DEVELOPMENT AND EFFICACY ANALYSIS OF

LIQUID STATE RABIES VACCINE FOR HUMAN

USE

Submitted by:

Balaganesh.K , Prashanth Kumar. K. 1OX06BT002 1OX06BT013.

External Guide:

S.Jagannathan, M.Sc, PGDBI, MBA, Ph.D(Pursuing)Assistant Research Officer,

PIIC, Coonoor.

Contents:

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 anti-

sera , 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.

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)

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.

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.

REFIT Analysis

ESTIMATION OF MAGNESIUM CHLORIDE IN VACCINE

SAMPLE

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

And the process was carried out in the steps mentioned as

follows

Insilico analysis

Homology modeling of the Rabies Viral G-

Protein

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 Swiss-

proteomics server using Spdbv

software.

Docking of the modeled protein with various

stabilizers.

Receptor : Modeled Rabies Viral G Protein.

Ligands being –

Trehalose, Lactose,

Aluminum Phosphate,

Magnesium Chloride,

Human Albumin.

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

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

SRID Potency evaluation

A B

C D

A: 5th day

samples ( both

+4 and +34˚C)

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)

Chromatogra

m

code

Sample code Quantity

A Sample A 20µg in 1µL

T1Trehalose

standard150ng in 1.5µL

T2Trehalose

standard300ng in 3µL

T3Trehalose

standard450ng in 4.5µL

T4Trehalose

standard600ng in 6µL

T5Trehalose

standard750ng in 7.5µL

C Sample C 20µg in 1µL

D Sample D 20µg in 1µL

HPTLC ANALYSIS OF TREHALOSE CONTENTTable shows : Sample code for HPTLC

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.

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

Chromatogram

Fig showing Chromatogram for

TCARLV A TCARLV C TCARLV

D

QUANTIFICATION OF RESULTS

Area of Sample x Concentration of StandardThe % of Trehalose =

_____________________________________ x 95Area 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%

Magnesium chloride estimation

Samples Volume

of

std/unkn

own

taken

(ml)

Conc. Of

MgCl2

(µg)

Volume

of

distilled

H2O (ml)

Volume

of

Gumgha

patti (ml)

Volume

of Titon

yellow

(ml)

Volume

of 4N

NaOH

(ml)

Volume

of CaCl2

(ml)

OD at

540nm

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

Sample

B

1.0 1.0 1.0 2.0 1.0 0.708Concentration of MgCl2 = (Test OD/ Std. OD) x Concentration of standard x

(1/1.0)

= (0.708/0.608) x 800

=832.94 µg

=0.83 mg/SHD.

Docking Results

Ligand

Name

E Total E Shape E force E air V shape V clash

Human

Albumin

-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

From which, it can be conferred

that Human Albumin acts a

better stabilizing agent in the

formulated samples, and

followed by the rest.

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, TCARLV-B, 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.

Bibliography

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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).

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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.

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.