Sheep Pox Vaccine And Process Thereof

FIELD OF INVENTION The present invention relates to a live attenuated vaccine against sheep pox virus (SPV). In particular the present invention provides a process for producing live attenuated sheep pox virus by passaging the Romanian strain of the virus adapted to lamb testicular cells (primary cell line) in Vero cell line. BACKGROUND OF THE INVENTION Sheep pox infections can be effectively controlled because of stable nature of the virus. In an endemic situation, control of the disease is possible through immunization with a potent vaccine. Immunity to poxvirus is considered predominantly through cell mediated and the immune status of animals does not correspond to serum neutralizing antibody titers (Hosamani et al., 1993). However, neutralizing antibodies may be detected in immimized animals by Serum neutralization test (SNT), showing an anamnestic response to the challenge and therefore humoral response could also have a role in protecting animals. Heterologous cell systems like bovine fetal muscle cells (Cam, 1993) and baby hamster kidney cells (Kitching et al., 1986) have been used for development of attenuated animal pox vaccines, which were safe, stable and potent in animals. SUMMARY OF THE INVNETION The present invention describes a process of production of a live attenuated vaccine against sheep pox virus (SPV). The process of production of the vaccine involves adaptation and modification of the virus in Vero cell line. The virus formulated as a vaccine of the present invention has been shown to be safe and protected sheep on virulent challenge.

One aspect of the present invention relates to a vaccine composition comprising a live attenuated sheep pox virus population adapted and attenuated to the Vero cell line, wherein said virus was serially passaged in vero cell culture. Another aspect of the present invention relates to a process of production of vaccine composition comprising live attenuated sheep pox virus, said process comprising adapting said virus to the lamb testicular cells and attenuating said adapted virus by serial passaging in vero cells. DETAILED DESCRIPTION OF THE INVENTION The present invention describes a process of production of a live attenuated vaccine against sheep pox virus (SPV). The process disclosed in the present invention involves adaptation and modification i. e. further attenuation of the virus in Vero cell line. The vaccine composition produced by the process disclosed in the present invention has been shown to be safe and protected sheep on virulent challenge. The present invention also provides a process for immunizing a subject using the vaccine composition disclosed. On embodiment of the present invention is to develop a safe and efficacious live and attenuated vaccine against Sheep pox virus (SPV). Another embodiment of the present invention is to develop a safe and efficacious live and attenuated vaccine against Sheep pox Romanian strain, wherein the Romanian strain of the sheep pox virus adapted to Lamb testicular cells (LT) was further adapted and attenuated to the Vero cell line. Yet another embodiment of the present invention provides a process of sheep pox virus production in Vero cell as a vaccine against sheep pox virus, wherein the process offers several advantages like free from adventitious agents and

pathogens. In accordance with the present invention, one embodiment provides a process of production of a live attenuated vaccine against sheep pox virus, wherein the process involves adaptation and modification of the Romanian strain of sheep pox virus (SPV) adapted to lamb testicular cells, wherein the virus is further attenuated by serial passaging in Vero cell cultures and its immunogenicity was tested in sheep. In one embodiment of the present invention, live attenuated sheep pox virus was prepared passaging the virus in Vero cell line. The virus was obtained by passaging the lamb testicular derived Romanian strain in Vero cell line. The virus was passaged about 15 to about 30 times in Vero cell line. The virus was found to be safe and protected sheep (the target species) on virulent challenge. One embodiment of the present invention related to passaging the Romanian strain of sheep pox virus (SPV) adapted to lamb testicular cells for about 15 to about 30 times in Vero cell line. Another embodiment of the present invention provides a vaccine composition against the Sheep Pox virus (SPV), wherein the vaccine comprises a live attenuated sheep pox virus population adapted and attenuated to the Vero cell line, wherein said virus was serially passaged in vero cell culture. Yet another embodiment of the present invention provides a process immunizing a subject using the vaccine composition disclosed herein. One embodiment of the present invention is to provide a vaccine composition comprising a live attenuated sheep pox virus population adapted and attenuated to the Vero cell line, wherein said virus was serially passaged in vero cell culture.

Another embodiment of the ;sent invention provides the vaccine composition comprising a live attenuated sheep pox virus population, wherein said sheep pox virus is Romanitin virus strain. Another embodiment of the present invention provides the vaccine composition comprising a live attenuated sheep pox virus population, wherein said virus is adapted to the lamb testicular cells. Another embodiment of the present invention provides the vaccine composition comprising a live attenuated sheep pox virus population, wherein said composition is capable of immunizing a subject against sheep pox virus. Yet another embodiment of the present invention provides a process of production of vaccine composition comprising live attenuated sheep pox virus, said process comprising adapting said virus to the lamb testicular cells and attenuating said adapted virus by serial passaging in vero cells. Still yet another embodiment of the present invention provides a process of production of vaccine composition comprising live attenuated sheep pox virus, wherein attenuating of said virus was carried out by passaging 15- 30 times in vero cells. In further embodiment, the present invention provides a process of immunizing the subject against sheep pox virus comprising administering the vaccine composition comprising a live attenuated sheep pox virus population adapted and attenuated to the Vero cell line, wherein said virus was serially passaged in vero cell culture.

Live attenuated sheep pox virus by passaging the Romanian strain of the virus adapted to lamb testicular cells (primary cell line) in Vero. The virus was passaged continuously 15 times in Vero cell line and the safety and potency of the vaccine was tested at intervals of 5 passages i.e. at the passage 5, passage 10 and passage 15 (P5, PIO and PI5). Though the virus was potent at all passages and protected the sheep on virulent challenge with a protective units greater than 3.5 i.e. PU>3.5. The virus failed safety at P5 and PIO as was indicated by high rise in temperature. With respect to PI 5 of the virus, Vero cell-attenuated SPV provided complete protection against virulent challenge. It produced no adverse reaction at high dosage of 10"*^ TCID50 indicating safety of the vaccine, and at the same time, it protects sheep at even as low as 10 TCID50 against challenge with a high dose (10'' SRD50) of virulent virus. Such an exposure to virus is unlikely under natural conditions. Since Vero cells is a heterologous host system as compared to sheep derived cells in primary cultures, the attenuation is more effective. Virus production in Vero cells offers several advantages such as cost-effective production and freedom from adventitious agents. The animals showed no rise in temperature in spite of being injected with -lOOx dose of the vaccine composition disclosed in the present invention indicating the safety of the vaccine. Problems associated with culturing the virus in lamb testicular cells (LTC) were overcome by the use of Vero cells with respect to using cells free of pathogens and other adventitious contaminants. The live attenuated sheep pox virus vaccine can now be used to vaccinate sheep in the field and can also be developed as a recombinant viral vector expressing antigenic genes of various diseases of small ruminants for example incorporating the genes F and H ofPeste des petits ruminants.

It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. EXAMPLES It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. Example 1 Preparation of live Attenuated sheep pox virus Romanian strain Sheep pox virus was passaged continuously 15 times in Vero cell line and the safety and potency of the vaccine was tested at intervals of 5 passages i.e. at the passage 5, passage 10 and passage 15 (P5, PIO and P15). Though the virus was potent at all passages and protected the sheep on virulent challenge with a protective units greater than 3.5 i.e. PU>3.5. The virus failed safety at P5 and PIO as was indicated by high rise in temperature. With respect to PI5 of the virus, Vero cell-attenuated SPV provided complete protection against virulent challenge. It produced no adverse reaction at high dosage of 10 TCID50 indicating safety of the vaccine, and at the same time, it protects sheep at even as

low as 10 TCID50 against challenge with a high dose (10^ SRD50) of virulent virus. Such an exposure to virus is unlikely under natural conditions.

We Claim: 1. A vaccine composition comprising a live attenuated sheep pox virus population adapted and attenuated to the Vero cell line, wherein said virus was serially passage in vero cell culture. 2. The vaccine composition according to claim 1, wherein said sheep pox virus is Romanian virus strain. 3. The vaccine composition according to claim 1, wherein said virus is adapted to the lamb testicular cells. 4. The vaccine composition according to claim 1, wherein said composition is capable of immunizing a subject against sheep pox virus 5. A process of production of vaccine composition comprising live attenuated sheep pox virus, said process comprising adapting said virus to the lamb testicular cells and attenuating said adapted virus by serial passaging in vero cells. 6. A process as claimed in claim 5, wherein attenuating of said virus was carried out by passaging 15- 30 times in vero cells. 7. A process of immunizing the subject against sheep pox virus comprising administering the vaccine composition as claimed in claim 1.