04 - Viral Uses in Biotechnology

1. 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

2. Viruses: Basic Structure

3. Viruses: Basic Structure ssDNA dsDNA ssRNA dsRNA (+)ssRNA (-)ssRNA Nature of the nucleic acid in the virion • 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

4. Viruses: Basic Function – Lytic Cycle

5. Viruses: Basic Function – Lysogenic Cycle

6. Viruses: Basic Function - Budding

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

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

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

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

11. Viruses: Classification Taxonomy

12. 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

13. Gene therapy: Viruses as gene delivery vehicles • 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

14. 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

15. 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. Vaccinology.

16. 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, hepb.,occellular vaccines), Conjugate polysacaride vaccines linked with suitable carrier proteins (Hib). Also single or polyvalent vaccines. Vaccinology.

17. Viral Vaccines Vaccinology.

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

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

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

21. Vector Vaccines for HIV-1 Live AttenuatedViral Vectors Adenovirus Modified Vaccinia (MVA) DNA from HIV is Cloned into Various Vectors

22. Live Attenuated Viral Vectors Modified Vaccinia Ankara (MVA) • 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 DNA prime rMVA boost

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

24. Bacteriophage: viral antibacterial agents • 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 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 • Also use for detecting pathogenic bacteria - phage infects bacterial lawn - assay plaques by antibody or by phage-encoded marker gene expression