DESC:FIELD OF THE INVENTION
The present invention relates to the field of vaccines. The invention particularly relates to providing an efficient and effective combination of vaccines that can prevent multiple diseases. In particular, the invention provides a trivalent live attenuated combination vaccine composition against Goat Pox, Sheep Pox and Peste-des-petits-ruminants (PPR) infection and diseases with an improved thermostability.

BACKGROUND OF THE INVENTION
The animal husbandry sector in India is rapidly growing and fast-moving towards attaining self-sufficiency in the production of livestock products. Animal husbandry is a major contributor to the Indian economy with a share of 28-32% in agricultural GDP and 4 to 6% of the national GDP. It also provides employment to 8-10% of the population in India. India has a huge livestock population of about 71.5 million sheep and about 140 million goats. The rearing of sheep and goats is generally done by poor population living in developing countries including India. Therefore, controlling animal death caused by the infectious diseases is considered as an important step for alleviating poverty in areas where such infectious diseases are endemic and cause heavy losses due to disease and deaths. For example, PPR alone caused a loss of about USD 160 million in 2014-15 globally.

Peste-des-petits-ruminants (PPR) is a highly contagious, acute and transboundary viral disease of goats and sheeps. It is caused by the genus Morbillivirus belonging to the family Paramyxoviridae. The clinical symptoms of the disease are conjunctivitis, high fever, occulonasal discharge, necrotizing and erosive stomatitis, enteritis and bronchopneumonia. Owing to a very high morbidity (50-90%) and mortality (60-85%) rates, PPR has become a cause of concern for goat and sheep population.

Sheep Pox virus (SPV) and Goat Pox virus (GPV) belong to a member of the genus Capripox virus of the family Poxviridae. The clinical symptoms of Goat Pox and Sheep Pox are almost similar. The disease affects animals of all age, but younger population is more vulnerable to these viruses. The common symptoms are Occulonasal discharge, sialorrhea, skin lesions, allurated papules on mucus membrane and hyperpyrexia. SPV and GPV primarily spread through contact, sexual transmission, through bioaerosol and through the fomites of the infected animal. The Sheep Pox and Goat Pox diseases are notifiable to Office Internationale des Epizooties (OIE) due to their economic impact. In Indian subcontinent, Sheep Pox and Goat Pox diseases are found at an endemic level and pose serious economic losses to small ruminant husbandry as mortality in young animals can exceed 50%.

Goat capripox virus (Goat Pox virus) that causes Goat Pox has clinical symptoms those are similar to Sheep Pox. Goat Pox is mainly manifested with the occurrence of blebs on the skin and mucous membrane and also forms acne and swells. Further progression of disease causes suppuration, generalized vaccinia and systemic reaction. Clinically swollen acnes are seen without maturation process, i.e. so-called “stone acne”. Severe infection results in internal organs lesions and death. This disease is prevalent in Indian subcontinent, Mediterranean region, Africa and some other countries. Cases have also been recorded in Europe and Australia. Due to the differences in infecting strain and hosts, variations in the symptoms and death rate due to the disease is observed. The death rate reported for Turkey is 3%~ 40%, while the death rate reported for Morocco is 5%~70%.

Improved technologies are being developed to increase efficacy of veterinary vaccines that can reduce vaccination cost and eradicate diseases. Though it is challenging to develop an effective and stable vaccine for different animal diseases, it is even more challenging to immunize the animal against multiple diseases due to economical, logistics and other issues. Therefore, there is need of efficient and effective combination of live attenuated vaccine in order to provide a desirable option for effective and long-lasting immunization against multiple diseases. The approach of using combination live attenuated vaccines is not only cost effective but would also provide solution to logistics difficulties. There are specific vaccines available for Goat Pox, Sheep Pox and PPR and often the animals are partially immunized against one or the other diseases, but not against all these diseases. Thus, such animals, although protected against one of the diseases, are vulnerable to the remaining diseases.

Therefore, availability of a single shot live attenuated vaccine combining GPV, SPV and PPR virus would be particularly beneficial in getting the animal completely immunized against a group of diseases. Further, in general, veterinary vaccines suffer serious deterioration in vaccination campaigns, especially in far reaching areas due to the difficulties in maintaining cold chain during the storage and transport of vaccine, and inevitably resulting in loss of vaccine potency particularly in tropical and subtropical environments. Thus, to ensure viability of the vaccine in tropical and subtropical environments, thermostable vaccines are more suitable option.

To address the problem known in the art, inventors of present invention have developed a one-shot cost-effective live attenuated combination vaccine with improved thermostability. The vaccine will provide a cost-effective solution for immunization and protection of small ruminants against a group of diseases (Sheep Pox, Goat Pox, and PPR).

Combination vaccines
Vaccine that protects a subject against various diseases in a single shot are highly desirable, since it reduces the administration and production costs. Combination vaccines also result in an improved compliance of the vaccination schedule and are generally better accepted. However, the development of multivalent vaccines is hindered by the well documented phenomenon of the antigenic competition. Antigenic competition refers to the observation that administering multiple antigens together often results in a diminished response to certain antigens vis-à-vis the immune response to these antigens when administered separately.

Earlier, the research has been focused on the development of the bivalent pox vaccines for the small ruminants like sheep and goats and protecting them from a combination of diseases like sheep pox and PPR, sheep pox and goat pox or goat pox and PPR.

Chinese Patent No. CN104800842A discloses a bivalent vaccine comprising goat pox and sheep pox antigens. This reference relates to a live attenuated combination vaccine in which the antigens are adsorbed on to gelatine.

Thus, it is desirable to add other antigens to such a combination vaccine that would give protection against diseases caused by GPV, SPV, PPR virus. It is also desirable to have antigens providing protection against other diseases added to the above said combination vaccines.

Currently, there are no known live-attenuated vaccines with appropriate formulations of trivalent antigens consisting of GPV, SPV and PPR virus in appropriate immunogenic forms for achieving desired levels of efficacy and immunogenicity in the subject animal population in a single shot. Therefore, a multi component vaccine that provides a continued protection against goat pox, sheep pox and PPR and is in a form that provides ease of administration and cost-effectiveness is the need of the hour. It is also desirable to provide for a stable and efficacious trivalent live attenuated vaccine against these three diseases caused by GPV, SPV and PPR virus. For such vaccines to be effective, the criterion of sero protection for each of the antigens of the vaccine needs to be ensured. For this, there is a need to overcome the hurdles and the challenges posed by antigenic competition and interference.

The present invention overcomes the limitations of prior arts and solves the related problems by providing a fully lyophilized combination live attenuated vaccine formulation protecting against a plurality of diseases.

SUMMARY OF THE INVENTION
The present invention relates to the field of vaccines. The invention particularly relates to providing an efficient and effective combination of vaccines that can prevent multiple diseases. In particular, the invention provides a trivalent live attenuated vaccine composition against Goat Pox, Sheep Pox and PPR having improved thermostability. One aspect of the invention is related to a live attenuated lyophilized trivalent combination vaccine for Goat Pox, Sheep Pox and PPR. In yet another aspect, the present invention provides live attenuated lyophilized trivalent combination vaccine with improved thermostability for protection against Sheep Pox Virus, Goat Pox Virus and PPR Virus.

In an aspect of the present invention, the present invention provides a vaccine composition with improved thermostability, comprising live attenuated Sheep Pox virus, live attenuated Goat Pox virus, live attenuated Peste-des-petits-ruminants (PPR) virus, and excipients selected from stabilisers, preservatives, tonicity modifying agents, and pH modifiers and buffers.

In another aspect, the present invention provides a vaccine composition, wherein each of the live attenuated Sheep Pox virus, Goat Pox virus, and PPR virus are present in an amount of at least 3 log10 CCID50 per ml of the vaccine composition.

In yet another aspect, the present invention provides a vaccine composition, wherein the excipients comprise 200-218 mM Sucrose, 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), 5.2-6.0 mM Glutamate, 2.5 % Lactalbumin Hydrolysate (LAH), 5% Sucrose, 5% Trehalose dehydrate and 0.5% Gelatin prepared in Dulbecco's phosphate-buffered saline (DPBS).

In another aspect, the present invention provides a vaccine composition, wherein the pH of the vaccine composition is in a range of 7.0 to 7.4.

In another aspect, a process for preparation of vaccine composition of the present invention is provided. The process for preparation of the vaccine composition comprises: (a) Retrieving live attenuated Sheep Pox virus, Goat Pox virus and PPR virus from deep freezer and thawing at 25°C ± 1°C, (b) Optionally pooling the processed Sheep Pox virus, Goat Pox virus, and PPR virus aseptically in a biosafety cabinet, (c) Separately mixing excipients comprising Sucrose, 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), 5.2-6.0 mM Glutamate, 2.5% LAH, 5% Sucrose, 5% Trehalose dehydrate and 0.5% Gelatin prepared in DPBS to obtain a formulation mixture with the pH in a range of 7.0-7.4, (d) Adding the virus in an amount of not less than 3 log10 CCID50/ml each, to the formulation mixture to obtain the vaccine composition, (e) Mixing the processed Sheep Pox virus, Goat Pox virus, and PPR virus with the formulation mixture and lyophilizing it to a temperature ranging upto (-95°C) to obtain lyophilized mixture, (f) Optionally transferring the vaccine composition into glass vials for storing at about 4°C.
In another aspect, the present invention provides the vaccine composition as and when used for immunization against Sheep Pox, Goat Pox and PPR.

DETAILED DESCRIPTION OF THE INVENTION
For the purpose of promoting an understanding of the principles of the invention, reference made to the embodiments in the specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated composition, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The composition, methods, and examples provided herein are illustrative only and not intended to be limiting.

The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.

The terminology and structure employed herein is for describing, teaching, and illuminating some embodiments and their specific features and elements, and does not limit, restrict, or reduce the spirit and scope of the invention.

While the invention is susceptible to variations and modifications other than those specifically described herein by specific embodiments and examples, it is to be understood that the present disclosure includes all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims.

The figures and protocols have been provided merely for representing the specific details that are pertinent to understanding the embodiments of the present invention and not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the disclosure.

The present invention is directed towards the development of a live attenuated lyophilised vaccine with improved thermostability and comprises vaccine components that are suitable for prevention, amelioration and treatment of multiple diseases and confers sero-protection for each of the said vaccine components.

The present invention provides a multivalent vaccine which confers protection against multiple diseases and infections in a safe and efficacious manner. The vaccine of the present invention provides immunogenicity to the most common viral diseases and infections of small ruminants without any interference in the activities of any of the antigens that are present in the vaccine. Since a single shot would confer immunogenicity against multiple diseases and infections, it will make the vaccine more compliant, cost effective, user friendly, and would eventually improve the success rate of immunization. Vaccine of the present invention would be beneficial in the sense that it will reduce the multiple immunizations to be given separately for each disease. This will prevent multiple rounds of travel required by the farming community to bring their animals repeatedly for vaccination from the remote areas where they live. Thus, the present invention provides a vaccine that is more acceptable and commercially competitive.

Non-antigenic components of the vaccine
In addition to the antigenic components, the vaccine may comprise a number of non-antigenic components that are pharmaceutically or veterinary acceptable excipients. These include but are not restricted to pH modifiers, buffers, Stabilisers, preservatives, carriers and tonicity modifying agents.

Stabilisers
Studies have shown that freeze drying protectant have good heat resistance, and stabilizers can protect the degradation of antigenic component, formula components. Most preferably, the formula of described heat-resisting lyophilized protecting agent is: Trehalose 50g/L, defatted milk powder 20g/L, Polyvinylpyrrolidone 20g/L, Gelatine 10g/L, Arginine 1.5g/L, vitamin C 2.5g/L, and residual components is ultra-pure water.

Preservatives
The vaccines are prone to contamination by microbes. Thus, to avoid the potentially life-threatening contamination with harmful microbes, which may be introduced during the event of accidental contamination, a preservative may be included in the vaccine composition while formulating it. In one of the embodiments of the invention sucrose is a preferred preservative in a combined trivalent GP, SP and PPR vaccines.

Tonicity modifying agents
The tonicity of the vaccine composition can be controlled by including a tonicity modifying agent in the vaccine formulation. These agents include but are not limited to salts (examples- NaCl, KC1, CaC12 etc.), sugars (examples- dextrose, mannitol, lactose), amino acids (examples- Arginine, Glycine, Histidine) and Polyols (examples- Sucrose, Glycerol, Sorbitol). In a preferred embodiment, salt such as sodium salt is used in the formulation of the vaccine. Sodium chloride (NaCl) is most preferably included in the vaccine composition of the invention.

pH modifiers and/or buffers
Various pH modifiers known to person skilled in the art may be used to adjust the pH of the vaccine composition as desired, such as sodium hydroxide or hydrochloric acid. Various buffers such as sodium phosphate, potassium phosphate and citrate buffers may be used in the formulation of the vaccine.

Vaccine composition of the invention
The vaccine composition of the present invention is rendered immunogenic by virtue of specific amount of each antigen contained therein. Each of the antigen in the vaccine of the invention is preferably in amounts so that the combination vaccine when administered to a subject, elicits immune response in the subject, against the antigens of the composition. The combination vaccine of the present invention comprises Goat Pox (GP) Antigen, Sheep Pox (SP) Antigen and Peste-des-Petits-Ruminants (PPR) Antigen.

In another embodiment of the invention the trivalent live vaccine comprises of live GP, SP, PPR antigen each present in an amount of about not less than 3 log10 CCID50 per 1.0 ml to form a trivalent vaccine which would be stable, immunogenic and in the form of a lyophilized formulation. Such a vaccine would be a stable vaccine capable of eliciting protective immune response against all the antigens of the composition, when administered to a subject.

The invention further relates to a method of inducing immunological response against any of the antigen selected from the group of GP, SP and PPR comprising administering immunologically active amount of the vaccine of the present invention.

According to yet another aspect of the invention, the preferred amount of excipients is: Sucrose 218 mM, Potassium dihydrogen phosphate (KH2PO4) 3.8 mM, Di-potassium hydrogen phosphate (K2HPO4) 7.2 mM, Glutamate 6.0 mM.

Process for manufacturing of the vaccine of the invention
In one of the aspects, the invention relates to a process for manufacturing of the vaccine of the invention. The immunogenicity, the stability and the maintenance of the right form of the antigens in the immunogenic composition is determined by the way the composition has been formulated. This may include the sequence of addition of the antigens, the use of the specific stabiliser for certain antigens, the use of various parameters including agitation, temperature and/or pH.

One of the embodiments of the invention relates to the process of manufacturing a fully lyophilized combination vaccine comprising goat pox (GP), sheep pox (SP) and Pestes des ruminant (PPR) antigens which comprises the steps of:
a) preparing a component I comprising a mixture of Live antigens of i) Goat Pox (GP), ii) Sheep Pox (SP) and iii) Pestes des ruminant antigens,
b) Optionally, preparing a component II comprising separately each of the individual antigen adsorbed on to LAH, 5% Sucrose, 5% Trehalose dehydrate and 0.5% Gelatin,
c) Optionally, mixing the component II of each specific antigen to component I to obtain a mixture.

According to one of the preferred embodiments of the invention, the GP, SP and PPR antigens are adsorbed on to LAH, 4-6% Sucrose, 3-5% Trehalose dehydrate and 0.5-1.0% Gelatin.

Another preferred embodiment of the invention relates to the preparation of the component-I, which comprises the following steps:
a) Transferring LAH, 5% Sucrose, 5% Trehalose dehydrate and 0.5% Gelatin into a vessel,
b) Transferring Live Goat Pox, Sheep Pox and PPR Virus antigen into the above vessel,
c) Transferring excipients, Potassium dihydrogen phosphate (KH2PO4), Di-potassium hydrogen phosphate (K2HPO4), Glutamate, MEM Medium in the said vessel,
d) Transferring sodium chloride solution to the above said vessel under constant stirring,
e) Checking the pH and adjusting it in the range of 7.0-7.4.

The foregoing description teaches generally how to make a live attenuated trivalent lyophilized combination vaccine for Goat Pox, Sheep Pox and PPR with improved thermostability. The following examples are given to teach more particularly how this invention may be carried out. However, the inventions should not be construed as being limited to the specific examples.

EXAMPLES
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1: Preparation of a live attenuated trivalent lyophilized vaccine
(A) Preparation of vaccine antigens.
Strains of Goat Pox, Sheep Pox and PPR virus were separately grown following the vaccine production protocol. Well adapted Vero cell were seeded into cell factories at a predefined concentration. Once desired confluence in the monolayer was achieved, the flasks were infected with respective live viruses and were incubated at a temperature of 33-37°C. Following the protocol, media changes were made, and cells were observed for cytopathic effect (CPE). Once 80-90% CPE were achieved, viruses were harvested from the respective experiments to maintain the uniformity in the virus titre. Virus harvests, were pooled and aliquoted in different bottles and preserved at -80°C. More specifically preparation of Goat Pox, Sheep Pox and PPR Virus antigens are detailed below:
(i) Propagation of Vero cells: The Vero cells were routinely grown in a T-175 tissue culture flask in MEM with 10% FBS medium till 90-100% confluency. At this stage, cells were washed once with Dulbecco's phosphate-buffered saline (DPBS) and trypsinized using TrypLE select. Trypsinized cells were diluted in the MEM with 10% FBS medium and pelleted down by centrifugation. A cell count was done, and cells were seeded either in a 6-well plate or Tissue culture flask in MEM with 10% FBS medium.

(ii) Propagation of Goat Pox virus:
Stepwise 1 million to 10 million Vero cells were grown to seed the flasks of different capacities starting with T-25, T-75, and finally T-175 flask seeded with 10 million Vero cells in MEM with 10% FBS medium. Experiments were also carried out to optimize the multiplicity of infection (MoI) at each stage of scale up i.e., T-25, T-75, and T-175. Of the different MoI tried, clearly MoI 0.05 was found optimal for infectivity and was used in further repeat T-175 Flask.

After approximately 24 hours, cells were infected with Goat Pox virus. For infection, the growth medium was decanted off the cells. 5 ml of virus inoculum was used to infect the cells. Virus was adsorbed onto the cells by incubation at 37°C and 5% CO2 for one hour. During this one-hour, intermittent mixing was given at 15-minute interval to avoid drying of the monolayer. After incubation for 1 hour, cells were fed with another 30 ml of MEM with 2% FBS medium. The infected cells were incubated at 37°C and 5% CO2 for next 2 days to allow virus propagation. Complete media on Day 2 post infection was replaced using serum-free media (MEM without FBS) and there after incubation was continued in a serum-free media at 37°C, and 5% CO2. Viral supernatant was harvested on Day 6 and stabilized with addition of 1x stabilization buffer SPG (218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM KH2PO4, 7.2 mM K2HPO4, pH 7.2) and stored at minus 70°C or below. Experiments were repeated to see the consistency in results obtained. The process used in T-175 flask has yielded virus potency log10 5.4-5.8 CCID50/ml for the pooled harvest.

(iii) Propagation of Sheep Pox virus:
Stepwise 1 million to 10 million Vero cells were grown to seed the flasks of different capacities starting with T-25, T-75, and finally T-175 flask were seeded with 10 million Vero cells in MEM with 10% FBS medium. Experiments were also carried out to optimize the multiplicity of infection (MoI) at each stage of scale up i.e., T-25, T-75, and T-175. Of the different MoI tried, MoI 0.05 was found optimal for infectivity and was used in further repeat T-175 Flask.

After approximately 24 hours, cells were infected with Sheep Pox virus. For infection, the growth medium was decanted off the cells. 5 ml of virus inoculum was used to infect the cells. Virus was adsorbed onto the cells by incubation at 37°C and 5% CO2 for one hour. During this one-hour, intermittent mixing was given at 15-minute interval to avoid drying of the monolayer. After incubation for 1 hour, cells were fed with another 30 ml of MEM with 2% FBS medium. The infected cells were incubated at 37°C and 5% CO2 for next 2 days to allow virus propagation. Complete media on Day 2 post infection is replaced using serum-free media (MEM without FBS) and there after incubation was continued in a serum-free media at 37°C, and 5% CO2. Viral supernatant was harvested on Day 6 and stabilized with addition of 1x SPG and stored at minus 70°C or below. Experiments were repeated to see the consistency in results obtained. The process used in T-175 flask has yielded virus potency log10 5.0 -5.6 CCID50/ml for the pooled harvest.

(iv) Propagation of PPR virus:
Stepwise 1 million to 10 million Vero cells were grown to seed the flasks of different capacities starting with T-25, T-75, and finally T-175 flask seeded with 10 million Vero cells in MEM with 10% FBS medium. Experiments were also carried out to optimize the multiplicity of infection (MoI) at each stage of scale up i.e., T-25, T-75, and T-175. Of the different MoI tried, MoI 0.05 was found optimal for infectivity and was used in further repeat T-175 Flask.

After approximately 24 hours. Cells were infected with PPR virus at 0.05 MoI. For infection, the growth medium was decanted off the cells. 5 ml of virus inoculum was used to infect the cells. Virus was adsorbed onto the cells by incubation at 37°C and 5% CO2 for one hour. During this one-hour, intermittent mixing was given at 15-minute interval to avoid drying of the monolayer. After incubation for 1 hour, cells were fed with another 30 ml of MEM with 2% FBS medium. The infected cells were incubated at 37°C and 5% CO2 for next 2 days to allow virus propagation. Complete media on Day 2 post infection is replaced using serum-free media (MEM without FBS) and thereafter incubation was continued in a serum-free media at 37°C, and 5% CO2. Viral supernatant was harvested on Day 5 and stabilized with addition of 1 x SPG and stored at minus 70°C or below. Experiments were repeated to see the consistency in results obtained. The process used in T-175 flask has yielded virus potency log10 5.7-6.0 CCID50/ml for the pooled harvest.

(B) Production Steps for Drug Substance (antigen) production
(i) Revival and propagation of Vero cells:
• Revive the Vero cells in complete MEM media (supplemented with 10% FBS).
• Remove the spent media from the flasks on second day and add fresh complete MEM media.
• Propagate Vero cells further to achieve sufficient cells for seeding in T-75 cm2 tissue culture flask.
• Incubate the Tissue culture flask at 37°C for 1-2 days.

(ii) Infection, Adsorption, Media top-up:
• Infect the Vero cells at a MoI (multiplicity of infection) of 0.05.
• Vero cells can be trypsinized from one of the control tissue culture flasks to determine the cell count, in order to calculate the required pfu of PPR virus or Sheep pox virus to set up infection at a MoI between 0.05 as follows:
• CCID50 or pfu required = Number of cells to be infected × required MoI
• Volume of virus required (mL)=CCID50 or pfu required/ titre of virus stock (in CCID50/mL)
• Dilute the calculated virus inoculum in the MEM media with 2% FBS and add into tissue culture flask after decanting the spent media. Similarly, mock-infection can be set up in the control flask by adding only complete MEM medium + 2% FBS.
• Incubate both infected and control flask for 60 minutes to allow adsorption of virus.
• After 60 minutes, add MEM media (with 2% FBS) to both infected and control flask.
• Incubate both infected and control flask at 37°C ± 1°C with 5% CO2.

(iii) Media change on Day 2 post-infection: On day 2 post infection, replace 100% of the spent media with MEM media (serum free) in the flask and incubate it at 37°C ± 1°C with 5% CO2.
(iv) Harvesting of virus from infected flask: 100% harvesting of culture supernatant on Day 5 or Day 6, addition of stabilizers (1x SPG) to the harvests and storage at minus 70°C or below.

(C) Preparation of formulated vaccine
This example gives the composition and the process of manufacturing of a combined Sheep Pox, Goat Pox and PPR Vaccine as per one of the aspects of the invention.

1] Composition of the combined Sheep Pox, Goat Pox and PPR vaccine as per the invention is as under:

Each 1 ml vaccine comprises the following:
Component I: Vaccine Antigens
Sheep Pox Antigen: Not less than 3 log10 CCID50
Goat Pox Antigen: Not less than 3 log10 CCID50
PPR Antigen: Not less than 3 log10 CCID50

Component II: Other Ingredients: -
The excipients, 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), 5.2-6.0 mM Glutamate, MEM Medium and 2.5-3.5 % Lactalbumin Hydrolysate (LAH), 4-6% Sucrose, 3-5% Trehalose dehydrate and 0.5% Gelatin were prepared in DPBS Solution. The pH of the mixture was checked and adjusted to fall in the range of 7.0-7.4.

2] The process of manufacturing of a combined live attenuated lyophilised Goat Pox, Sheep Pox and PPR Vaccine as per the invention is as under:

Individual antigen of the Goat Pox, Sheep Pox and PPR virus (component I), stored at -70°C were retrieved from the deep freezer and thawed at 25°C ± 1°C in a water bath. The bottles were removed from the water bath and were wiped with disinfectant. The processed Goat Pox antigen, Sheep Pox antigen and PPR antigen were then pooled aseptically in a biosafety cabinet.

Excipients, 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), 5.2-6.0 mM Glutamate, MEM Medium and 2.5-3.5 % Lactalbumin Hydrolysate (LAH), 4-6% Sucrose, 3-5% Trehalose dehydrate and 0.5-1.0 % Gelatin were added in DPBS Solution. The pH of the mixture was checked and adjusted to fall in the range of 7.0-7.4.

To prepare the combined formulation each individually formulated antigen not less than 103 CCID50 was transferred into a vessel then gently mixed (to form combined Vaccine) maintaining a pH of 7.2-7.4 and transferred into glass vials, and then stoppering was done.

Lyophilization was carried out using Scan Vac freeze-drier. The vaccine vials of virus were first chilled to -95°C for 2 hour and drying was conducted at that self-temperature and pressure for 16 hr. Stoppering was done of vaccine vial and stored at 2-8°C.

Alternatively, each of live and purified antigens of GPV, SPV and PPR virus, not less than103 CCID50 were separately added in a vessel. The mixture of the purified antigen was then suspended in Dulbecco’s Phosphate Buffered Saline (DPBS) solution and further formulated by adding the excipients 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), 5.2-6.0 mM Glutamate, MEM Medium and 2.5-3.5 % LAH, 4-6 % Sucrose, 3-5% trehalose dehydrate and 0.5-1.0% Gelatin prepared in DPBS Solution. The pH of the mixture was checked and adjusted to fall in the range of 7.0-7.4.

EXAMPLE 2: This example gives a brief on the Stability or the Potency of the vaccine of the present invention
The stability studies were performed to evaluate the shelf life of Sheep Pox, Goat Pox and PPR Drug Product (formulated antigen) at real-time (5ºC ± 3ºC), accelerated (25ºC ± 2ºC), stress (37ºC ± 2ºC) and thermostability conditions (43ºC ± 2ºC). Lyophilized vials were exposed at 4°C in refrigerator and titrated at monthly intervals up to 1, 3, 6, 9 and 12 months. The exposures of lyophilized vials at 25°C, 37°C, and 43°C in incubators and titrated at days interval up to 30 days for 25ºC, 7 days at 37ºC and up to 2 days for 45ºC.

Based on the evaluation of the stability study data it can be concluded:
1. The vaccine composition of Sheep Pox, Goat Pox and PPR virus is stable at least for 12 months at 5° ± 3°C (real time stability) as per specification.
2. The vaccine composition of Sheep Pox, Goat Pox and PPR virus is stable for 15 Days at 25° ± 2°C (accelerated stability) as per specification.
3. The vaccine composition of Sheep Pox, Goat Pox and PPR virus is stable for 7 days at 37° ± 2°C (stress stability) as per specification.
4. The vaccine composition of Sheep Pox, Goat Pox and PPR virus is stable upto 2 days at 43° ± 2°C (thermostability) as per specification.

EXAMPLE 3: This example gives a brief on the in-vivo Safety, Potency and Immunogenicity carried out for GPV, SPV and PPRV

A] In-vivo safety testing carried out for Combined Sheep Pox, Goat Pox, and PPR Vaccine
Animal species taken: BALB/c mice
No. of animals taken for 1 batch: 5
Route of vaccine administration: intraperitoneally
Volume of injection: 0.2 ml
No. of days animals were housed: 56

The safety test was carried out in rodents to detect any non-specific toxicity associated with the vaccine as per the procedure laid out in OIE manual.

Freshly reconstituted 0.2 mL of vaccine was injected intraperitoneally (i/p) to five BALB/c mice each and 0.2 mL of placebo was injected intraperitoneally into five mice as un-inoculated 20 controls. The animals were observed for 3 weeks for any signs including death that can be attributed to a living organism in the vaccine. The vaccine was considered satisfactory if at least 80 % of animals remain in good health during the period of observation.

Result: Based on the results obtained, it is found that “Combined Sheep Pox, Goat Pox and PPR Vaccine” did not produce any systemic toxicity when administered through intraperitoneal injection route at different occasions in BALB/c mice, under the conditions and procedures followed in the present study.

B] In-vivo Immunogenicity testing carried out for Combined Sheep Pox, Goat Pox, and PPR Vaccine
Animal species taken: BALB/c mice
No. of animals taken for 1 batch: 5
Route of vaccine administration: intraperitoneally
Volume of injection: 0.2 ml
No. of days animals were housed: 56

Immune Responses of Mice Vaccinated with a Combined SPV, GPV, PPRV & Individual Vaccine
Vaccine Groups (N=5) Immunization Virus neutralization antibody titres
SPV GPV PPR
Combination Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) 5.7-18.28
(P < 0.001) 5.7-16.7
(P < 0.001) 5.7-18.2
(P < 0.001)
Post dose-2
(Day 42 & Day 56) 14.8-19.9
(P < 0.001) 13.18-16.7
(P < 0.001) 13.14-19.98
(P < 0.001)
SPV Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) 5.7-18.2
(P < 0.001) = 5 = 5
Post dose-2
(Day 42 & Day 56) 14.65-15.02
(P < 0.001) = 5 = 5
GPV Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) = 5 5.73-19.6
(P < 0.001) = 5
Post dose-2
(Day 42 & Day 56) = 5 14.65-15.02
(P < 0.001) = 5
PPR Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) = 5 = 5 5.7-18.2
(P < 0.001)
Post dose-2
(Day 42 & Day 56) = 5 = 5 14.65-15.02
(P < 0.001)
Placebo Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) = 5 = 5 = 5
Post dose-2
(Day 42 & Day 56) = 5 = 5 = 5

The immunogenicity studies were carried out in BALB/c mice aged 6 to 8 weeks and were randomly divided into five groups (n = 5 for each group). Mice was immunized intraperitoneally with 0.2 mL of Vaccine. Blood samples were collected by retro-orbital plexus puncture, at various time-points. Thereafter the blood samples were allowed to clot and then centrifuged, serum samples were collected and stored at -70°C required for antibody titration.

Result: The sera samples were tested for antibody titre using Virus Neutralization assay and overall study result from the given set of data shows that combined vaccine is capable of eliciting immunogenic response by having the neutralizing antibody.

C] Comparative study of immunogenicity testing carried out for individual Sheep Pox, Goat Pox, or PPR Vaccine and Combined Sheep Pox, Goat Pox, and PPR Vaccine of the present invention
BALB/c mice were immunized with Live attenuated Lyophilized Combined Vaccine, Live attenuated Lyophilized Sheep Pox Vaccine, Live attenuated Lyophilized Goat Pox Vaccine, and Live attenuated Lyophilized PPR Vaccine at 0 days and 28 days. Seroconversion is defined as a neutralizing antibody titre of =10. Data were analyzed in GraphPad Prism with two-way ANOVA test compared to all groups *P < 0.001.

Advantages of the present invention
The present invention provides an efficient and effective combination of vaccine that can prevent multiple diseases to ameliorate issues related with immunization against multiple diseases. The invention also provides a trivalent vaccine composition against Goat Pox, Sheep Pox and PPR having improved thermostability. The approach of present invention is not only cost effective but is also helpful in leading to successful immunization considering logistics difficulties.
,CLAIMS:A vaccine composition with improved thermostability, comprising live attenuated Sheep Pox virus, live attenuated Goat Pox virus, live attenuated Peste-des-petits-ruminants (PPR) virus, and excipients selected from stabilisers, preservatives, tonicity modifying agents, pH modifiers and buffers.

2. The vaccine composition as claimed in claim 1, wherein each of the live attenuated Sheep Pox virus, Goat Pox virus, and PPR virus are present in an amount of at least 3 log10 CCID50 per ml of the vaccine composition.

3. The vaccine composition, as claimed in claim 1, wherein the excipients comprise about 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), about 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), about 5.2-6.0 mM Glutamate, about 2.5% Lactalbumin Hydrolysate (LAH), about 5% Sucrose, about 5% Trehalose dehydrate and about 0.5% Gelatin prepared in Dulbecco's phosphate-buffered saline (DPBS).

4. The vaccine composition as claimed in any of the preceding claims, wherein the pH of the vaccine composition is in a range of 7.0 to 7.4.

5. A process for preparation of vaccine composition of any of the preceding claims comprising:
a. Retrieving live attenuated Sheep Pox virus, Goat Pox virus and PPR virus from deep freezer and thawing at 25°C ± 1°C,
b. Optionally pooling the processed Sheep Pox virus, Goat Pox virus, and PPR virus aseptically in a biosafety cabinet,
c. Separately mixing excipients comprising, about 3.1-3.8 mM Potassium dihydrogen phosphate (KH2PO4), about 6.8-7.2 mM Di-potassium hydrogen phosphate (K2HPO4), about 5.2-6.0 mM Glutamate, about 2.5-3.5% LAH, about 4-6% Sucrose, about 3-5% Trehalose dehydrate and about 0.5-1.0 % Gelatin prepared in DPBS to obtain a formulation mixture with the pH in the range of 7.0-7.4,
d. Adding the virus an amount of not less than 3 log10 CCID50/ml each, to the formulation mixture to obtain the vaccine composition,
e. Mixing the processed Sheep Pox virus, Goat Pox virus, and PPR virus with the formulation mixture and lyophilizing it to a temperature ranging upto (-95°C) to obtain lyophilized mixture,
f. Optionally transferring the vaccine composition into glass vials for storing at about 4°C.
6. The vaccine composition, as claimed in any of the preceding claims 1 to 4, as and when used for immunization against Sheep Pox, Goat Pox and PPR.