Title of Invention

AN IMPROVED METHOD OF PRODUCING PLANT BASED RABIES VACCINE

Abstract

An improved method of producing plant based rabies vaccine by introducing the rabies glycoprotein gene in plants by Agrobacterium method or microprojectile bombardment using growth regulators in a tissue culture medium, collecting the plant produce of the genetically modified crops, extracting the rabies glycoprotein by affinity chromatographic method and purified by column purification method to provide immunity effective treatment against rabies infection.

Full Text

A= Area- background of the invention. This invention relates to an improvement in the already existing rabies vaccine. The research comes under Biotechnology/ genetic engineering. The rabies vaccine has never before been produced in the plant system, which could create immunization in animal system. The available vaccines have a number of problems, which have been improved upon. Rabies is one of the most fearsome diseases known and is highly prevalent. Most of the death occurs because of the inability to meet the expenses for taking the anti rabies vaccine or because of the side effects of the rabies vaccine presently available in the market. This invention primarily aims to erase the side effects and secondarily aims to make it as inexpensive as possible so that any standard of people can avail the vaccine without having to spend a lot. B= Status of the prior art. So far there are no reports on rabies vaccine produced in plant system, which has generated the immunity in animal system like mice. Currently available rabies vaccines include the neural tissue derived sample vaccines and cell culture derived vaccines. The vaccines developed in the animal systems like sheep brain are highly contaminated and neurotoxic. The sheep brain derived vaccines contain lot of chemicals, which are not completely removed and thus causes the side effects in the animal system. The cell culture derived vaccines are much safer than the neural tissue derived sample vaccines but the cost of the vaccine are very high besides it requires refrigeration for storage. The recombinant vaccines produced in yeast have got a problem of contamination while purification and also sometimes mutation may occur in the fermentor. Hence, it is not very safe and is also costly. Dr. McGarvey and others from Thomas Jefferson University, USA has reported the transformation of tomato with rabies vaccine gene. He could not prove the immunogenicity of the glycoprotein, since it was produced in very low quantities. There are reports on other vaccines like Hepatitis B, Cholera vaccine produced in plant system . like banana by Charles J Arntzen and his associates and they have successfully proved the immunization capability in mice and human beings. C=> Problem with the prior art 1. The vaccines produced by fermentation method or by animals system is costly and there is also the danger of other virus contamination or mutation of the recombinant bacteria/virus. Hence it may cause severe problems like allergicity. It also is very painful. These vaccines need refrigeration for storage. Most of the dispensaries in the village lack the facilities for storage of vaccines. The neural tissue sample vaccine is still administered to a large number of people in developing countries, though it causes a lot of serious side effects. Cell -culture vaccines are very expensive for routine use in developing countries. The production of vaccines in bulk quantities is difficult and hence soraetimes the vaccines are not available in the market. The presently available commercial vaccines needs cold^ chain and also stringent GMP regulations etc. the infrastructure for the production of the fermentation based or cell culture vaccines are very high. The vaccines produced in the cell culture, neural tissue cultured vaccines and other presently available vaccines are not edible and are also painful, D=> How this invention seeks to solve the problem The plant vaccines can be produced in bulk in wide range of hosts at a low cost. The plant growth is fast and they can be grown within the existing infrastructure, which will be very low when compared to the cell culture vaccines and neural tissue vaccines. Once the transgenics are produced, the seeds can be multiplied at a very faster rate and can be grown in large areas, According to this technique, the vaccines were produced in the above plant systems and were used as injectable, oral and edible forms to induce immunity. The vaccines produced in the plant systems are edible which are not painful like the other available vaccines, Since the plant produced vaccines contain a cell wall coating, the glycoprotein is well preserved inside the cell, hence there is no need for the refrigeration. The plants producing the desired gene product also produce normally synthesized proteins. In the case of edible vaccines the derived proteins need no purification. I« What features achieve these problems solving process. The rabies vaccine production in plants is a new technique and it involves the transformation of the gene into the plant system and the purification of the protein and testing on the animal system. The plant system selected in the study like Muskmelon, tobacco and groundnut is a new original idea and it has a lot potential in commercial production of rabies vaccine in plants. This method overcomes the problem of production of vaccines by fermentation or other methods, which are costly, need refrigeration and purification of the protein. F= How is the prese3nt Invention constitutes an improvement over what is already known. The present invention is an unique invention. This is for the first time we have produced rabies vaccine in tobacco, Muskmelon and groundnut. We have shown that the plant system can produce enough of glycoprotein, which can induce antibodies in mice and also survive the challenge of the virus. Hence the system can be commercially utilized for the production of rabies vaccine in plants. Earlier the fermentation, cell culture and other methods used were very expensive to start with and also they need refrigeration and there are problems of contamination. These problems can be overcome by using this new method. G= Statement of inventions. Keywords: MS- Murashige and Skoog Medium BAP- Benzyl Adeno Purine NAA- Naphthalene Acetic Acid IBA-Indole Butyric Acid PCR-Polymerase Chain Reaction FCv-Flue Cured Tobacco Variety PAGE-Polyacrylamide Gel electrophoresis CVS- Challenge Virus Strain PRGSPRgpKDEL-Rabies Plasmid Construct. Disclosed here is the novel process, which facilitates the cheaper and safer production of rabies vaccine in the plant systems like tobacco, muskmelon. Groundnut and other plants. The novel process involves the choice of the plant system, the transformation of rabies gene into the plant and extraction of the glycoprotein from plants, feeding the animals oral, edible/injectable form for immunization against rabies. This invention contemplates producing an improved anti rabies vaccine which comprises the following steps:- a) Insertion of anti rabies glycoprotein gene in plants by agrobactria method or micro particle bombardment using growth regulators in a tissue culture medium. b) Collecting the plant produce of genetically modified crops. c) Extracting the anti rabies glycoprotein by affinity chromatographic method. d) Purifying the said extract by column purification method, that can be used as an injectable and e) Optionally, lyophilising the said extract for oral administration. In this process, the desired growth regulators employed are BAP-Benzyl Adeno Purine, NAA-Napthalene Acetic Acid, IBA-Indole Butyric Acid. In the current invention, evaluation of the transgenic Muskmelon and Tobacco with rabies glycoprotein gene has been carried out. Observations on growth and yield parameters of the transgenic and untransformed Muskmelon and Tobacco were recorded. In case of Muskmelon the transgenic plant showed poor growth when compared to the untransformed plant whereas in Tobacco, the transgenic plant showed better growth than the untransformed plant. The expression of the rabies glycoprotein in the transgenic Muskmelon and Tobacco were confirmed by SDS-PAGE, western blot and ELISA, Affinity chromatography for the crude protein from transgenic Muskmelon and Tobacco gave partially purified rabies glycoprotein, which was tested by ELISA, western blot and SDS-PAGE. The transgenic Muskmelon and Tobacco plants showed higher total protein based on fresh weight when compared to control plants which may be due to the over expression of rabies glycoprotein. The Muskmelon fruits confirmed to contain the rabies glycoprotein were used for protein extraction and protein was injected into Female Swiss albino mice by intramuscular and intraperitoneal route to test for immunogenic nature. The results showed that the crude protein along with Freund's adjuvant when administered intramuscularly gave a higher antibody titre of 1.6 lU/ml. The challenge experiments with CVS strain of virus resulted in the protection of mice from rabies virus. This study confirms the potential of transgenic Muskmelon and Tobacco plants with rabies glycoprotein as vaccine candidates. In a similar experiment on Groundnut also confirmed the expression of rabies glycoprotein in groundnut seeds. The purified column protein of these plants by oral and injections gave better results than the crude protein. The lyophilized powders of the plants also have shown the immunogenicity in animal systems. This invention teaches the following new features and solves existing problems in a better way in following areas. 1. Increased creation of Rabies glycoprotein 2. Improvements of extraction of Rabies glycoprotein from plants in comparison to animal based vaccines 3. Improvement in terms of absorption by body if administered orally and stability without refrigeration. The inventors have used growth regulators for hither by hither to unknown methods. More particularly, the growth regulators BAP and NAA in the concentration of 1 to 5 mg/1 and 0. 1 to 5 mg/1 respectively when incorporated to the tissue culture medium of the plant tissues of Muskmelon and Tobacco having the rabies anti glycoprotein gene, facilitates optimum multiplication of the tissues with the inserted gene and accumulates the Rabies glycoprotein in all parts of the plant. (While the normal plants do not produce any Rabies glycoprotein). The innovative step of this invention lies in the identifying the best growth regulator combination for the enhancement of multiplication of the transgenic tissues with Rabies glycoprotein gene. / Table 1 illustrates the successful integration and the expression of the inserted gene in Tobacco and Muskmelon plants. The results indicated in table 2 show the ratio of accumulation of Rabies glycoproteins in Muskmelon ranging from 0.18 to 0.40. Table 3 illustrates the percentage of total protein content in Muskmelon seeds, which ranges between 4.55% in control varities and 7.3 to 9.16 % in the transgenic variety disclosed under the invention. In other words, the inventive steps lies in increasing the overall protein content in the seeds, which is attributable to the insertion of the Rabies glycoprotein gene. Table 4^dicates the results of ELISA, by measuring protein content by optical density at 492 nanometers. The table 4 shows that normal verities recorded OD 0.028 where as the transgenic leaves under the invention recorded 1.154 to 1.782, showing increased protein presence. Table 'Sy&vt tobacco plants under invention, produce the glycoprotein in the range of 0.8 % to 1.2%of weight of the percentage total proteins. / Table 6/is the molecular characterization of the transgenic tobacco plants. This table sows the successful integration and expression of the anti rabies glycoprotein in Tobacco plants, when treated with the growth regulators. Table 7/indicates the results of neutralizing antibodies titers in Mice immunization, when the column purified rabies glycoprotein obtained from Muskmelon fruit sample is injected to the mice through intra muscular and intra peritoneal routes. The neutralizing antibody titers ranged from 0.7 to 1.6 international units. Table 8 indicates the results of Mouse protection tests. The results indicate that 66% of the Mice administered with this vaccine survived the infection.. Table 9 shows the results of immunization of mice with rabies glycoprotein obtained from transgenic and control muskmelon fruits. The transgenic and control fruits of muskmelon were squeezed and the extracted Juice was lyophilized. The protein concentration was estimated in tiie lyophilized powder. The fruit powder was made in the form of pellet was fed to the mice orally. The oralfeeding induced 0.5IU/ml of antibodies on 60tii day where as the control did not produce any antibodies. As per the WHO recommendation 0.5IU/ml antibody is sufficient to immunize the animals. Table 10 depicts the percent total protein in lyophilized transgenic and control muskmelon fruit at 0 and 30 days after storage at room temperature. The protein content of the lyophilized powder was estimated on 0 and 30th day. The percentage total protein was 0.13 on 0 day in control and 0.35 in transgenic lyophilized powder. On 30th day again the lyophilised muskmelon fruit protein was estimated in control and found to be 0.13 percent and in transgenic it was 0.35 percent. This data shows that the fruit protein was conserved in the lyophilized powder without any degradation even at room temperature. The extraction of recombinant Rabies glycoprotein from yeast, bacteria and other animal systems involve cumbersome procedure, having high capital investments such as fermenters, fermentation ponds, which needs investment of millions of dollars, whereas the rabies glycoprotein from transgenic plants can be grown and glycoprotein extracted at negligible cost. Besides, the plant based vaccine also avoids contamination by other animal virus, which is inherent in any animal based glycoprotein extraction. The animal based vaccine, the protein extraction requires several steps including the use of enzymes and chemicals, where as our plant based rabies vaccine, we have extracted the protein by simple grinding of the transgenic tissues with water. The invention also discloses a better method of absorption of glycoprotein when administered orally. The glycoproteins encapsulated in the cell wall and the cell wall is made up of cellulose, which is hydrophilic in nature (water soluble), gets hydrolyzed in saliva and releases the glycoprotein into system. Since the rabies vaccine under invention triggered by mucosol immunity, the digestive system absorbs the protein and evokes the immunity. The presence of the cell wall in the plant tissue offers an advantage of storing the lyophilize powder under room temperatures, where as the lyophilized powder available in the market requires continuous cold storage facilities from the date of manufacture to the delivery. The invention for the first time teaches that column purification of plant based vaccines are possible using con A- column. The rabies glycoprotein produced implants passed through the con A-column resulted in high purity vaccine. There are no reports of use of con A- column for purification of Rabies glycoprotein produced in plants. We claim, KAn improved method of producing plant based rabies vaccine comprising the following steps of; A) introducing the rabies glycoprotein gene in plants, either by Agrobacterium method or by micro projectile bombardment, using growth regulators either alone or in combination with others in tissue culture medium. B) Collecting the plant produce of the genetically modified crops. C) Extracting the antirabies glycoprotein by without rupturing the cell wall D) Optionally the produce of step C is subjected to affinity chromatographic method and further purified by column purification method with or without use of freunds adjuvant to obtain injectable vaccine and optionally ,recovering the desired glycoprotein from the extract step C by lyophilizing the produce for oral administration. 2. The method as per claim 1 where in the plants suitable for transformation are Muskmelon, Groundnut and Tobacco. 3. The method of claim I where in the plant produce include fruit, leaves ,stem, shoots etc. 4,The mehod as claimed in claim 1 where the growth regulator used are recovering the desired glycoprotein by lyophilizing the produce for oral administration. E) Or, alternatively the produce , step C is subjected to affinity chromatographic method and further purified by column purification method with or without use of freunds adjuvant, if it is to be used as an injectible vaccine. 2. A method as per claim 1 where in the plants suitable for transformation are Muskmelon, Groundnut, and Tobacco. 3. A method of claim, where in the plant produce includes, fruit ,leaves ,stem shoots etc. 4. A method as claimed in Claim 1, where the growth regulators used are Benzyl Adino Purine (BAP), Napthaline Acetic Acid (NAA), Indole Buteric Acid (IBA), 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) and Thiodiozuron (TDZ). 6. A method as per Claim 1, where the optimum growth regulator to be used is BAP 1 to 5mg/L and NAA at 0. 1 to 5mg/L, to facilitate direct regeneration of transgenic plants especially in Tobacco and Muskmelon. 7. A method as claimed in Claim 1, where the optimum growth regulator is IBA in the range of 0.1 to 5mg/L to facilitate rooting of transgenic Muskmelon and Tobacco plants. 8. A method as claimed in Claim 1, where 2, 4 -D at 3 to 5mg/L in combination with Thiodiozuron (TDZ) 0.1 mg to 0.5mg/L is used for the production of transgenic Muskmelon plants . Table 5: Percent total protein based on fresh weight in R2 generation Transgenic and control tobacco plants* LEGEND : Ti- T4 = Transgenic Tobacco C = Untransformed Tobacco Table 6: Molecular charactersization of R2 generation transgenic plants of Tobacco. Table-9: Immunization of mice with rabies glycoprotein obtained from transgenic and control muskmelon fiiiits (oral immunization) Table-10: Percent total protein content in lyophilized transgenic and control muskmelon fruit at zero and 30 day after storage at room temperature. Note: No degradation of lyophilized protein at room temperature.

Documents:

0036-che-2003 claims duplicate.pdf

0036-che-2003 description (complete) duplicate.pdf

0036-che-2008 abstract duplicate.pdf

0036-che-2008 others duplicate.pdf

036-che-2003-abstract.pdf

036-che-2003-claims.pdf

036-che-2003-correspondnece-others.pdf

036-che-2003-correspondnece-po.pdf

036-che-2003-description(complete).pdf

036-che-2003-form 1.pdf

036-che-2003-form 3.pdf