Rabies is a highly fatal epidemic disease in the world with high mortality rate in the infected individuals. According to the survey conducted by WHO across different parts of the globe, every year 50000 people die because of Rabies. And most of the vaccines are produced as solid-state vaccines. Before formulation the purified PV 11 derived concentrated, infected and chromatographically purified rabies antigens are checked for their efficiency, potency by invitro methods. Four different combinations of stabilizers, additives and adjuvants are blended with rabies antigen. Those are labelled as TCARLV-A, TCARLV-B, TCARLV-C, TCARLV-D. And find estimate the constituents in single Human dose.

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.

7. REFIT Analysis

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.