WO2005109991B1

WO2005109991B1 - Anti-theileriosis vaccine

Info

Publication number: WO2005109991B1
Application number: PCT/IN2005/000154
Authority: WO
Filing date: 2005-05-12
Publication date: 2006-04-27

Abstract

A process for the preparation of anti-theileriosis vaccine comprising the steps of isolating lymphoid cells infected with schizonts of Theileria annulata present in an infected animal, culturing the infected cells in a growth medium, subjecting said cultured cells to the step of in-vitro propagation by-serial passaging, developing cloned curltures from a single cell by limiting dilution technique and further passaging these, karyotyping the cells in the medium to ascertain the diploid character, and obtaining viable cells with maximum infectivity rates, determining macroschizont nuclear number, subjecting a number of viable cells with diploid character to the step of cryoprotectant in liquid nitrogen in the presence of a cryoprotectant in the said growth medium to obtain a suspension of viable cells, testing these cultures at different passages and in varying dose levels in groups of calves for ascertaining their pathogencity / antigenicity, determining passage level and dose for the safety and efficacy of candidate vaccine, its safe use in new born calves and pregnant cows, time taken to induce immunity, serological responses and erect on haematological parameters, and testing its efficacy under wide field conditions in the susceptible stock.

Claims

AMENDED CLAIMS received by the International Bureau on 14 December 2005 (14.12.05)

1. A process for the preparation of anti-theileriosis vaccine comprising the steps of isolating lymphoid cells infected with schizonts of Theileria annulate present in an infected animal, culturing the infected cells in a growth medium, subjecting said cultured cells to the step of in-vitro propagation by serial passaging twice / thrice-weekly or as static cultures, developing cloned cultures from a single cell of twice/thrice-weekly cultures by limiting dilution technique in cupark plates and further passaging these, karyotyping the cells in the medium to ascertain the diploid character, and obtaining viable cells with maximum infectivity rates, determining macroschizont nuclear number, subjecting a number of viable cells with diploid character to the step of cryopreservation in liquid nitrogen in the presence of DMSO (Dimethylsulphoxide) as a cryoprotectant in the said growth medium to obtain a suspension of viable cells, testing the original cultures as well as cloned cultures at different passage levels and in varying doses in groups of calves for ascertaining their pathogenicity / antigenicity, determining passage level and dose for the safety and efficacy of candidate vaccine, its safe use in new born calves and pregnant cows, time taken to induce immunity, serological responses and effect on haematological parameters, and testing its efficacy under wide field conditions in the susceptible stock.

2. The process as claimed in claim 1 wherein the said growth medium used is RPMI 1640: 10 to 20 % selected from foetal bovine serum, neonatal or normal bovine serum; antibiotics solution containing benzyl penicillin sodium @100 i. u. per ml, streptomycin sulphate @ 100 μg per ml. and mycostatin @10 i.u. per ml; and also 200 mM of L- glutamine per ml. All the contents should be pre-sterilised by filtration and should be free from any micro¬ organism contamination by standard procedures.

3. A process as claimed in claim 1 , wherein said twice-weekly passaging consists of adding 8-9 ml of growth medium to 1-2 ml of old culture in a new flask to make a total volume of approximately 10 ml and thrice-weekly passaging consists of adding 7-8 ml of fresh growth medium to 2-3 ml of old culture in a new flask and static culture by adding 8-9 ml fresh medium to 1-2 ml of old culture twice weekly in the same flask to retain a total volume of approximately 10 ml for a period upto one year, followed by passaging to a bigger flask, bulking up as herein described and cryopreservation at different passages of twice/ thrice weekly cultures and time intervals in case of static cultures.

4. . A Process as claimed in claim 2, wherein initially used serum is taken as foetal bovine serum which was substituted subsequently by normal bovine serum or neonatal calf serum.

5. A process for development of cloned cultures from a single cell: subjecting the original cultures in the step of passaging twice/thrice weekly between passage 90 to 100 to obtain cell population of 1X10³ cells per ml; pouring said culture into cupark plates and observing under microscope; marking the wells of the cupark plate containing a single cell and monitoring daily and allowing it to multiply; transferring the said mass of cells alongwith the medium into bigger well erf costar plate and allowing them to grow further; adding fresh medium and transferring the cells into a flask; allowing the culture to grow and again transferring them to bigger and bigger flasks and subjecting the said culture to the step of bulking up and cryopreserved in 10%»

DMSO as cryoprotectant in liquid nitrogen container at next passage level.

6. . A process as claimed in claim 1 , wherein said bulking up of culture consists of⁷ either thawing of seed culture to 37⁰C from .liquid nitrogen, washing by resuspending in 9-10 mi of growth medium, centrifuging at 40Og for 1O minutes, discarding the supernatant, seeding in a tissue culture flask taking approximately 10 ml growth medium and incubating at 37⁰C, subsequently seeding in 80 or 250 cm² tissue culture flasks at 1.5-2X 10⁵ cells / ml in 25 or 100 ml growth medium, respectively and incubating at 37⁰C and after 4S

19 hours adding equal amount of growth medium in each flask, or by bulking up of the on-going culture in the same way,

7. The process as claimed in claim 1 , wherein the step of in vitro propagation of various cell lines of the parasite developed by twice or thrice weekly passaging were tested at various passage levels i.e. 10-15, 20-25, 50-55 as original cultures and 100-105, 150-155 & 200-205 as cloned cultures with varying dose levels and it was found that the cloned culture at its passage 200-205 with 4.5-5x10⁶ cells per ml as a single dose was safe and immunogenic as a vaccine.

8. The process as claimed in claim 1 , wherein the cell concentration in said cell suspension is adjusted to 9-11x10⁶ viable cells per ml, the total volume is measured and mixed with equal quantity of growth medium containing 20% DMSO so as to make the final concentration of cells at 4.5-5.5x10⁶ viable cells per ml in growth medium containing 10% DMSO.

9. An anti-theileriosis vaccine comprising schizonts of T. annulata in a cryoprotectant and in growth medium.

10. The anti-theileriosis vaccine as claimed in claim 9, wherein the growth medium contains 20% DMSO so as to make the final concentration of 4.5- 5.5x10⁶ viable cells per ml in growth medium containing 10% DMSO.

11. The anti-theileriosis vaccine as claimed in claim 9 is protective and safe for the exotic breeds of cattle i.e. Jersey, Holstein-Fressian, Brown Swiss, Red Dane etc. and their cross-breds with indigenous Indian breeds of cattle including new born calves and pregnant cows.

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