DESC:FIELD OF INVENTION
The present invention relates to the field of vaccine compositions. In particular, the invention relates to providing an efficient and effective combination of vaccines that can prevent multiple diseases to ameliorate issues related to immunization against multiple diseases. In particular, the invention provides a trivalent inactivated vaccine composition against Goat Pox, Sheep Pox, and Peste-des-petits-ruminants (PPR) infection and diseases, with improved thermostability.
BACKGROUND OF THE INVENTION
India has a rapidly growing animal husbandry sector and is fast moving towards attaining self-sufficiency in the production of livestock products. Animal husbandry is a major contributor to the Indian economy and its overall contribution is 28-32% of the agricultural GDP and 4 to 6% of the national GDP. It also contributes to about 8-10% of the country’s labor power. India has a very huge livestock population of which about 71.5 million are sheep and about 140 million are goats. Sheep and goats are reared generally by poor population in developing countries including India. Therefore, prevention of animal death from the infectious diseases is considered as an important step for poverty alleviation in areas where such infectious diseases are endemic and cause heavy losses. For example, PPR alone caused a loss of Rs.12.04 billion (around USD 180 million) in 2014-15.
PPR is a highly contagious, acute, and transboundary viral disease of goats and sheep, caused by the genus Morbillivirus belonging to the family Paramyxoviridae. Clinically, the disease is manifested as 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 is a huge 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. Goat Pox and Sheep Pox have more or less similar clinical symptoms. The disease affects animals of all ages, but young population is mostly affected. Occulonasal discharge, sialorrhea, skin lesions, allurated papules on mucus membrane and hyperpyrexia are the common symptoms. SPV and GPV primarily spreads 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 impacts. In Indian subcontinent, Sheep Pox and Goat Pox diseases are widespread to an endemic level, and are a cause of serious economic losses to small ruminant husbandry as mortality in young animals can exceed 50%.
Goat Pox is caused by goat capripox virus (Goat Pox virus) and the clinical symptoms of it are somewhat 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 the disease leads to suppuration and causes generalized vaccinia and systemic reaction. Clinically swollen acne are seen without maturation process, i.e. so-called "stone acne". Severe infection results into internal organs lesions and death. This disease is prevalent in Indian subcontinent, Mediterranean region, Africa, and some other countries. Accidentally cases are also recorded in Europe and Australia. There are variations in the symptoms and death rate of the disease, because of differences in infecting strain and the hosts. The death reported in Turkey is called 3%~ 40%, while the death rate reported in Morocco is 5%~70% (Grimpret).
Novel technologies are being developed to improve efficacy of veterinary vaccines, reduce vaccination cost and eradicate diseases. Although, developing an effective and stable vaccine for different animal diseases is challenging, it is even more challenging to immunize the animal against multiple diseases due to economical, logistics and other issues. Therefore, efficient and effective combination vaccine is a desirable option. Combination vaccines are required worldwide for effective and practical immunization against multiple diseases. The approach of combination vaccines is not only cost effectiveness but would also lead to successful immunization considering logistics difficulties.
There are specific vaccines available for Goat Pox, Sheep Pox and PPR. Mostly, the animals are partially vaccinated against one or the other disease, but not all the diseases. Thus, such animals although protected against one of the diseases, are vulnerable to be affected by remaining diseases. Therefore, availability of a single shot vaccine combining GPV, SPV and PPRV would be particularly beneficial in getting the animal completely protected against group of diseases. Further, in general veterinary vaccines suffer serious deterioration in vaccination campaigns due to the difficulties in maintaining cold chain during the storage and transport of vaccine in far reaching areas, which inevitably result in loss of vaccine potency in tropical and subtropical environments. Therefore, the vaccine with improved thermostability, would be considered more suitable option in field situations to ensure viability of the vaccine in tropical and subtropical environments.
Accordingly, to address the problem known in the art, inventors of the present invention have devised a one-shot cost-effective combination vaccine with improved thermostability, which is highly effective and provides a one-shot cost-effective solution for immunization and protection of small ruminants against group of diseases, like Sheep Pox, Goat Pox, and PPR.
SUMMARY OF THE INVENTION
The present invention provides a vaccine composition with improved thermostability comprising, Sheep Pox antigen, Goat Pox antigen, PPR antigen, and excipients selected from adjuvants, preservatives, tonicity modifying agents, and pH modifiers or buffers. The antigens of Sheep Pox virus, Goat Pox virus, and PPR virus are inactivated antigens.
In a preferred aspect, the vaccine composition comprises each of the Sheep Pox antigen, Goat Pox antigen, and PPR antigen in an amount of not less than 5 log10 CCID50 (prior to inactivation) per ml of the vaccine composition.
In another aspect, the vaccine composition of the present invention, the excipients comprise Sucrose, Potassium Dihydrogen Phosphate (KH2PO4), Di-potassium Hydrogen Phosphate (K2HPO4), Glutamate, and Aluminum Hydroxide 2-2.5 mg per ml (Adjuvants) in DPBS.
In yet another aspect, in the vaccine composition, the excipients per ml comprises of 200-218 mM Sucrose, 3.1-3.8 mM Potassium dihydrogen phosphate, 6.8-7.2 mM Di-potassium hydrogen phosphate, 5.2-6.0 mM Glutamate, and 2-2.5% Aluminum Hydroxide 2-2.5 mg per ml (Adjuvants) in Dulbecco's Phosphate Buffered Saline.
In a preferred aspect, the pH of the vaccine composition is between 7.2 and 7.4.
The present invention also provides a process for preparation of the vaccine composition which comprises of:
(i) Retrieving inactivated antigens of Sheep Pox virus, Goat Pox virus and PPR Virus from deep freezer and thawing at 25°C ± 1°C,
(ii) Optionally pooling the processed Sheep Pox virus, Goat Pox virus, and PPR Virus antigens aseptically in a biosafety cabinet,
(iii) Separately mixing excipients comprising Sucrose, Potassium dihydrogen phosphate (KH2PO4), Di-potassium hydrogen phosphate (K2HPO4), Glutamate, MEM Medium and Alhydrogel 2% (Aluminum Hydroxide Gel Adjuvants) in DPBS to obtain a formulation mixture,
(iv) Adjusting the pH of the formulation mixture in the range of 7.0-7.4,
(v) Adding the Sheep Pox virus, Goat Pox virus and PPR Virus antigens, separately, or the pooled mixture of antigens, in an amount of not less than 5 log10 CCID50/ml each, to the formulation mixture to obtain the vaccine composition,
(vi) Optionally transferring the vaccine composition into glass vials for storing at about 4°C.
DETAILED DECRIPTION 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.
Vaccine that protects a subject against various diseases in a single go or single shot are highly desirable products, since it reduces the number of shots required to be given and thus 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 well documented phenomenon of the antigenic competition has complicated and hindered the development of the multivalent vaccines. This phenomenon refers to the observation that administering multiple antigens together often results in a diminished response to certain antigens relative to the immune response to these antigens when administered separately.
The earlier 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 Viruses.
It is desirable to add other antigens to such a combination vaccine that would give protection against diseases caused by GPV, SPV, and PPR viruses. It is also desirable to have antigens providing protection against other diseases added to the above said combination vaccines.
Reference is made to Chinese Patent No. CN104800842A, which discloses a bivalent vaccine comprising Goat Pox and Sheep Pox antigens. The invention relates to a live attenuated combination vaccine in which the antigens are adsorbed on to gelatine.
As Goat pox, Sheep pox and PPR are prevalent, it is desirable to develop a combination vaccine that would give protection against diseases caused by GPV, SPV, and PPR viruses. It is also desirable to have antigens providing protection against other diseases added to the above said combination vaccines.
Currently, there are no known combinations of vaccines, either inactivated or live-attenuated that contain appropriate formulations of trivalent antigens consisting of GPV, SPV and PPRV in appropriate immunogenic forms for achieving desired levels of efficacy and immunogenicity in the susceptible subject animal population, for multiple diseases in one shot. Therefore, there is a need for a multiple component vaccine that provides a continued protection against Goat Pox, Sheep Pox and PPR virus and is in a form that provides ease of administration and is cost-effective. It would be desirable to provide for a stable and efficacious trivalent vaccine against these three diseases caused by GPV, SPV and PPRV. For such vaccines to be effective, the criterion of seroprotection for each of the antigens of the vaccine needs to be fulfilled. 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 liquid combination vaccine formulation protecting against a plurality of diseases.
The term “multivalent vaccine” or “trivalent vaccine” refers to a combination vaccine comprising antigens for GPV, SPV and PPRV, which has the ability to provide protection or immunization against multiple diseases caused by GPV, SPV and PPRV, or against diseases caused by several strains of a single virus/pathogen.
The term “vero cells” refers to a lineage of cells used in cell cultures that are isolated from kidney epithelial cells extracted from an African green monkey.
The present invention is directed towards the development of a liquid vaccine with improved thermostability and comprises vaccine components that are suitable for prevention, amelioration and/or treatment of multiple diseases and confers seroprotection for each of the said vaccine components.
The present invention provides a trivalent vaccine, which confers protection against multiple diseases and infections in a safe and efficacious manner. The vaccine of the invention provides immunogenicity to the most common viral diseases and infections to small ruminants without any interference to any of the antigens that are present in the vaccine. Thus, a single shot would confer immunogenicity against multiple diseases and infections, making the vaccine more compliant. Since a single shot would induce immunity against three diseases simultaneously, the cost of vaccination would be substantially reduced, which would eventually improve the 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 aspect of the invention would make it more useful and advantageous especially for the farming community who would not be required to bring their animals repeatedly for vaccination from the far-reaching areas where they live. Thus, the present invention provides a vaccine that is more acceptable.
More specifically, the combination vaccine of the present invention is thermostable and retains the potency for a reasonable time even at higher temperatures, like 40-45?. Hence, the vaccine of the present invention is suitable for use even in remote village areas where there is no facility of storage at low temperatures. The vaccine composition of the present invention enables reduction of storage and transportation costs and also helps in administration of vaccine at the stie, instead to moving the cattle (goat or sheep) to vaccination centers.
In one aspect, the combination vaccine of the present invention comprises Sheep Pox Antigen, Goat Pox Antigen, PPR Antigen and adjuvants selected from Sucrose, Potassium Dihydrogen Phosphate (KH2PO4), Di-potassium Hydrogen Phosphate (K2HPO4), Glutamate, and Aluminum Hydroxide 2-2.5 mg per ml (Adjuvants) in DPBS, or a combination thereof. In a preferred aspect, the pH of the combined vaccine is about 7.2.
In another aspect, each 1 ml of the combination vaccine comprises vaccine antigens and adjuvants, wherein the vaccine antigens comprise Sheep Pox Antigen in an amount of not less than 5 log10 CCID50 (prior to inactivation), Goat Pox Antigen in an amount of not less than 5 log10 CCID50 (prior to inactivation), and PPR Antigen in an amount of not less than 5 log10 CCID50 (prior to inactivation); in combination with excipients, stabilizers and adjuvant comprising of Sucrose, Potassium Dihydrogen Phosphate (KH2PO4), Di-potassium Hydrogen Phosphate (K2HPO4), Glutamate, and Aluminum Hydroxide 2-2.5 mg per ml (Adjuvants) in DPBS, and the vaccine has a pH of about 7.2.
Non-antigenic components of the vaccine
Along with the antigenic components the vaccine may comprise a number of non-antigenic components that are pharmaceutically acceptable excipients. These include but are not restricted to pH modifiers, buffers, adjuvants, preservatives, carrier and tonicity modifying agents.
Adjuvants
The antigens of the final formulation may or may not be adsorbed on to an adjuvant. An adjuvant functions to stimulate stronger immune response against the pathogen thus making the vaccine more effective.
Adjuvants may also serve to:
• Bring the antigen in contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration);
• Decrease the toxicity of certain antigens; and
• Provide solubility to some vaccine components.
Studies have shown that many aluminum containing vaccines, like Aluminum Hydroxide, causes higher and more prolonged antibody responses than comparable vaccines without the adjuvant. The benefit of adjuvants has usually been observed during the initial immunization series rather than with booster doses. Further:
• it brings the antigen in contact with the immune system and influences the type of immunity produced, as well as the quality of the immune response (magnitude or duration);
• decreases the toxicity of certain antigens; and
• provides solubility to some vaccine components.
These adjuvants have also been approved by FDA for use in vaccines. There are three general types of aluminum-containing adjuvants:
• Aluminum hydroxide,
• Aluminum phosphate, and
• Potassium aluminum sulfate (often called "Alum")
In one embodiment, the invention relates to selection of a specific adjuvant to be used against a range of available options, for example, Aluminum hydroxide has been found to be more effective with the inactivated virus antigens of the present invention as compared to gelatine. Aluminum hydroxide when adsorbed to inactivated viruses of trivalent vaccine yielded a better immunogenic response. The selection of the adjuvant for the adsorption to a specific type of antigens gives the vaccine of the invention its characteristics. The selected adjuvant and specific use of antigen in this invention has been described in more details in the section on 'Process for manufacturing of the vaccine of the invention'.
Preservatives
The vaccines are prone to contamination by bacteria. Thus, to avoid the potentially life-threatening contamination with harmful microbes, that 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 Goat pox, Sheep pox and PPR vaccine.

Tonicity modifying agents
To control the tonicity of the vaccine composition, it is preferred to include 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, salts 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 a 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 in the vaccine. 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 inactivated vaccine comprises of inactivated GP, SP, PPR antigen each present in an amount of about not less than 5 log10 CCID50 (prior to inactivation) per 1.0 ml to form a trivalent vaccine which is stable, immunogenic and in the form of fully liquid formulation. Such a vaccine is 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 invention.
According to another aspect of the invention, the aluminium content (Al+3) in the vaccine of the invention may not be more than preferably, 2.5 mg per ml, more preferably 1 mg per 0.5 ml.
According to one another aspect of the invention, the preferred amount of excipients is as: Sucrose 200-218 mM, Potassium dihydrogen phosphate (KH2PO4) 3.1-3.8 mM, Di-potassium hydrogen phosphate (K2HPO4) 6.8-7.2 mM, Glutamate 5.2-6.0 mM.
The present invention also provides a process for the preparation of vaccine composition of the present invention. In one aspect, the process of preparation comprises the preparation of vaccine antigens and adding suitable adjuvants to prepare the vaccine composition.
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 depends upon the way the composition has been formulated. This may include the sequence of addition of the antigens, the use of the specific adjuvants for certain antigens, the use of various parameters including agitation, temperature and pH.
One of the embodiments of the invention relates to the process of manufacturing a fully liquid combination vaccine comprising antigens of Goat Pox (GP), Sheep Pox (SP) and Pestes des ruminant (PPR) virus, which comprises the steps of:
a) preparing a component I comprising a mixture of inactivated antigens of i) Goat Pox (GP), ii) Sheep Pox (SP) and iii) Pestes des ruminant (PPR),
b) optionally, preparing a component II comprising separately each of the individual antigens adsorbed on to aluminium salts,
c) optionally, mixing the component II 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 aluminium hydroxide.
Another preferred embodiment of the invention relates to the preparation of the component I which comprises the following steps:
a) transferring Aluminium hydroxide gel into a vessel,
b) transferring inactivated Goat Pox, Sheep Pox and PPR Virus antigen preparation into the above vessel,
c) transferring excipients, Sucrose, 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 an inactivated trivalent fully liquid 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 invention 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 trivalent inactivated vaccine
(A) Preparation of vaccine antigens.
Approved Vaccine 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 is 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 was achieved, viruses were harvested from the respective experiments to maintain the uniformity in the virus titre. Virus harvests were polled 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-75 tissue culture flask in Modified Eagle Medium or Minimum Essential Medium (MEM) with 10 % Fetal Bovine Serum (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, 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 about 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 about 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 thereafter the incubation was continued in a serum-free media at about 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 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 about 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 about 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 thereafter 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 the results obtained. The process used in T-175 flask yielded virus potency of 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 about 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 about 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 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 1x SPG and stored at minus 70°C or below. Experiments were repeated to see the consistency in the results obtained. The process used in T-175 flask yielded virus potency of log10 5.7-6.0 CCID50/ml for the pooled harvest.
B. Production Steps for Drug Substance (antigen)
(i) Revival and propagation of Vero cells:
• Revival of Vero cells in complete minimum essential media (MEM) (supplemented with 10% FBS).
• Removal of the spent media from the flasks on second day and adding fresh complete MEM.
• Propagation of Vero cells to achieve sufficient cells for seeding in T-75 cm2 tissue culture flask.
• Incubation of the Tissue culture flask at about 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/ titer 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 shall 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 and addition of stabilizers (1x SPG) to the harvests and storage at minus 70°C or below.

(v) Inactivation of Virus and Storage:
Vero cells, infected with strains of SPV, GPV and PPR at a multiplicity of infection of 0.05 (MoI = 0.05), were grown in serum-free medium for 5-6 days. Supernatant having virion particles were clarified by centrifugation at about 10,000 rpm for 30 min. 37% formaldehyde was diluted with 1X PBS to 0.5% and the pH was adjusted to 7.2 with 10 N NaOH. The mixture was added to viruses Supernatant to have a final formalin concentration of 0.05%. The formalin-treated virus (FIV) or untreated virus (untreated control virus) were incubated at about 4°C for 7 days, or at 25°C for 2 days to check the viral inactivation. Samples were stored at -70°C for analysis. The residual infectious viral titers of FIV viruses were assessed by micro CCID50 assay.
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.
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 5 log10 CCID50 (prior to inactivation)
Goat Pox Antigen: Not less than 5 log10 CCID50 (prior to inactivation)
PPR Antigen: Not less than 5 log10 CCID50 (prior to inactivation)
Component II: Other Ingredients: -
Sucrose, Potassium Dihydrogen Phosphate (KH2PO4), Di-potassium Hydrogen Phosphate (K2HPO4), Glutamate, and Aluminium Hydroxide 2.0-2.5 mg per ml (Adjuvant) in DPBS, pH 7.2.
EXAMPLE 2: Process of manufacturing a combined inactivated Goat Pox, Sheep Pox and PPR Vaccine
The process of manufacturing of a combined inactivated 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 antigens (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 (component II) i.e., Sucrose, Potassium dihydrogen phosphate (KH2PO4), Di-potassium hydrogen phosphate (K2HPO4), Glutamate were then added to the pooled antigen.
To inactivate the live virus harvest, an amount of 37% formaldehyde was diluted with 1X PBS to 0.5% and adjusted to pH 7.2. The mixture was added to viruses Supernatant to obtain a final formalin concentration of 0.05%. The formalin-treated virus or untreated virus (untreated control virus) was incubated at 4°C for 7 days, or at 25°C for 2 days. The inactivated antigen was further purified using a 300 kDa molecular weight cut off membrane and sterilized through 0.2 µm filtration. The purified antigen was suspended in Dulbecco’s Phosphate Buffered Saline (DPBS) solution and further formulated by adding Aluminium Hydroxide 2.0-2.5 mg per ml (Adjuvant). The pH of each formulated antigen/vaccine (GPV, SPV and PPRV) 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 105 CCID50 (prior to inactivation) was transferred into a vessel then gently mixed (combined Vaccine) maintaining a pH of 7.2-7.4 and transferred into glass vials, stoppering was done, and the final formulated vaccine was stored at 2-8°C.
Alternatively, each of inactivated and purified antigens of GPV, SPV and PPRV, not less than 105 CCID50 (prior to inactivation) 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 Aluminium Hydroxide 2.0-2.5 mg per ml (Adjuvant). The pH of the formulated combined vaccine (GPV, SPV and PPRV) was checked and adjusted to fall in the range of 7.0- 7.4. the above mixture (combined Vaccine) was then transferred into glass vials, stoppering was done and the final formulated vaccine was stored at 2-8°C.
EXAMPLE 3: This example gives the composition and the process of manufacturing of an inactivated trivalent liquid vaccine as per one of the aspects of the invention
A] Composition of the inactivated trivalent liquid vaccine as per the invention is as under:
Each 1.0 ml vaccine comprises the following:
COMPONENT I AMOUNT
Sheep Pox virus in an amount = 5 log10 CCID50
Goat Pox virus in an amount = 5 log10 CCID50
PPR virus in an amount = than 5 log10 CCID50
Other Ingredients: -
Sucrose 200-218 mM, Potassium dihydrogen phosphate (KH2PO4) 3.1-3.8 mM, Di-potassium hydrogen phosphate (K2HPO4) 6.8-7.2 mM, Glutamate 5.2-6.0 mM, and Aluminium Hydroxide 2.0-2.5 mg per ml (Adjuvant) in DPBS. The vaccine composition has a pH of about 7.2.
B] The process of manufacturing of Combined Sheep Pox, Goat Pox, and PPR Vaccine as per the invention is as under:
Formulation procedure for Component I
One batch of Sheep Pox virus, Goat Pox virus and PPR Virus antigens 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 Sheep Pox virus, Goat Pox virus, and PPR Virus antigens were then optionally pooled aseptically in a biosafety cabinet.
The excipients Sucrose 200-218 mM, Potassium dihydrogen phosphate (KH2PO4) 3.1-3.8 mM, Di-potassium hydrogen phosphate (K2HPO4) 6.8-7.2 mM, Glutamate 5.2-6.0 mM, and Aluminium Hydroxide 2.0-2.5 mg per ml (Adjuvant) in DPBS. The pH of the formulation mixture was checked and adjusted to fall in the range of 7.0-7.4.
The formulation mixture was transferred into a vessel, followed by addition of Sheep Pox virus, Goat Pox virus and PPR Virus antigens, separately, or the pooled mixture of antigens, in an amount of not less than 5 log10 CCID50/ml each, and then mixed and transferred into glass vials stored at 4°C.
EXAMPLE 4 This example gives a brief on the Stability or the Potency of the vaccine of the Invention.
The stability studies were performed to evaluate the shelf life of the vaccine composition comprising Sheep Pox, Goat Pox and PPR antigens at real-time (4ºC ± 2ºC), accelerated (25ºC ± 2ºC), stress (37ºC ± 2ºC) and thermostability conditions (43ºC ± 2ºC). Liquid vials were exposed at 4°C in refrigerator and titrated at monthly intervals up to 1, 3, 6, 9 and 12 months. The Liquid vials were exposed to 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 43ºC.
Based on the evaluation of the stability study data it can be concluded that:
1. Vaccine composition of Sheep Pox, Goat Pox, and PPR Virus antigens is stable for more than 12 month at 4° ± 2°C (real time stability) as per specification.
2. Vaccine composition of Sheep Pox, Goat Pox, and PPR Virus antigens is stable for 15 days at 25° ± 2°C (accelerated stability) as per specification.
3. Vaccine composition of Sheep Pox, Goat Pox, and PPR Virus antigens is stable for 7 days at 37° ± 2°C (stress stability) as per specification.
4. Vaccine composition of Sheep Pox, Goat Pox, and PPR Virus antigens is stable upto 2 days at 43° ± 2°C (thermostability) as per specification.

EXAMPLE 5 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-speci?c 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 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
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 titer 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

Immune Responses of Mice Vaccinated with a Combined SPV, GPV, PPRV & Individual Vaccine
Vaccine Groups(N=5) Immunization Virus neutralization antibody titers
SPV GPV PPR
Combination Pre-Vaccination
(Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) 10.29-18.28
(P < 0.001) 7.5-17.79
(P < 0.001) 7.78-17.17
(P < 0.001)
Post dose-2
(Day 42 & Day 56) 13.14-19.98
(P < 0.001) 13.14-19.98
(P < 0.001) 14.41-9.98
(P <0.001)
SPV Pre-Vaccination (Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) 15.7-18.28
(P < 0.001) = 5 = 5
Post dose-2
(Day 42 & Day 56) 13.14-17.08
(P < 0.001) = 5 = 5
GPV Pre-Vaccination
(Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) = 5 15.72-19.86
(P < 0.001) = 5
Post dose-2
(Day 42 & Day 56) = 5 13.14-16.51
(P < 0.001) = 5
PPR Pre-Vaccination
(Day 0) = 5 = 5 = 5
Post dose-1
(Day 14 & Day 21) = 5 = 5 15.72-19.86
(P < 0.001)
Post dose-2
(Day 42 & Day 56) = 5 = 5 14.41-17.08
(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

Result: From the above comparability study data it is clear that the values of neutralizing antibody titer produced against the specific virus in the combination vaccine was no inferior to the quantity of antibody produced in the animal vaccinated with monovalent vaccine, which is in fact a precondition for the success for an effective combination vaccine.
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:1. A vaccine composition with improved thermostability comprising, Sheep Pox antigen, Goat Pox antigen, PPR antigen, and excipients selected from adjuvants, preservatives, tonicity modifying agents, and pH modifiers and buffers.
2. The vaccine composition as claimed in claim 1, wherein each of the Sheep Pox antigen, Goat Pox antigen, and PPR antigen are present in an amount of not less than 5 log10 CCID50 (prior to inactivation) per ml of the vaccine composition.
3. The vaccine composition as claimed in claim 1, wherein the excipients comprise Sucrose, Potassium Dihydrogen Phosphate (KH2PO4), Di-potassium Hydrogen Phosphate (K2HPO4), Glutamate, and 2-2.5 mg per ml Aluminium Hydroxide (Adjuvant) in DPBS.
4. The vaccine composition as claimed in any of the preceding claims, wherein the excipients comprise 200-218 mM Sucrose, 3.1-3.8 mM Potassium dihydrogen phosphate, 6.8-7.2 mM Di-potassium hydrogen phosphate, 5.2-6.0 mM Glutamate, and 2-2.5 mg per ml Aluminium Hydroxide (Adjuvant) in Dulbecco's Phosphate Buffered Saline.
5. The vaccine composition as claimed in any of the preceding claims, wherein the pH of the vaccine composition is between 7.2 and 7.4.
6. A process for preparation of vaccine composition of any of the preceding claims comprising:
(i) Retrieving inactivated antigens of Sheep Pox virus, Goat Pox virus and PPR Virus from deep freezer and thawing at 25°C ± 1°C,
(ii) Optionally pooling the processed Sheep Pox virus, Goat Pox virus, and PPR Virus antigens aseptically in a biosafety cabinet,
(iii) Separately mixing excipients comprising Sucrose, Potassium dihydrogen phosphate (KH2PO4), Di-potassium hydrogen phosphate (K2HPO4), Glutamate, MEM Medium and 2-2.5 mg per ml Aluminium Hydroxide (Adjuvant) in DPBS to obtain a formulation mixture,
(iv) Adjusting the pH of the formulation mixture in the range of 7.0-7.4,
(v) Adding the Sheep Pox virus, Goat Pox virus and PPR Virus antigens, separately, or the pooled mixture of antigens, in an amount of not less than 5 log10 CCID50/ml each, to the formulation mixture to obtain the vaccine composition,
(vi) Optionally transferring the vaccine composition into glass vials for storing at about 4°C.