Making a vaccine

Question 1

Making a vaccine

Answer 1

The disease-causing agent must be isolated and attenuated, so it is no longer pathogenic. The correlates of production (i.e. antibodies & cytotoxic T lymphocytes involved in pathogenesis) must be determined, and so the targets antigens can be identified.

Question 2

Vaccines must be sterile

Answer 2

Therefore, they should be made in a clean room, using laminar flow cabinets under positive pressure conditions. There should be separation of personnel and equipment. There are many challenges facing formulation of vaccines, as soluble proteins used in vaccines are often poorly immunogenic when used on their own as a vaccine.

Question 3

Microbe culture

Answer 3

The pH is optimised, and the antigen is cultured.
* Viruses are grown on primary cells (cell line or cultivating virus into chicken embryo)
* Bacteria are cultured in bioreactors by fermentation
* Proteins are grown inside cells (yeast/ bacteria) using recombinant technology

Question 4

Harvest

Answer 4

Chemical inactivators remove pathogenicity and act as a disinfectant. The most commonly used agent is formaldehyde, which becomes formalin in water. It cross-links lysine in protein molecules, which means the antigen is not active but is present in the vaccine. This means the vaccine is immunogenic but does not cause harm.
Heat (> 60oC for 10 hours) causes irreversible denaturation, so the antigen is inactive.
Low pH (acid-denaturation, where pH < 4 for 6 hrs – 21 days) can be used, but there is a possibility that the antigen reactivates upon reverting back to normal pH.

Question 5

Purification of active

Answer 5

Antigens are purified by a multi-step process: 1. Differential precipitation/ solubilisation 2. Ultracentrifugation
3. Final purification by:
a. Ultrafiltration/nanofiltration (porous membrane molecular weight cut-off) b. Gel permeation (or SEC) chromatography (GPC)
c. Hydrophobic interaction chromatography (HIC)
4. Testing for purification uses ELISA, using colour as an indicator of the concentration.Formulation (includes adjuvant preparation)

Question 6

Classes of adjuvants
Water soluable

Answer 6

1)Water soluble/ dispersible
-Soluble lipid derivative version of E. coli - Monophosphoryl lipid A (MPL)
- Poly(I:C) is a synthetic analogues of double-stranded RNA (dsRNA) that is lipid soluble o Saponins (e.g. Quil A detergent)
-Toxins

Question 7

Particulate vaccine-delivery systems moA

Answer 7

o They target the antigen to antigen presenting cells (APCs), by converting the soluble protein into particles which APCs recognise and process.
§ Entrapment/absorptionagents(e.g.alum)adsorbsproteins
§ Mineraloils(e.g.MF59)emulsifiesproteins
§ Watersolubleagents(e.g.QuilA)formscolloidswithproteins

Question 8

Immunostimulatory moA

Answer 8

They directly activate APCs through Toll-like receptors (TLR) which results in inflammatory responses that amplify the innate immune response
§ MPL is a non-toxic component derived from lipopoly saccharide(LPS)of bacterial cell walls and interacts with TLR-4 and TLR-2, inducing a Th1-skewed response. MPL is thought to directly activate macrophages resulting in the induction of IFN-γ and IL-2. However, it is not as potent at inducing antibody responses.

Question 9

Stabilisers ensure the vaccine remains unchanged when the vaccine is exposed to heat, light, acidity, or humidity:

Answer 9

• Freeze-drying cryoprotectants e.g. sugars such as, sucrose and lactose
• Buffers e.g. amino acids such as, glycine or Na glutamate
• Complexing and suspending agents e.g. proteins such as, human serum albumin or gelatin or EDTA
• Relics e.g. foetal bovine serum remnant from cell culture, deactivation substrates
• Delivery aids e.g. gums and viscosifiers e.g. gelatin
• Preservatives e.g. BHA – butylated hydroxyanisole: prevents oils becoming oxidised and rancid
• Co-solvent e.g. 2-phenoxyethanol
• Emulsifiers e.g. Span, Tween, Brij

Question 10

Antibiotics may be used in multi-use formulations

Answer 10

to prevent fungal and bacterial infection as consecutive aliquots are removed from the same bulk container. They can also be part of the manufacturing process, where low concentrations remain in the final product.

Question 11

Vaccines are sterilised by pressured steam:

Answer 11

121oC for 6 minutes. This results in > 10-log reduction of microorganisms, killing vegetative cells & spores, denaturing enzymes and causing the thermo-destruction of macromolecules.

Question 12

Stability testing & Quality assurance

Answer 12

Potency, safety, cost, preparation, usage, stability in cold-chain conditions (5oC) and room temperature are determined.

Question 13

Vaccines are frequently sensitive to light and temperature (heat or freezing)

Answer 13

Most vaccines are damaged at freezing temperatures, producing agglomerates and precipitates. Cold-chain is a system of storage and transport, where temperature has an effect on potency, so refrigeration is required to maintain the efficacy of the vaccine. As a result, the vaccine must be stored and transported at 5 ± 3oC at all times. Refrigeration facilities must be calibrated to ensure accuracy within 1oC. Bubble- wrap insulants and cool-box interim transfer systems should be used. Multi-dose format vaccines must be returned to the fridge after the required dose is withdrawn, and aseptic non-touch techniques should be implemented. Data loggers ensure the storage temperature is within limits required.

Question 14

Where vaccines are sensitive to light (fluorescent/ UV),

Answer 14

they should be stored in their original container until use.

Question 15

Demands on manufacturing

Answer 15

Antigenic drift and antigenic shift are terms used to describe ways in which the flu virus changes over time.

Question 16

Drift –

Answer 16

a natural, minor mutation of the surface antigen.
When a virus mutates or changes slightly, it looks different to our immune system. The antibodies that your body created last year in response to the flu virus, or the flu vaccine no longer recognize the new virus.

Question 17

Shift –

Answer 17

a major change occurs to the surface antigens of the same strain, altering its virulence. This typically occurs when a human virus crosses with a virus that usually affects animals. Sub-types combine to make a new form with hybrid surface antigens that are not recognized by the human immune system.

Question 18

Vaccine producers must

Answer 18

be able to respond to seasonal & global changes in antigens quickly, to avoid pandemics.

Question 19

Vaccine shortages can occur for a number of reasons:

Answer 19

• Cold chain failures
• Aggregation and degradation (poor storage)
• Higher-than-normal demand
• Production interruption (natural disasters, lack of resources)

Question 20

Adjuvants

Answer 20

Adjuvants are used to enhance the immunogenicity of protein antigens; they are immune-potentiators. Adjuvants often act as a depot for the antigen, presenting the antigen to the body over a longer duration maximising the immune response prior to clearance.
Aluminium-based mineral salts (Alum), mineral oil (MF59), Monophosphoryl lipid A (MPL) and Virosomes are used as adjuvants. Aluminium toxicity needn’t be a concern; toxicity occurs at high concentrations (150 ± 50 mg/kg/day) and vaccinated children are exposed to only 4-8 mg Al in first 2 years.

Question 21

Oil-in-water/ water-in-oil emulsions ADJUVANTS

Answer 21

Oil-in-water/ water-in-oil emulsions, where 1-10% of volume of vaccines are emulsifiers (oils). They are used to disperse various agents
o Oil in water: mineral oil/ squalene (e.g. MF59) – Ebola, Rabies, Smallpox, HIV

Question 22

Water-in-oil

Answer 22

o Water-in-oil: vegetable oil base – Pneumococcus, Rotavirus
o Freund’s complete adjuvant is most commonly used; it usually contains inactivated and dried M. tuberculosis & MPL. It
can be problematic as it is reactogenic.
* Entrapment or absorption agents aid immune promotor
o Alum is the most commonly used agent, but it’s cell-mediated immunity is often poorly stimulated. These are aluminium salts: aluminium hydroxide/aluminium sulphate, aluminium phosphate; usually Al(OH)3
* Polymer particles
o Liposomes are self-assembled lipid particles which allow entry into cells o Virosomes are virus structured liposomes

Question 23

Saponins

Answer 23

Saponins are derived from the bark of a Chilean tree, Quillaia Saponaria, so unlike other immunostimulatory adjuvants, is not pathogen derived. A highly purified fraction called QS21 is a potent adjuvant for the induction of a Th1-dominated response, including CTLs. Saponins are thought to form pores in cell membranes that allow antigens to gain access to the endogenous presentation pathway resulting in presentation by MHC class I and hence CTL activation.