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Synthetic Biology in Vaccine Development Julius Ho Biol1220 4/20/10 Outline Historical overview of vaccines Genetic methods in vaccine development Use influenza as an example for new synthetic bio approaches Traditional vaccines Use of inactive or weakened compounds from the microorganism causing disease Different approaches Heterologous Attenuated Inactivated Toxoid Heterologous 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 Attenuated 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 Inactivated 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 Toxoid 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 Next generation of vaccines A genetic approach: Determining the immunogenic portion of a microorganism Producing subunits without the harmful or replicative portions of pathogen Examples: 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 Influenza: A Case Study Traditional methods: 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 Current shortcomings Takes around six months to prepare annual vaccine Only a prediction of the expected strains Variable quality of egg product Contamination Difficult to control amount of virus Live-attenuated vaccine is unsuitable in immunocompromised patients Synthetic biology solutions Virus-like particles DNA vaccine Synthetic attenuation Virus-Like Particles 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 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 Quan et al. 2010 continued Mice injected with isolated VLPs The future: 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 DNA vaccine Inserting plasmid DNA for immunogenic portions directly into human cells; producing antigens on-site Advantages: 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 DNA vaccine to H1N1 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 Synthesis of attenuated viruses Problem: Attenuated virus reverting to wild type Directed mutations Macadam et al. 2006, “Rational Design of Genetically Stable, LiveAttenuated Poliovirus Vaccines of All Three Serotypes: Relevance to Poliomyelitis Eradication” Altering thermodynamic stability of virus domain with point mutations (using splicing segments with RE) Synthetic attenuation cont. Using knowledge of codon pair bias 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 Sources http://discovermagazine.com/2009/jul-aug/27-vaccine-production-horribly outdated-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