04 - Viral Uses in Biotechnology Gene therapy Vaccines and vaccine carrier / delivery vehicles Antibacterial agents Basic knowledge of cell metabolic processes Vectors for mammalian, plant and insect cell protein expression systems Peptide display – industrial /pharmaceutical / medical reagent development

Viruses: Basic Structure

Viruses: Basic Structure Nature of the nucleic acid in the virion ssDNA dsDNA ssRNA dsRNA (+)ssRNA (-)ssRNA ss - single stranded; ex. parvovirus ds - double stranded; ex. Herpesviruses (+)ssRNA - codes for the mRNA strand once inside the cell; ex. Coronaviruses (-)ssRNA - codes for the complimentary strand to mRNA once inside the cell; ex. Paramyxoviruses dsRNA – codes for both template and complementary strands; ex. Retroviruses like HIV

Viruses: Basic Function – Lytic Cycle

Viruses: Basic Function – Lysogenic Cycle

Viruses: Basic Function - Budding

Viruses: Classification Four characteristics used for the classification of all viruses: 1. Nature of the nucleic acid in the virion ssDNA – dsDNA – ssRNA - dsRNA

Viruses: Classification Four characteristics used for the classification of all viruses: 2. Symmetry of the protein shell

Viruses: Classification Four characteristics used for the classification of all viruses: 3. Presence or absence of a lipid membrane

Viruses: Classification Four characteristics used for the classification of all viruses: 4. Dimensions of the virion and capsid

Viruses: Classification Taxonomy

Gene therapy: Viruses as gene delivery vehicles  Necessary viral properties –Use a viral vector that persists in humans, and shows moderate level of long-term gene expression –Need to clone in foreign DNA (where, how much) – develop cloning sites, need large cloning capacity –Virus must target specific cell-type – cell-type-specific receptor mediated attachment and uptake – can genetically engineer for receptor

 Manufacture –propagate virus in specific mammalian cell line – expensive –Biosafety - mutate to produce attenuated virus (low virulence) or crippled virus (reduced pathogenicity) - but generally low titre –To circumvent low titre – Helper virus or transgenic cell line provides packaging / replication functions in trans for non-replicating delivery virus –viral packaging - protects and stabilises DNA from degradation Gene therapy: Viruses as gene delivery vehicles

 Host response –Do not want a strong immune response to viral vector (& its associated payload) – leads to rapid clearance of delivery virus  Thus, use viruses with –rare serotypes –low seroprevalence –low-level replication or non-replicating virus –E.g. retroviruses such as lentiviruses Gene therapy: Viruses as gene delivery vehicles

Viruses: Direct use in vaccines a substance used to stimulate the production of antibodies and provide immunity against one or several diseases, prepared from the causative agent of a disease, its products, or a synthetic substitute, treated to act as an antigen without inducing the disease. 10/10/2011Vaccinology.15

Vaccines: Current Categorization Live attenuated: Viruses (Sabin polio, measles, mumps, rubella, yellow fever), Bacteria (cholera)- Long lasting immunity, very fragile (cold chain), mutation to pathogenicity Killed Vaccines: Viruses (hep. A, Salk polio, flu) Bacteria (pertussis, cholera)-intermediate immunity, several doses may be required Sub-unit vaccines: Toxoids: (tetanus, hep b.,occellular vaccines), Conjugate polysacaride vaccines linked with suitable carrier proteins (Hib). Also single or polyvalent vaccines. 10/10/2011Vaccinology.16

Viral Vaccines 10/10/2011Vaccinology.17

Plasmid DNA makes encoded HIV protein in cells of the body Epitope Display Vectors Live Attenuated Viral Vectors Replicon Vaccines: Virally encapsidated plasmid vaccine DNA from HIV is Cloned into Various Vectors Vector Vaccines for HIV-1 AdenovirusModified Vaccinia (MVA) Virus-like particle with outer surface display of epitopes

Plasmid DNA makes encoded HIV protein in cells of the body DNA from HIV is Cloned into Various Vectors Vector Vaccines for HIV-1

Epitope Display Vectors DNA from HIV is Cloned into Various Vectors Vector Vaccines for HIV-1 Virus-like particle with outer surface display of epitopes

Live Attenuated Viral Vectors DNA from HIV is Cloned into Various Vectors Vector Vaccines for HIV-1 AdenovirusModified Vaccinia (MVA)

DNA prime Live Attenuated Viral Vectors Modified Vaccinia Ankara (MVA) HIV-1 vaccine development at University Cape Town  HIV-1 vaccine development at University Cape Town  Recombinant MVA (rMVA) expressing HIV-1C gag and env genes  Used in a Prime-Boost immunisation regimen  prime immune response with plasmid vaccine expressing gag and env  boost to broaden / increase response with rMVA expressing gag and env rMVA boost

Replicon Vaccines: Virally encapsidated plasmid vaccine DNA from HIV is Cloned into Various Vectors Vector Vaccines for HIV-1

Bacteriophage: viral antibacterial agents Advantages:Advantages: –Useful where multiple antibiotic resistance has developed –host specific - won't kill off commensal bacteria –Rapid action – exponential replication –self-limiting infection once pathogenic bacteria are killed –cheap - single dose - self propagates Disadvantage - strain specificDisadvantage - strain specific –need to generate, keep and archive large bank of phage serotypes –need accurate diagnosis –must give cocktail of phage types to prevent bacterial escape detecting pathogenic bacteriaAlso use for detecting pathogenic bacteria - phage infects bacterial lawn - assay plaques by antibody or by phage- encoded marker gene expression