RABIES VACCINE

RABIES VACCINE

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

• 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)

Rabies -Introduction

Longitudinal Section of Rabies Virus

Sequential Steps of Rabies Transmission

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.

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

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

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

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.

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.

Rabies vaccine

PRINCIPLE:

Potency of rabies vaccine is determined bycomparing a lethal intra-cerebral dose of arabies virus with the dose of the standardpreparation of rabies vaccines necessary togive for same protection.

Rabies Vaccine AssayTest 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

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 clotsMethod 1 Method 2

10% suspension brain homogenised Brain homogenised with diluentgives 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)

Rabies Vaccine AssayVirus 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.

Rabies Vaccine Assay cont’nPrepare 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.

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.

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.

DET. OF TEST DOSE OF VIRUS:Virus dilutionequal 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.

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

↓

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

Rabies Vaccine Assay cont’nVaccine 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.