RABIES VACCINE By: J.S.K. NAGARAJAN, ASST. PROFESSOR, JSS UNIVERSITY, (OFF CAMPUS, JSS COLLEGE OF PHARMACY), OOTY- 643 001. E-mail: firstname.lastname@example.org Ph: 09443149945
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)
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 vaccinePRINCIPLE: 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.
Rabies Vaccine AssayTest Animals:Mice – 3 -4 weeks old11 – 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 clots Method 1 Method 2 10% suspension brain homogenised Brain homogenised with diluent gives 10% suspension Centrifuge lightly & collect the supernatant liquidDistribute into sterile vials Distribute in sterile ampoules/vialsFreeze 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 toprotect mice against a lethal intracerebral dose of rabies vaccine necessary toprovide the same protection.
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 sameAfter 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 bycomparing a lethal intracerebral dose of rabies virus with thedose of standard preparation of rabies antiserum necessary togive 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 potencyis 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 ofthe 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 liesbetween 5 to 50 LD50.