Julius Ho
Biol1220
4/20/10

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Historical overview of vaccines
Genetic methods in vaccine development
Use influenza as an example for new synthetic bio approaches

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 Heterologous
 Attenuated
 Inactivated
 Toxoid

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Immune response from a non-pathogenic relative of the organism
Smallpox vaccine (1796):
 Edward Jenner uses coxpox virus to induce resistance to smallpox http://en.wikipedia.org/wiki/File:Edward_Jenner2.jpg
http://emergency.cdc.gov/agent/smallpox/images/hand_position_for_vaccination.jpg

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Reduce pathogenicity of virus/bacteria by repeated culturing
TB vaccine (1921):
 Discovery of Mycobacterium bovis, a relative of M. tuberculosis
 Selection of less-virulent strains over 10+ years http://feww.files.wordpress.com/2008/10/tb.jpg
http://www.bbc.co.uk/jersey/content/images/2005/11/24/jersey_cow_350x350.jpg

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Kill the actual pathogen but expose immune system to the remnants
Polio vaccine (1952):
 Polio virus grown in animal cell line, then inactivated by formalin http://en.wikipedia.org/wiki/File:Polio.jpg
http://americanthings.files.wordpress.com/2009/10/meisenproductionsdotcom.jpg

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Neutralize the toxin produced by pathogens and inject into patient
Tetanus vaccine (1924):
 Culturing Clostridium tetani, collecting tetanospasmin toxin and inactivating with formalin http://en.wikipedia.org/wiki/File:CBell1809.jpg
http://www.livestockpros.com/images/catttlevacs/87-20.jpg

 A genetic approach:
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Determining the immunogenic portion of a microorganism
Producing subunits without the harmful or replicative portions of pathogen
 Examples:
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Hepatitis B vaccine: surface antigen used to be isolated in human blood, transplanted into yeast in 1980s
HPV vaccine: surface antigens produced in yeast, approved 2006 http://en.wikipedia.org/wiki/File:Hepatitis_B_virus_v2.png
http://en.wikipedia.org/wiki/File:Gardasil_vaccine_and_box_new.jpg

 Traditional methods:
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Inactivated: Inject eggs with virus, incubate and allow virus to proliferate, apply formalin to “kill”
Attenuated: Expose virus to repeated cold adaption cycles, until it no longer can reproduce in body temp (directed evolution!) http://science.nationalgeographic.com/staticfiles/NGS/Shared/StaticFiles/
Science/Images/Content/cultivating-flu-vaccine-sf5473-lw.jpg
http://beta.thehindu.com/multimedia/dynamic/00001/SWINE_FLU_VACCINE_1_1628f.jpg

 Takes around six months to prepare annual vaccine
 Only a prediction of the expected strains
 Variable quality of egg product
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Contamination
Difficult to control amount of virus
 Live-attenuated vaccine is unsuitable in immunocompromised patients

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Quan et al. 2010, “Virus-Like Particle Vaccine Protects against
2009 H1N1 Pandemic Influenza Virus in Mice”
The HA and M1 genes were converted to cDNA, PCR amplification, insertion into pFastBac vector
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Plasmids placed into a baculovirus, infected into insect cells
Structural proteins aggregate and form empty capsules in supernatant
Western blot to confirm HA and M1 in VLP Electron micrograph of VLP

 Mice injected with isolated VLPs

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A universal flu vaccine
Adar et al. 2009, “A universal epitope-based influenza vaccine and its efficacy against H5N1”
 Insert a variety of flu epitopes on flagellin chassis
 Flagellin detectable by TLR5 in innate immune system

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Inserting plasmid DNA for immunogenic portions directly into human cells; producing antigens on-site
Advantages:
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Easy to synthesize and adapt
Stable storage
Prolonged exposure to immunogen
Most similar expression/structure to actual infection http://www.niaid.nih.gov/news/newsreleases/2005/pages/wnvvaxtrial.aspx

 R.J. Drape et al. 2006, “Epidermal DNA vaccine for influenza is immunogenic in humans”
 HA coding sequence isolated by RT-PCR, inserted into DNA plasmid
 DNA coated on 1-3um gold particles, delivered by gene gun

 Problem: Attenuated virus reverting to wild type
 Directed mutations
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Macadam et al. 2006, “Rational Design of Genetically Stable, Live-
Attenuated Poliovirus Vaccines of All Three Serotypes: Relevance to Poliomyelitis Eradication”
Altering thermodynamic stability of virus domain with point mutations (using splicing segments with RE)

 Using knowledge of codon pair bias
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Coleman et al. 2008, “Virus Attenuation by Genome-Scale
Changes in Codon Pair Bias”
Preserving AA sequence of P1 structural domain (2643bp), but modifying synonymous codons (500-600 mutations)
 Changes in translation

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 http://discovermagazine.com/2009/jul-aug/27-vaccine-production-horriblyoutdated-3-ways-fix-it http://www.i-sis.org.uk/LASIVCSQ.php
http://www.who.int/vaccines/en/hepatitisa.shtml
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820088/ http://www.who.int/biologicals/areas/vaccines/dna/en/index.html
http://www.cdc.gov/mmwr/preview/mmwrhtml/00041645.htm