1. RABIES VACCINE

2. RABIES VACCINE
By:
J.S.K. NAGARAJAN,
ASST. PROFESSOR,
JSS UNIVERSITY,
(OFF CAMPUS, JSS COLLEGE OF PHARMACY),
OOTY- 643 001.
E-mail: nagasaki2001@rediffmail.com
Ph: 09443149945

3. Rabies -Introduction
• Viral disease - cause acute encephalitis (inflammation of the
brain) in warm-blooded animals.
• Zoonotic (animals to humans).
• Bite from an infected animal.
• For a human, rabies is almost invariably fatal if post-exposure
prophylaxis is not administered prior to the onset of severe
symptoms.
• Virus infects the CNS, cause disease in the brain & death.
• Causative Organism – Lyssavirus genus(Family Rhabdoviridae)

4. Longitudinal Section of Rabies Virus

5. Sequential Steps of Rabies Transmission

6. Virus lifestyle
• Virus replicated in muscle cells near site of bite for most of
incubation time.
– Incubation time 30-90 days.
• Then ascends along motor and sensory axons at rate of 12-
100mm/day and has predilection for brainstem and medulla
• Enters salivary glands after replication in CNS.

7. Animal Behavior
• Classic Picture of rabid, mangy dog foaming at the
mouth…not often seen, signs frequently more subtle.
• Animals - display aggressive behavior, ataxia, irritability,
anorexia, lethargy/excessive salivation.
• Cats are more likely to be aggressive than dogs
• Animals exhibit change in instinctive behavior: nocturnal
animal walking around in daylight (i.e. raccoons)
• Unprovoked bites

8. Transmission
• Almost all transmission is by bite
• 50 times greater risk than a scratch
• One human case may have been acquired in a
laboratory transmitted by aerosol
• In wild animals: Rabies can be transmitted
transplacentally
• Transplants in human- possible
• Human-to-human: Never has been confirmed
• Rabies virus never isolated from blood

9. Rabies virus
• Risk of developing rabies after a bite: 5-80%.
– Depends upon….
• Severity of exposure
• Location of the bite
• The biting animal
• Bites on head and neck have shorter incubation time
(as short as 15 days) because of rich peripheral nerve
supply

10. Encephalitic Form
• Hydrophobia: Patient can’t swallow because violent jerky contraction of
diaphragm and accessory muscles of inspiration when pt attempts to
swallow liquids
- Patients will be terrified during this reaction and may even
experience this at the sight of water or if water touches their face.
• Aerophobia: an extreme fear of air in motion can be elicited from some
patients. This can also cause violent muscle spasms in the neck and
pharynx.
• Hallucinations, seizures, ataxia, focal weakness and arrhythmias can
occur.

11. Paralytic Rabies
• Other form is “dumb” or paralytic rabies. Similar
to Guillain-Barre.
– Prominent limb weakness. Consciousness
initially spared
• Two forms can overlap or progress from one to the
other
• Coma after one week of neuro symptoms with
death a few days after.

12. Rabies vaccine
PRINCIPLE:
Potency of rabies vaccine is determined by
comparing a lethal intra-cerebral dose of a
rabies virus with the dose of the standard
preparation of rabies vaccines necessary to
give for same protection.

13. Rabies Vaccine Assay
Test Animals:
Mice – 3 -4 weeks old
11 – 15 gm
6 X 16 and 4 X 10
Calculate LD 50 For Virus suspension
Estimation Of Potency

14. Preparation of Standard Challenge Virus Suspension:
Inject the Virus (0.3ml of 10 fold diln. -Intra Cerebrally)
Sacrifice the animal(after getting signs)
Harvest the brain aseptically
Wash with saline to remove blood clots
Method 1 Method 2
10% suspension brain homogenised Brain homogenised with diluent
gives 10% suspension
Centrifuge lightly & collect the supernatant liquid
Distribute into sterile vials Distribute in sterile ampoules/vials
Freeze dry and store @ -200 C Store @ -600 C ( maintain NLT 1 Yr.)
(maintain for NLT 3yrs)

15. Rabies Vaccine Assay
Virus Tire of the Challenge Virus:
Prepare 3-10 fold dilution of std.challenge virus suspension.
↓
0.03ml inject intracerebrally to 4 X 10 mice.
↓
Observe mice for 14 days.
↓
Count the number of mice surviving in each group.
↓
Calculate the virus titre of std challenge vurus suspension by statistical
method.
Determination of Potency:
Potency of rabies vaccine is determined by comparing the dose necessary to
protect mice against a lethal intracerebral dose of rabies vaccine necessary to
provide the same protection.

16. Rabies Vaccine Assay cont’n
Prepare 3-5 fold serial dilution of standard and test soln of vaccines
Separate mice in 6 groups of 16each
Inject the vaccine 0.03ml(Intra-Peritoneal)
After 7 days inject the same
After further 7 days inject 0.03ml Standard challenge virus suspension
by 5 to 50 LD50
Observe for 14 days and record the number of survivals
Calculate the potency of the preparation under examination by
standard statistical methods.

17. RABIES ANTI SERUM
• Rabies anti serum is a preparation containing the specific
globulin [or] its derivatives obtaines by purification of hyper
immune serum[or] plasma of healthy horses [or] other
animals having the specific activity of neutralizing the
rabies virus.
• BIOLOGICAL ASSAY OF RABIES ANTISERUM:
The potency of rabies antiserum is determined by
comparing a lethal intracerebral dose of rabies virus with the
dose of standard preparation of rabies antiserum necessary to
give same protection.

18. PROCEDURE:
STANDARD PREPARATION:
Standard preparation is dried serum (or)
Other preparation , the potency of which has been
determined in relation to international standard.
TEST ANIMALS:
Mice, 10g-14g-animal same sex.
TEST VIRUS:
Any suitable strain rabies virus of known potency, such as the
CVS strain may be used.
TEST DOSE OF VIRUS:
20-1000 LD50 intracerebral injection to each mouse.

19. DET. OF TEST DOSE OF VIRUS:
Virus dilutionequal quantity of 2%v/v solution of heat
inactivated horse serum in water .
↓
Maintain at 37⁰c at one hour.
2) Prepare is 10 fold dilution in a 2%v/v solution of heat –
inactivated normal horse serum
Inject into mouse.
↓
The test is not valid unless the quantity of virus used lies
between 20-1000 LD50.

20. DETERMINATION POTENCY OF RABIES
ANTISERUM:
Prepare 2 fold dilution of std preparation and test
preparation with 2% v/v heat inactivated
normal horse serum in water.
↓
To each dilution add a quantity of suspension of
test virus
↓
Keep the mix at 37⁰c for 1hr
↓
Inject 0.03ml intracerebrally to mice
↓

21. Observe mice for 14days
↓
Mice dying before 5th day after inoculation of virus are eliminate
from test, all the mice dying between 5th -14th day after
showing signs of rabies
↓
Count the no of mice surviving
↓
Calculate potency of the test preparation by std
statisticalmethod
– The preparation pass the test it found to have 80units /ml
– The preparation for the test also same as standard

22. Rabies Vaccine Assay cont’n
Vaccine complies with the test if the estimated potency
is NLT 2.5 IU/single dose.
- The test is not valid unless
(a) for both the ppn under examination and the std
ppn, the 50% protective dose lies between the
largest and smallest doses given to the mice;
(b) there is not deviation from linearity or parallelism of
the dose response lines, the confidence limit (P= 0.95)
are NLT 25% & NMT 400% of the estimated potency;
(c) the titre of the challenge virus suspension lies
between 5 to 50 LD50.