Development, safety and efficacy analysis of liquid state rabies
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The document details the development and analysis of a liquid state rabies vaccine for human use, focusing on biochemical estimations, stability, and potency evaluations of vaccine samples. Specific analytical techniques such as HPTLC and SRID were applied to assess the vaccine's stabilizers and immune response. The findings suggest that certain formulations demonstrate significant potency and safety, paving the way for future research in rabies vaccine efficacy.
Development, safety and efficacy analysis of liquid state rabies
- 1. DEVELOPMENT AND EFFICACY ANALYSIS OF LIQUID STATE RABIES VACCINE FOR HUMAN USE External Guide: S.Jagannathan, M.Sc, PGDBI, MBA, Ph.D(Pursuing) Assistant Research Officer, PIIC, Coonoor. Submitted by: Balaganesh.K , Prashanth Kumar. K. 1OX06BT002 1OX06BT013.
- 2. Contents:
- 3. Abstract In this set of review, the biochemical estimation of magnesium chloride and Trehalose by the D-HPTLC method will be dealt along with the REFIT analysis of the antisera , stability analysis of the samples and the docking studies carried out in identifying the component acting as a suitable stabilizer in maintaining the native state of Ag will be found out.
- 4. Topics covered in last reviews SRID has been performed for different samples which are obtained from the TCARV section of the PIIC The potency of the samples was estimated quantitatively from the results of the SRID Based on this potency value the sample which is having more potency was selected for the further formulation Haemagglutination , Formulation. Biochemical estimations of all components used. Animal studies (Production of primary & secondary antibodies,abnormal toxicity tests)
- 5. Methods and Materials Further experimental studies conducted upon the formulated vaccine samples were as follows – Biochemical estimation (contd) - Estimation of magnesium chloride HPTLC of Trehalose REFIT analysis In silico analysis of stabilizers used.
- 6. SRID potency Check of the formulated Vaccine samples. The formulated Vaccine samples were subjected for stability analysis via the SRID method , conducted on a periodical basis in the alternate days till the 7th day and later on once in 5 days,. The potency evaluation will be carried out on a long term basis to ensure the efficacy of the formulated vaccine samples.
- 8. ESTIMATION OF MAGNESIUM CHLORIDE IN VACCINE SAMPLE
- 9. HPTLC Analysis of Trehalose content ANALYSIS DETAILS MOBILE PHASE : n-butanol-pyridine-water ( 8:4:3) SPRAY REAGENT: N-(1-naphthyl)-ethylenediamine dihydrochloride in methanol containing 3% sulphuric acid reagent (Bournias, 1980) DETECTION : Bluish white colored fluorescent zones at UV 366nm QUANTIFICATION OF RESULTS Area of Sample x Concentration of Standard The % of Trehalose = _____________________________________ x 95 Area of Standard x Concentration of Sample
- 10. And the process was carried out in the steps mentioned as follows
- 11. Insilico analysis Homology modeling of the Rabies Viral GProtein G – Viral protein sequence was obtained and was process for various steps and the appropriate template was selected and whose pdb id was retrieved and the modeling request was submitted through Swissproteomics server using Spdbv software.
- 12. Docking of the modeled protein with various stabilizers. Receptor : Modeled Rabies Viral G Protein. Ligands being – Trehalose, Lactose, Aluminum Phosphate, Magnesium Chloride, Human Albumin.
- 13. Results and Discussion REFIT REFIT Analysis Table shows: Immune response analysis Sl.N o Name of the Vaccines Booster dosage RFFIT (Rabies Neutralizing antibodies in IU/ml) 14th Day 21st Day 1. PRVA 02/08 On 7th day 0.5 8.0 2. Local Reference (HO23) On 7th day ≥32.0 ≥32.0 3. TCARLV-A On 7th day 8.0 8.0 4. TCARLV-B On 7th day 2.0 32.0 5. TCARLV-C On 7th day 16.0 16.0 6. TCARLV-D On 7th day ≥32.0 32.0
- 14. Fig. shows: Positive fluorescence indicating presence of antibody The positive fluorescence indicating the presence of antibodies against the administered Formulated Vaccine samples in the anti sera collected from the animals subjected for immunization
- 15. SRID Potency evaluation A: 5th day samples ( both +4 and +34˚C) A B B: 10th day samples ( both +4 and +34˚C) C: 15th day samples ( both +4 and +34˚C) D: 20th day samples ( both +4 and +34˚C) C D
- 16. HPTLC ANALYSIS OF TREHALOSE CONTENT Table shows : Sample code for HPTLC Chromatogra m code Sample code Quantity A Sample A 20µg in 1µL T1 Trehalose standard 150ng in 1.5µL T2 Trehalose standard 300ng in 3µL T3 Trehalose standard 450ng in 4.5µL T4 Trehalose standard 600ng in 6µL T5 Trehalose standard 750ng in 7.5µL C Sample C 20µg in 1µL D Sample D 20µg in 1µL
- 17. 3D Peak densitogram of all tracks scanned at 366nm Using which the concentration of the trehalose was determined in the formulated samples A and D.
- 18. Table shows: Peak table for the analysed samples Track Peak Rf Height Area Assigned substance A 1 0.19 48.3 874.4 Trehalose T1 1 0.19 46.5 1057.7 Trehalose standard T2 1 0.19 47.6 948.1 Trehalose standard T3 1 0.19 59.4 1046.1 Trehalose standard T4 1 0.19 33.2 483.8 Trehalose standard T5 1 0.19 40.7 554.0 Trehalose standard C 1 0.19 44.2 777.8 Trehalose D 1 0.20 42.5 632.5 Trehalose
- 19. Chromatogram Fig showing Chromatogram for TCARLV A D TCARLV C TCARLV
- 20. QUANTIFICATION OF RESULTS Area of Sample x Concentration of Standard The % of Trehalose = _____________________________________ x 95 Area of Standard x Concentration of Sample 874.4 0.45 Sample A = ________x _____ x 99 = 1.86% 1046.1 20 Sample C = 1.65% Sample D = 1.35%
- 21. Magnesium chloride estimation Samples Volume Conc. Of Volume Volume Volume Volume Volume OD at of MgCl2 of of of Titon of 4N of CaCl2 540nm std/unkn (µg) distilled Gumgha yellow NaOH (ml) H2O (ml) patti (ml) (ml) (ml) own taken (ml) Blank - - 2.5 1.0 1.0 2.0 1.0 0.00 S1 0.2 200 2.3 1.0 1.0 2.0 1.0 0.152 S2 0.4 400 2.1 1.0 1.0 2.0 1.0 0.311 S3 0.6 600 1.9 1.0 1.0 2.0 1.0 0.445 S4 0.8 800 1.7 1.0 1.0 2.0 1.0 0.680 S5 1.0 1000 1.5 1.0 1.0 2.0 1.0 0.780 Concentration of MgCl2 = (Test OD/ Std. OD) x1.0 Concentration of standard x Sample 1.0 1.0 2.0 1.0 0.708 (1/1.0) B = (0.708/0.608) x 800 =832.94 µg =0.83 mg/SHD.
- 22. Docking Results Ligand E Total E Shape E force E air V shape V clash -499.03 -499.03 0.00 0.00 0.00 0.00 Lactose -183.81 -183.81 0.00 0.00 0.00 0.00 Trehalose -171.18 -171.18 0.00 0.00 0.00 0.00 MgCl2 - 144.37 -144.37 0.00 0.00 0.00 0.00 AlPO4 -110.33 -110.33 0.00 0.00 0.00 0.00 Name Human Albumin From which, it can be conferred that Human Albumin acts a better stabilizing agent in the formulated samples, and followed by the rest.
- 23. CONCLUSION Purified rabies viral antigens obtained from the TCARV section of Pasteur Institute of India, Coonoor, were analysed for their safety, potency and purity by various IPQC tests. Based on the values sample PRVA 02/08 shows some very good results when compared to the other samples. PRVA 02/08 was used to formulate four kind of liquid rabies vaccines with different stabilizers and additives and are named as TCARLV-A, TCARLVB, TCARLV-C and TCARLV-D. All the formulated vaccine samples were analysed for their safety by abnormal toxicity studies in Mice and Guinea pigs. The constituents of the vaccines were estimated biochemically. The potency of the vaccines was estimated by RIFFIT method and all the vaccine samples shows good amount of potency, especially vaccine TCARLV-B and TCARLV-D shows greater amount of potency. Our contribution however opens the door for further investigation and hopefully might also paved the way for further research. It is a valuable tool to fight against rabies in man.
- 24. Bibliography Morefield GL, sokolovska A, Jiang D, et al., 2005. Role of aluminumcontaining adjuvants in antigen internalization by dendritic cells in vitro. Vaccine 23; 1588-1595. Nickerson, et al (1976) Colorimetric estimation of Lactose and its hydrolytic products, J. Dairy Science 59, No 3, page386. PathScan® Sandwich ELISA Protocol. Tsiang H; (1993).Pathophysiology of rabies virus infection of the nervous systemIn: Adv. Virus Res; 42: 375-412.Cited: Plotkin S.A; Rupprecht C.E. and Koprowski M; (1999). WHO; (1992).Expert committee on Rabies. Diagnosis, 8th Report.WHO, Geneva.In: Tech. Rep. Ser.NO.824; 1-84. Wiktor. T.J.; Macfarlan, R.I.; Forggin, C.M.; Koprowiski, H.; (1984).Antigenic analysis of rabies and Mokola Virus from Zimbabwe using monoclonal antibodies.In: Dev. Biol.Stand;57: 199-211Cited from: Dietzschold, B.; Rupprecht, C.E.; Fu, Z.F.; and Koprowiski, H.(1996). Wunner, W.H.; (1992).Rabies. In: Encyclopedia of Microbiology, 3:497-508. Jagan nathan.S. et al., 2008. Filtration techniques in Vaccine manufacturing., Journal of advanced Biotechnology 07(02); 37-41.
- 25. Acknowledgement For the first we would like to thank The principal, The Oxford College of Engineering and Head of Department, Biotechnology and all the faculty for providing us the permission and support as well as the opportunity to carry out this project at PIIC. Followed by Pasteur Institute of India, Coonoor for providing us with all the facilities required for the execution of project and for its stupendous cooperation as well.
- 26. And also we would like to thank, Dalmia Centre for Research and Development Coimbatore, India. For their co-operation and full-fledged assistance in quantifying the amount of Trehalose present in the formulated samples A, C and D. And finally, would like to thank all my friends for all their support and for being an inspiration forever.