Presentaci

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Avian Flu Detection, Research and Vaccine
Production
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Influenza Virus Avian Flu BSL-3 Detection,
Research and Vaccine Production
Orthomyxoviridae family (RNA) Two genera 3
types - influenza A and B viruses - influenza C
virus

• Influenza A infect a range of animals (humans,
  swine, birds, seals, horses)
• Influenza B infect only humans
• Influenza C infects humans and swine.
• Influenza A subtypes (surface glycoproteins
  antigenicity)
• hemagglutinin (H1-H15)
• neuraminidase (N1-N9)

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Mutations Avian Flu BSL-3 Detection, Research
and Vaccine Production
Antigenic Shift
Antigenic Drift
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Avian Influenza Viruses Avian Flu BSL-3
Detection, Research and Vaccine Production
Avian strains of influenza A viruses
Wild water birds (natural reservoir) infection
avirulent, little/no symptoms. To poultry
species (chickens, turkeys) mild clinical
symptoms Transmision direct physical contact,
through contact with surfaces, water or feed
contaminated with virus.
LPAIV low pathogenic avian influenza virus
HPAIV high pathogenic avian influenza virus
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WHO Detection Protocol Avian Flu BSL-3
Detection, Research and Vaccine Production

• The WHO is co-ordinating the Detection of Avian
  Flu strains in the area, through national
  networks of laboratories.
• The procedure approved by the FDA on 3rd of
  February 2006 is called the Influenza A/H5 (Asian
  lineage) Virus Real-time RT PCR
• Primer and Probe Set The test provides
  preliminary results on suspected H5 influenza
  samples within four hours once a sample arrives
  at the lab and testing begins. If the presence of
  the H5 strain is identified, then further testing
  is conducted to identify the specific H5 subtype
  (e.g., H5N1)

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Avian Flu virus handling WHO guidelines for
BSL-3 Avian Flu BSL-3 Detection, Research and
Vaccine Production
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Avian Flu virus handling WHO guidelines for
BSL-3 Avian Flu BSL-3 Detection, Research and
Vaccine Production
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Avian Flu virus handling WHO guidelines for
BSL-3 Avian Flu BSL-3 Detection, Research and
Vaccine Production
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Modular BSL-3 by TPRO Avian Flu BSL-3 Detection,
Research and Vaccine Production
Autoclave
Introducing the sample Biological SAS
Centrifugal Thermal Cycler Real Time RT-PCR. In
Biosafety Cabinet Class II - A
Biowaste Treatment
Bag-In Bag-Out HEPA
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Modular BSL-3 by TPRO Avian Flu BSL-3 Detection,
Research and Vaccine Production
Cool down, storage
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4
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Entry undressing
Exit degowning
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5
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Entry gowning
Water shower
BSL-3 Technical area
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6
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Exit dressing
Working lab
Non critical technical area
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Influenza Vaccine Production types of
vaccines Avian Flu BSL-3 Detection, Research and
Vaccine Production
Influenza vaccines Trivalent inactivated virus
strains, either whole viruses or parts or
subunits of them.
Vaccine Types

1. Whole inactivated viruses.
2. Split inactivated virus particles disrupted
   (detergent treatment)
3. Subunit or surface antigen purified
   hemagglutinin and neuraminidase
4. Live attenuated whole virus (cold-adapted
   weakened/non-pathogenic).

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Influenza Vaccines production procedures Avian
Flu BSL-3 Detection, Research and Vaccine
Production
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Avian Flu vaccine production procedures Avian
Flu BSL-3 Detection, Research and Vaccine
Production

• Vaccines produced in fertilized chicken eggs
• Eggs 11 days after fertilization
• Each strain is grown separately
• Embryo infected ? virus multiplication
• Harvest, purification, inactivation and
  production
• 1-2 eggs to produce 1 dose of vaccine
• Entire production process lasts at least six
  months

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Avian Flu vaccine production procedures Avian
Flu BSL-3 Detection, Research and Vaccine
Production

• Flu vaccine based on cell or tissue cultures
• Mammalian kidney cells are preferably used for
  these cell cultures.
• Virus is injected into these cells, multiply
  before cells outer walls are removed
• Harvest, purification, inactivation and
  production
• Process similar to biotechnological fermentation,
  in which you move from small liter jars to huge
  fermenters during production.
• Described in the mid-nineties and is still in its
  experimental stage
• Sanofi Pasteur Cell Culture-Based Seasonal
  Influenza Vaccine Enters First Clinical Trial (27
  Sep 2006) PER.C6 cell line in 20,000 liters
  bioreactor.

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Avian Flu vaccine production procedures Avian
Flu BSL-3 Detection, Research and Vaccine
Production

• EGG-BASED PRODUCTION
• Advantages
• Well established and cost-effective
• Lower cost
• Disadvantages
• Extensive planning long timeline for million
  eggs procurement
• Limited flexibility in case of exponentially
  increasing demand (pandemic not contained
  defeated)
• production takes too long
• eggs dont grow on demand
• Potential impurities in eggs (antibiotics, other
  viruses) may cause sterility problems
• Risk of allergies against egg albumin
• Growth of epidemic viruses in eggs result in
  variants that are antigenically distinct from the
  original viruses
• Emerging endemic viruses sometimes do not grow
  at all in eggs

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Avian Flu vaccine egg based production
stages Avian Flu BSL-3 Detection, Research and
Vaccine Production
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Avian Flu vaccine egg based production Avian
Flu BSL-3 Detection, Research and Vaccine
Production
Reassortment
To maximize virus yield from eggs, reassortants
of influenza A strains are often utilized
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Avian Flu vaccine egg based production Avian
Flu BSL-3 Detection, Research and Vaccine
Production
Large scale ? not viable to use SPF eggs High
quality production eggs are obtained from
accredited flocks certified to rigorous quality
control standards. Eggs are incubated for 10-11
days before inoculation
Inoculation of Working Seed Virus
(manually/automated machinery) into the allantoic
cavity of the egg Virus cultivation Eggs
incubation at 35-37oC at a relative humidity of
around 65 for 48 to 72 hours to allow the virus
to multiply
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Avian Flu vaccine production procedures Avian
Flu BSL-3 Detection, Research and Vaccine
Production
Harvest Allantoic fluid. Manual or automated)
vacuum systems Then usually clarified by
centrifugation to remove cellular debris.

• Production process will vary from manufacturer to
  manufacturer, but the next stages normally
  include
• chemical inactivation formaldehyde and
  betapropiolactone
• concentration
• purification centrifugation on a density
  gradient or by column chromatography

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Avian Flu vaccine production facility
design Avian Flu BSL-3 Detection, Research and
Vaccine Production

• Production process manually vs automatic
  machines
• Aseptic manufacturing ? IM vials suspension
• BSL-2
• Pharmaceutical critical utilities
• HVAC Clean Room
• Pharmaceutical waters PW WFI
• Pure Steam
• CIP/SIP system
• Virus contaminated waste treatment
• Plastics, glass, paper ? Autoclave (121ºC
  30min)
• Liquids in container ? Autoclave (121ºC 30min)
• Liquid effluent (drains) ? BW (chemical)
• Solids eggs waste products ? Pasteurization
  (90secs, gt70ºC)

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