (Covid-19).
In one embodiment of the present invention is to provide a stable recombinant adenovirus vaccine compositions based oh recombinant human adenovirus of the 5th serotype (Ad5) or recombinant human adenovirus of the 26th serotype (Ad26).
The present invention relates adenoviral vectors based vaccine research to develop thermo-stable formulation(s) which are stable for longer periods of time within a useful temperature range (about 2-8°C).

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The invention also provides the process of manufacturing stable recombinant adenovirus vaccine compositions using different stabilizers is also disclosed.
The another aspect of the present invention features a method of manufacturing a vaccine for treating or reducing the risk of a viral infection of novel coronavirus (Covid-19) in a mammal, such as a human.
The present invention provides a pharmaceutical composition, which includes a recombinant adenovirus vaccine and pharmaceutically acceptable excipients.
In yet another embodiment, stable recombinant adenovirus vaccine compositions in combination with one or more pharmaceutical ly acceptable excipients, diluents, buffers, preservatives, stabilizers, adjuvant, lyophilization excipients or other acceptable carriers.
In one embodiment, stable recombinant adenovirus vaccine compositions comprising
(i) a buffered solution (ii) a chelating agent (iii) a divalent cation (iv) a stabilizer/ stability modifiers
(v) Inclusion of one or more additional excipients that act as inhibitors of free radical oxidation which enhance thermal stability of the adenovirus.
The invention formulated to enhance thermal stability of the adenovirus. Components which may enhance the stability of a vaccine formulation include salts, buffers, non- ionic surfactants and other stabilizers such as polymeric polyanion stabilizers.
Stabilizers suitable for use in vaccines are known in the art and may include, for example, buffers, sugars, sugar alcohols, and amino acids. Stabilizing buffers are preferably adjusted to a physiological pH range and may include phosphate buffers, Tris buffers, divalent cations, TE (Tris/EDTA), TEN (Tris/NaCl/EDTA) and Earle's salt solution wherein the buffer concentration is ranging between about 1 mg and 10 mg.

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Stabilizing sugars may include, for example, one or more of saccharose, glucose, sucrose, trehalose, fructose, dextranes, dextranesulphate, and trehalose. Stabilizing sugar alcohols may include, for example, mannitol, sorbitol and glycerol. Amino acids suitable for use in the invention include, for example, L-glutamine, arginine, cysteine, and lysine. Additional stabilizers which may be used in the invention include Tartaric acid, Pluronic F 68, and Tween 20/ Tween 80.
The stabilizer is mainly used for enhancing the stability of the vaccine formulation during storage wherein the concentration of stabilizer is ranging between about 1 mg and 100 mg. The stability enhancer/ modifiers can be selected from:
Complexing agents such as Cyclodextrin (2-Hydroxypropyl)-[3-cyclodextrin); Tertiary amine P-cyclodextrin (TMBCD) etc.
Water soluble Polymer(s) such as Polyvinylpyrrolidone, Polyvinyl alcohol, Polyethylene glycols (PEGs), Polyethylene oxide etc.
Proteinaceous stabilizers like Gelatin/Hydrolyzed gelatin/Collage/Hydrolyzed collagen/Albumin etc.
Cellulose and its derivative such as Hydroxypropyl methyl cellulose, Methylcellulose and Carboxy methylcellulose sodium etc.
Dextran/ Dextran Sulphate, hydroxyethyl starch, Span 20 ,Polysorbate 80, Poloxomerl88, chelating agent may be selected from the group of Alpha lipoic acid (ALA) Ethylenediamine tetraacetic acid (EDTA) Diethylene triamine pentaacetic acid (DTPA), Pluronic and protamine histidine, L-arginine etc wherein the chelating agent concentration is ranging between about 1 ug and 500 ug.
Divalent cations may be selected from the group of Magnesium chloride, Barium chloride, Zinc chloride and Calcium chloride wherein the concentration of divalent cation is ranging between about 1 ug and 500 ug.

Preservatives may be selected from the group of Pheoxyethanol, Methyl Paraben, Propyl Paraben, Butyl Paraben, Benzyl Alcohol, Chlorobutanol, Phenol, Meta cresol, Thiomersal, Phenylmercuric nitrate, Benzylkonium Chloride and Benzethonium Chloride etc.
In one embodiment, the vaccine of the invention will be selected from the following Adenoviruse serotypes; although it is appreciated that one or two other serotypes could be substituted depending on the age of the recipient receiving the vaccine and the geographical location where the vaccine will be administered.
Adenoviruses
Recombinant adenoviruses offer several significant advantages for use as vectors for the expression of one or more optimized viral polypeptides of the invention. Furthermore, adenoviruses do not integrate their DNA into the host genome. Thus, their use as expression vectors has a reduced risk of inducing spontaneous proliferative disorders.
The rare serotype and chimeric adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792 are particularly useful as vectors of the invention. For example, recombinant adenoviruses Ad5, Ad26, Ad34, Ad03 and Ad48 etc. can encode one or more optimized viral polypeptides of the invention. One or more recombinant viral vectors encoding optimized viral polypeptides of the invention can be administered to a subject to treat or prevent a viral infection.
In certain embodiments, the recombinant adenovirus in the compositions or methods of the invention, is a recombinant human adenovirus, and is preferably of recombinant human adenovirus of serotype 5, 26, 35, 49 or 50.
Preferably, the recombinant adenovirus is a recombinant human adenovirus of the 5th serotype (Ad05) or recombinant human adenovirus of the 26th serotype (Ad26).
In certain embodiments of the compositions or methods of the invention, the recombinant adenovirus is formulated into a pharmaceutical composition.

For administering to humans, the invention may employ pharmaceutical compositions comprising the recombinant adenovirus and a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art.
Administration of stable recombinant adenovirus vaccine compositions of the present invention can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as by injection, e.g. intradermal, intramuscular, etc, or subcutaneous or transcutaneous, or mucosal administration, e.g. intranasal, oral, and the like. In one embodiment a composition is administered by intramuscular injection, e.g. into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh. The skilled person knows the various possibilities to administer a composition, e.g. a vaccine in order to induce an immune response to the antigen(s) in the vaccine.
A composition according to the invention may be administered by any conventional route which is used in the field of vaccines, in particular by the systemic, i.e. parenteral route, e.g. by the subcutaneous, intramuscular, intradermal or intravenous route for simultaneous or sequential administration of the immunogenic compositions.
The dosage form of the pharmaceutical composition includes but is not limited to: liquid preparation, lyophilized preparation, emulsion, etc.
In a preferred embodiment, in the above application, the recombinant adenovirus vaccine composition is prepared as an injection, nasal drops or spray.
In a more preferred embodiment, the recombinant adenovirus vaccine composition is prepared as an intramuscular injection.
Preferably, the pharmaceutical compositions comprising the recombinant adenovirus vaccine is a liquid injection.

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Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions, which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
The composition of the present invention can be formulated in a form of a unit dose vial, multiple dose vial, dual chamber syringe, or pre-filled syringe.
Suitable containers for compositions of the invention (or kit components) include vials, syringes (e.g. disposable syringes), nasal sprays,, etc. These containers should be sterile. Where a composition/component is located in a vial, the vial is preferably made of a glass or plastic material. The vial is preferably sterilized before the composition is added to it. To avoid problems with latex-sensitive patients, vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred. The vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses. Preferred vials are made of colorless glass.
A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (e.g. to reconstitute lyophilized material therein), and the contents of the vial can be removed back into the syringe. After removal of the syringe from the vial, a needle can then be attached and the composition can be administered to a patient. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed. A vial may have a cap that permits aseptic removal of its contents, particularly for multi-dose vials.
Where a composition/component is packaged into a syringe, the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. Safety needles are preferred. 1-inch 23-gauge, 1 -inch 25-gauge and s/g-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels on which the lot number, influenza season and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the svrinee preferably has a stopper to prevent

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the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield.
A composition, immunotherapy, or vaccine described herein can be supplied in the form of a kit.
The kits of the present disclosure can further comprise instructions regarding the dosage and/or
administration regimen information.
In one aspect of the invention is provided in a vaccine kit; comprising a vial containing an
immunogenic composition of the invention in aqueous form, i.e. solutions or suspensions;
optionally, in a Iyophilized form, and further comprising a vial containing an adjuvant as described
herein. It is intended that in this aspect of the invention, the adjuvant will be used to reconstitute
the Iyophilized immunogenic composition.
In other embodiments, the compositions do not comprise adjuvants.
Kits
The invention provides kits that include a pharmaceutical composition containing a vaccine,
vector, or optimized viral polypeptide of the invention, and a pharmaceutical ly-acceptable carrier,
in a therapeutically effective amount for preventing or treating a viral infection. The kits include
instructions to allow a clinician (e.g., a physician or nurse) to administer the composition contained
therein.
Preferably, the kits include multiple packages of the single-dose pharmaceutical composition(s)
containing an effective amount of a vaccine, vector, or optimized viral polypeptide of the
invention. Optionally, instruments or devices necessary for administering the pharmaceutical
composition(s) may be included in the kits. For instance, a kit of this invention may provide one
or more pre-filled syringes containing an effective amount of a vaccine, vector, or optimized viral
polypeptide of the invention. Furthermore, the kits may also include additional components such
as instructions or administration schedules for a patient infected with or at risk of being infected
with a virus to use the pharmaceutical composition(s) containing a vaccine, vector, or optimized
viral polypeptide of the invention.

The vaccine formulations were evaluated for their stability at different storage conditions i.e., real time, accelerated, and stress by using following evaluation parameters: Identification, pH, Total protein content, Sterility, Abnormal toxicity, Bacterial endotoxin, Specific activity/potency etc.
The stable recombinant adenovirus vaccine compositions of the present invention improved the thermal stability of recombinant adenovirus vector based novel coronavirus (Covid-19) vaccine for longer periods of time within a useful temperature range, such as from about 2°C to 8°C.
The stable recombinant adenovirus vaccine compositions of the present invention are cost effective in terms of avoiding the storage at -18°C or below, as compared to current undergoing products and will further open new avenue in the treatment of the novel coronavirus (Covid-19). Hence this will promote an ensured and mass immunization and shall popularize owing to its easy storage handling.
It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, methods, and kits of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
The following example gives the composition and process of manufacturing of stable recombinant adenovirus vaccine compositions of the present invention.
Examples:
Example 1: Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2
The screening study for Stabilization of DP were carried out to finalize the stabilizer in the drug product for its storage at 2-8°C. We have screened, Cyclodextrin (HPpCD), PEG 6000, Dextran 40 for their effectiveness. For this Small Scale formulation were prepared and subjected to short term stability studies (37°C/7Days) for Physiochemical and immunological testing.
Composition: The composition of Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 vaccine is presented in table 1 below.

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Table 1

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

I II III IV
1. Monovalent component ofrAd26 1.0 ± 0.5 x 10" Viral particles 1.0±0.5x 10" Viral particles l.0±0.5x 10" Viral particles 1.0±0.5x 101 Viral particles
2. Tris (hydroxymethyl) aminomethane 1.25 mg 1.25 mg 1.25 mg 1.25 mg
3. Sodium chloride 2.25 mg 2.25 mg 2.25 mg 2.25 mg
4. Sucrose 27 mg 27 mg 27 mg 27 mg
5. Magnesium chloride hexahydrate 105 ug 105 ^g 105 ug 105 Mg
6. EDTA disodium salt dihydrate 20 ug 20 ug 20 ug 20 ug
7. Polysorbate-80 250 ug 250 Mg 250 ug 250 ug
8. Ethanol, 95% 2.5 uL 2.5 uL 2.5 uL 2.5 uL
9. Hydroxypropyl Beta-Cyclodextrin X 25 mg X X
10. Dextran 40 X X 25 mg X
11. PEG 6000 X X X 30 mg
12. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, stabilizer (Hydroxypropyl Beta-Cyclodextrin or Dextran 40 or PEG 6000) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2(j. pore. Afterwards the required quantity of Drug substance (Ad26) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were

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adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 2: Stability Studies of formulations of Adenovirus Based (Ad26) SARS CoV-2
The composition of example 1/ table 1 was subjected to stress stability studies conditions i.e. 37°C/7days and were evaluated for following physiochemical and immunological behavior.
Number of virus particles during stability studies
The impact of stabilizer on the number of virus particle at stress stability were measured by spectrophotometer test. The acceptance criteria for number of virus articles is 1 ±0.5 X 10" virus particle (VP)/dose (0.5 ml). The concentration of viral particles in Drug Product is calculated by the formula:
Number of Viral particles = 1.1 x 1012 x (A260-A320) x 0.5
Where 1.1 x 1012 is the number of particles in 1 ml at 260 nm; 0.5 is dose volume: A260 -absorption rate at 260 nm; A320 - absorption rate at 320 nm.
The results of stabilized and un- stabilized formulation (without stabilizer) at 37°C/7days are compared in figure 1.
pH Measurement
The impact of formulation composition on pH at stress stability was tested by Potentiometric measurement. The acceptance criteria for pH was 7.0± 0.75. The comparative results are presented in figure 2.
Example 3: Immunogenicity studies by Intramuscular route during stability studies
The immunological behavior was characterized by injecting 0.2 ml of sample (stabilized as well as control) to animals (Balb/C mice) at different stability time points by intramuscular route. The sera samples were analyzed for Anti S glycoprotein IgG antibody titre by ELISA methodology titer after 14 days of injecting the samples. The acceptance criteria for Anti S glycoprotein antibodies is "mean titre of SARS-CoV-2 S glycoprotein-specific antibodies in the serum of the vaccinated mice should be at least 1:250. The comparative results are presented in below figure 3.

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Example 4: Immunogenicity studies of stable recombinant adenovirus vaccine composition for intranasal administration
The immunogenic potential of this stabilized formulation were administered to Balb/C mice by intranasal route. For this purpose female Balb/C Mice were administered with 25 ul of drug product in each nostril followed by Bleeding on 14 day for sera collection. The Sera samples were analyzed for Anti S glycoprotein IgG antibody titre by ELISA methodology at all stability condition. The comparative results are presented in figure 4.
The HPpCD and Dextran 40 were selected as potential stabilizer as
• No change in the pH was observed upon storage at 7Days/37°C in comparisons to Control product
• The stabilizers helps to protect the fall in number of virus particles (by spectrophotometry) upon storage at 7Days/37°C in comparisons to Control product
• The stabilized vaccine was able to illicit immune response by intramuscular as well as by intranasal route upon storage at 7Days/37°C
Example 5: Stabilized formulation of Adenovirus Based (Ad26) SARS CoV-2
Following the screening study for Stabilization of DP a pilot scale batch using Hydroxypropyl Beta-Cyclodextrin were prepared and different temperature conditions like 2-8°C, 25°C and 37°C and were evaluated for physiochemical and immunological behavior.
Composition: The composition of Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 vaccine is presented in table 2 below.
Table 2

S. No. . Name of the component V Quantity (Per dose of 0.5 ml)
1. Monovalent component of rAd 26 1.0 ± 0.5 x 10" Viral particles
2. Tris (hydroxymethyl) aminomethane 1.21 mg
3. Sodium chloride 2.19mg
4. Sucrose 25 mg
5. Magnesium chloride hexahydrate 102 ug

6. EDTA disodium salt dihydrate 19 ug
7. Polysorbate-80 250 ug
8. Ethanol, 95% 2.52 uL
9. Hydroxypropyl Beta-Cyclodextrin 25 mg
10. Water for Injection q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, stabilizer (Hydroxypropyl Beta-Cyclodextrin or Dextran 40 or PEG 6000) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2u pore. Afterwards the required quantity of Drug substance (Ad26) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 6: Stability Studies of recombinant adenovirus vaccine composition
The composition of example 5/ table 2 was evaluated for following physiochemical and immunological behavior.
Number of virus particles during stability studies
The number of virus particle were tested by spectrophotometer measurement. The acceptance criteria for number of virus articles is 1±0.5 X 10" virus particle (VP)/dose (0.5 ml). The results of stabilized and un- stabilized formulation (without stabilizer) at 2-8°C, 25°C and 37°C are compared in figure 5.

It is evident from the above Figure 5 data that the Control product (un-stabilized) formulation shows a drastic drop in number of virus particle from initial value of 0.96 X 10 '' VP/dose
- to 0.17 X 10 '' VP/dose at 25°C/45 days
- to 0.11 X 10 '' VP/dose at 37°C/14 days.
However the number of viral particle remains unchanged at 2-8 °C after 45 days i.e 0.99 X 10 "
VP/dose
The stabilized formulation of table I shows the viral particles of :-
- 0.85 X 10 " VP/dose at 2-8°C /45 days,
- 0.56 X 10 " VP/dose at 25°C /45 days and
- 0.53 X 10 " VP/dose at 37 °C/14 days,
Thereby meeting the specification (1.0 ± 0.5 X 10" virus particle (VP)/dose (0.5 ml) throughout the studied stability condition.
Example 7: Immunogenicity studies by Intramuscular route during stability studies
The immunological behavior was characterized by injecting samples (stabilized as well as control) to animals (Balb/C mice) at all stability time points by intramuscular route as detailed in example 1. The comparative results are presented in figure 6.
It is evident from above Figure 6 data that the Control product (un-stabilized) formulation shows a drop in Anti S Glycoprotein antibody titre from initial value of 1: 6400
- to 1:800 at 25°C/45 days
- to 1:3200 at 37°C/14 days.
The stabilized formulation of table 2 has maintained higher Anti S Glycoprotein antibody titre
of 1: 6400 at 25°C /45 days and 1:12800 at 37 °C/14 days, thereby meeting the specification
(mean titre of SARS-CoV-2 S glycoprotein-specific antibodies in the serum of the vaccinated
mice should be at least 1:250) throughout the studied stability condition
The Anti-S glycoprotein titre remains high in the formulation at 2-8 °C /45 days at 1: 6400 and
1: 12800 respectively
Example 8: Immunogenicity studies of stable recombinant adenovirus vaccine composition for intranasal administration
The immunogenic potential of this stabilized formulation were administered to Balb/C mice by intranasal route and the Sera samples were analyzed for Anti S glycoprotein IgG antibody titre by

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ELISA methodology at all stability condition. The comparative results are presented below in figure 7.
It is evident from the above data in the Figure 7 that the Control product (un-stabilized) formulation shows a drastic drop in Anti S Glycoprotein antibody titre from initial value of 1: 6400
- to 1:400 at 25°C/45.days
- to 1:100 at 37°C/14 days.
The stabilized formulation of table 2 has maintained moderately high Anti S Glycoprotein antibody titre of 1: 3200 at 25°C /45 days and 1:800 at 37 °C/I4 days, thereby meeting the specification (mean titre of SARS-CoV-2 S glycoprotein-specific antibodies in the serum of the vaccinated mice should be at least 1:250) throughout the studied stability condition. The Anti-S glycoprotein titre remains high in both the formulation at 2-8 °C /45 days at 1: 6400 and 1: 12800 respectively.
Based on the above data it can be concluded that the stabilized formulations of recombinant adenovirus vaccine of this invention showing promising results and to be stable at 2-8 °C with good immunological response when subjected to animal studies.
Example 9; Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2
Composition: The composition of Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 vaccine is presented in table 3 below.
Table 3

S. No. Name of the component Quantity (Per dose of 0.5 ml)

VI VII VIII IX
1. Monovalent component ofrAd26 1.0±0.5x 1011 Viral particles 1.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles 1.0±0.5x 101 Viral particles
2. Tris (hydroxymethyl) aminomethane 1.5 mg 1.5 mg 1.5 mg 1.5 mg
3. Sodium chloride 3mg 3mg 3mg 3mg
4. Sucrose 30 mg 30 mg 30 mg 30 mg
5. Magnesium chloride hexahydrate 200 ug 200 ug 200 ^g 200 ug

6. EDTA disodium salt dihydrate 25 Mg 25 Mg 25 Mg 25 Mg
7. Polysorbate-80 500 ug 500 ug 500 ug 500 Mg
8. Ethanol, 95% ~ 2.5 uL 2.5 uL 2.5 uL 2.5 ML
9. Hydroxy propyl Beta-Cyclodextrin 50 mg X 50 mg 15 mg
10. Dextran 40 X 30 mg 50 mg 10 mg
II. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, Hydroxypropyl Beta-Cyclodextrin and or Dextran) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred forNLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2M pore. Afterwards the required quantity of Drug substance (Ad26) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10'' Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 10: Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 Composition: The composition of Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 vaccine is presented in table 4 below.
Table 4

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

X XI XII XIII

1. Monovalent component ofrAd26 1.0 ± 0.5 x 10" Viral particles 1.0 ± 0.5 x 10" Viral particles 1.0 ± 0.5 x 10" Viral particles l.0±0.5x 10" Viral particles
2. . Tris (hydroxymethyl) aminomethane 1.5 mg 1.5 mg 1.5 mg 1.5 mg
Sodium chloride 3 mg 3 mg 3 mg 3mg
4. Sucrose 50 mg 30 mg 30 mg 30 mg
5. Magnesium chloride hexahydrate 200 ug 200 ^ 200 ug 200 ug
6. EDTA disodium salt dihydrate 25 ug 25 ug 25 ug 50 ug
7. Polysorbate-80 250 ug 250 ug 250 ug 100 ug
8. Ethanol, 95% 2.5 uL 2.5 uL 2.5 uL 2.5 uL
9. Hydroxypropyl Beta-Cyclodextrin 50 mg X 50 mg 15 mg
10. Trehalose X 25 mg 50 mg 10 mg
11. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, Hydroxypropyl Beta-Cyclodextrin and or trehalose) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred forNLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2u pore. Afterwards the required quantity of Drug substance (Ad26) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.

f

Example 11: Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2
Composition: The composition of Stabilized formulations of Adenovirus Based (Ad26) SARS CoV-2 vaccine is presented in table 5 below.
Table 5

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

XIV XV XVI XVII
1. Monovalent component ofrAd26 1.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles l.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles
2. Sucrose 25 mg 27 mg 27 mg 27 mg
3. Magnesium chloride hexahydrate 102 ug 105 Mg 105 Mg 105 Mg
4. EDTA disodium salt di hydrate 19 Mg 20 Mg 20 Mg 20 Mg
5. Polysorbate-80 250 ^g 250 Mg 250 Mg 250 Mg
6. Ethanol, 95% 2.52 uL 2.5 ML 2.5 ML 2.5 ML
7. Hydroxypropyl Beta-Cyclodextrin 25 mg 25 mg X X
8. Dextran 40 X X 25 mg X
9. Trehalose X X X 30 mg
10. Phosphate Buffer Saline q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection, Phosphate buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, stabilizer (Hydroxypropyl Beta-Cyclodextrin and or Dextran 40 and or Trehalose) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2M Pore-Afterwards the required quantity of Drug substance (Ad26) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was

X-
carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of filial bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 12: Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 Composition: The composition of Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 vaccine is presented in table 6 below.
Table 6

S. No. Name of the component Quantity (Per dose of 0.5 ml)

I II III IV
1. Monovalent component of rAd 5 1.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles ,1.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles
2. Tris (hydroxymethyl) aminomethane 1.21 mg 1.25 mg 1.25 mg 1.25g
3. Sodium chloride 2.19mg 2.25 mg 2.25 mg 2.25g
4. Sucrose 25 mg 27 mg 27 mg 27 g
5. Magnesium chloride hexahydrate 102 ug 105 ug 105 ug 105
6. EDTA disodium salt dihydrate 19 ng 20 ug 20 ug 20 g
7. Polysorbate-80 250 ug 250 ug 250 ug 250
8. Ethanol, 95% 2.52 uL 2.5 uL 2.5 uL 2.5 uL
9. Hydroxypropyl Beta-Cyclodextrin 25'mg 25 mg X X
10. Dextran 40 X X 25 g X
11. PEG 6000 X X X 30 g
12. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred

M

to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, stabilizer (Hydroxypropyl Beta-Cyclodextrin or Dextran 40 or PEG 6000) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2u pore. Afterwards the required quantity of Drug substance (Ad5) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 13: Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 Composition: The composition of Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 vaccine is presented in table 7 below.
Table 7

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

V VI VII VIII
1. Monovalent component ofrAd5 1.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles 1.0 ± 0.5 x 10" Viral particles 1.0±0.5x 10" Viral particles
2. Tris (hydroxymethyl) aminomethane 1.5 mg 1.5 mg 1.5 mg 1.5 mg
3. Sodium chloride 3mg 3mg 3mg 2 mg
4. Sucrose 30 mg 30 mg 30 mg 30 mg
5. Magnesium chloride hexahydrate 200 ug 200 ^ 200 ^ 200 ug
6. EDTA disodium salt dihydrate 25 ug 25 ug 25 ug 25 ug
7. Polysorbate-80 500 ug 500 ug 500 ug 500 ug
8. Ethanol, 95% 2.5 uL 2.5 uL 2.5 uL 2.5 uL

9. Hydroxypropyl Beta-Cyclodextrin 50 mg X 50 mg 15 mg
10. Dextran 40 X 30 mg 50 mg lOmg
II. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, Hydroxypropyl Beta-Cyclodextrin and or Dextran) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2\i pore. Afterwards the required quantity of Drug substance (Ad5) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 14: Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 Composition: The composition of Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 vaccine is presented in table 8 below.
Table 8

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

IX X XI XII
1. Monovalent component ofrAd5 1.0±0.5xl0" Viral particles 1.0±0.5x 10M Viral particles 1.0 ±0.5x10" Viral particles 1.0±0.5xl0n Viral particles
2. Tris (hydroxymethyl) aminomethane 1.5 mg 1.5 mg 1.5 mg 1.5 mg

A

J. Sodium chloride 3 mg 3 mg 3mg 2 mg
4. Sucrose 50 mg 30 mg 30 mg 30 mg
5. Magnesium chloride hexahydrate 200 ug 200 ug 200 ^g 200 Mg
6. EDTA disodium salt di hydrate 25 ug 25 ug 25 Mg 50 Mg
7. Polysorbate-80 250 Mg 250 ug 250 ^ 100 Mg
8. Ethanol, 95% 2.5 uL 2.5 uL 2.5 uL 2.5 ML
9. Hydroxypropyl Beta-Gyclodextrin 50 mg X 50 mg 15mg
10. Trehalose X 25 mg 50 mg 10 mg
11. Water for Injection q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, Hydroxypropyl Beta-Cyclodextrin and or trehalose) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred forNLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2M pore. Afterwards the required quantity of Drug substance (Ad5) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.
The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.
Example 15: Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 Composition: The composition of Stabilized formulations of Adenovirus Based (Ad5) SARS CoV-2 vaccine is presented in table 9 below.

/..

Table 9

s.
No. Name of the component Quantity (Per dose of 0.5 ml)

XIII XIV XV XVI
1. Monovalent component ofrAd5 1.0 ± 0.5 x 10" Viral particles l.0±0.5x 10" Viral particles 1.0±0.5x 10" Viral particles l.0±0.5x 10" Viral particles
2. Sucrose 25 mg 27 mg 27 mg 27 mg
3. Magnesium chloride hexahydrate 102 ug 105 ug 105 ug 105 ug
4. EDTA disodium salt dihydrate 19 ng 20 ug 20 ug 20 ^
5, Polysorbate-80 250 ug 250 ^ 250 ug 250 ug
6. Ethanol, 95% 2.52 uL 2.5 uL 2.5 uL 2.5 uL
7. Hydroxypropyl Beta-Cyclodextrin 25 mg 25 mg X X
8. Dextran 40 X X 25 mg X
9. Trehalose X X X 30 mg
10. Phosphate Buffer Saline q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml q.s. to 0.5ml
Formulation and filling of bulk vaccine
The required quantity of drug substance and other excipients was calculated based on the batch size and the label claim. A calculated quantity water for injection, Phosphate buffer solution was transferred to formulation vessel to which the required quantity of excipients (Sodium chloride, Sucrose, Magnesium chloride hexahydrate, EDTA disodium salt dihyrate, Polysorbate-80, Ethanol, 95%, stabilizer (Hydroxypropyl Beta-Cyclodextrin and or Dextran 40 and or Trehalose) was added. The pH of bulk was adjusted to 7.0±0.5 and it was stirred for NLT4 hrs at 5°±3°C with stirring at 150 rpm. The buffer was sterilized by filtration using PES membrane filter of 0.2[i pore. Afterwards the required quantity of Drug substance (Ad5) was added under constant stirring. The volume of resultant blend was made up to required batch size with buffer Saline. The dilution was carried out based on OD measurement at A260, 280 and 320 nm and the number of virus particles were adjusted to 1.0 ± 0.5 x 10" Viral particles. The pH of final bulk was checked and adjusted to 7.0± 0.75. Post pH adjustment the final bulk vaccine was stored at 2-8°C for pending filling.

^

The final bulk vaccine was filled into USP type I, tubular glass vials, stoppered with bromobutyl rubber stopper and sealed with flip off aluminum seal and was Stored at 2-8°C.

Claims:
1. A stable composition of a recombinant adenoviral vector comprising
(i) a buffered solution (ii) a chelating agent (iii) a divalent cation (iv) a stabilizer/ stability modifiers, and
(v) one or more additional excipients that act as inhibitors of free radical oxidation which enhance thermal stability of the adenovirus.
2. The stable composition of claim 1, wherein the adenoviral vector is an recombinant human adenovirus of the 26th serotype (Ad26) or recombinant human adenovirus of the 5 th serotype (Ad5).
3. The stable composition of claim 1, wherein the buffer is selected from a group comprising of Phosphate buffers, Tris buffers, Earle's salt solution, TE (Tris/EDTA), TEN (Tris/NaCl/EDTA) and Tris (hydroxymethyl) aminomethane.
4. The stable composition of claim 3, wherein the buffer concentration is ranging between about 1 mg and 10 mg.
5. The stable composition of claim 1, wherein the chelating agent is selected from a group comprising of Alpha lipoic acid (ALA) Ethylenediamine tetraacetic acid (EDTA) Diethylene triamine pentaacetic acid (DTPA), Pluronic and protamine histidine and L-arginine.
6. The stable composition of claim 5, wherein the chelating agent concentration is ranging between about 1 ug and 500 ug.
7. The stable composition of claim 1, wherein the divalent cation is selected from a group comprising of Magnesium chloride, Barium chloride, Zinc chloride and Calcium chloride.
8. The stable composition of claim 7, wherein the concentration of divalent cation is ranging between about 1 jag and 500 ug.
9. The stable composition of claim 1, wherein the stabilizer is selected from a group comprising of Trehalose, Polyethylene glycols (PEGs), Dextrans, Cyclodextrin, Hydroxypropyl-P-cyclodextrin (HBCD) and Tertiary amine P-cyclodextrin (TMBCD).
10. The stable composition of claim 9, wherein the concentration of stabilizer is ranging between about 1 mg and 100 mg.

11. The stable composition of claim 1, wherein composition is stable for at least 12 months when stored at 2-8° C.
12. The stable composition of claim 1 for use in active immunization for the preventing and treating Coronavirus infection-SARS-COV-2/ Coronavirus disease (COVID-19).