Download
beneficial uses of viruses in biotechnology n.
Skip this Video
Loading SlideShow in 5 Seconds..
Beneficial Uses of Viruses in Biotechnology PowerPoint Presentation
Download Presentation
Beneficial Uses of Viruses in Biotechnology

Beneficial Uses of Viruses in Biotechnology

4084 Vues Download Presentation
Télécharger la présentation

Beneficial Uses of Viruses in Biotechnology

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Beneficial Uses of Viruses 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. Gene therapy: Viruses as gene delivery vehicles • Necessary viral properties • Use a virus 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

  3. 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

  4. 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. lentiviruses

  5. Viruses: Direct use in vaccines • Against autologous virus • attenuated or inactivated virus • viral subunits - usually structural proteins, genetic vaccines • cross-reactivity - study relatedness of virus strains • Against heterologous virus • Viral structural proteins or “virus-like particles” (VLPs) can be made to carry heterologous pathogen epitopes • Must produce proteins, VLPs in quantity to high purity • e.g. rabies virus glycoprotein, HPV

  6. Viruses as DNA vaccine delivery vehicles • Preferred Immunological Properties • Want weak immune response (IR) to viral delivery vehicle • Weak / moderate IR to delivery vehicle results in enhanced response to DNA payload • Strong IR to delivery vehicle may provoke toxic over-response, clear the vaccine too rapidly for a response to develop to the payload, or can swamp response to payload • Therefore: • Use viruses having rare serotypes (low seroprevalence) • low virulence or non-pathogenic viruses

  7. Viruses as DNA vaccine delivery vehicles • Develop a viral delivery vehicle • study gene function, engineer suitable cloning sites • amount of DNA vaccine that can be cloned and packaged is limited by capsid size / viral packaging mechanism • Cell targeting – DNA delivery • viral engulfment by antigen presenting cells • cell-specific receptor-mediated uptake • Manufacture • Prefer a virus that replicates to high titre • Prefer a virus with a long survival half life outside host cell • Viral packaging of DNA vaccine protects and stabilises DNA from degradation • Need suitable production-host cells – if using attenuated virus (eg vaccinia vectors) - may need to provide some packaging / replication functions in trans

  8. 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

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

  10. Viral genetic elements used to construct Eukaryotic expression plasmid vectors • Viruses are highly efficient replicators & viral gene expression is adapted to eukaryotic systems • very strong promoters (CMV immediate / early promoter) • small introns (CMV intron) • regulatory elements often constitutive - require only host factor binding (porcine circovirus (PCV) capsid promoter / enhancer) • Therefore mine regulatory elements from viruses • Promoters, enhancers, polyadenylation signals, introns, replication origins, IRES elements.

  11. 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

  12. Use of insect Baculovirus : Autographa californica nuclear polyhedrosis virus (AcNPV): Foreign gene (HIV-1 gag) inserted under Baculovirus strong late promoter, polh -transient production of HIV Virus-Like Particles in cultured insect cells A. Meyers, E.P. Rybicki.

  13. Tobacco mosaic virus Viruses for Peptide display: M13 Phage or plant virus (TMV) Coat Protein Fusions • Need : • non-enveloped virus • many repeat capsid subunits • ordered capsid array - amplified display • external loops or termini available for peptide addition via gene fusion Mass peptide display on outer surface of TMV particle N C 60S loop Assembly of mixed TMV capsids carrying epitope variants = useful vaccine vs highly variable pathogen TMV VIRION

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

  15. Live Attenuated Viral Vectors at UCT Modified Vaccinia Ankara (MVA) • HIV-1 vaccine development at UCT • 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

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

  17. Replicon Vaccines: Virally encapsidated plasmid vaccine • Adenovirus 5, Adeno-associated virus • Bacteriophage vectors e.g. Lambda or M13 • clone foreign DNA into Lambda genome • Large cloning capacity • passive uptake by immune cells and complement mediated uptake • Non-pathogenic for humans - safe • Highly stable vehicle - can dehydrate • Cheap to make – high titre production in E. coli

  18. Macrophage Dendritic cell Cell transcribes DNA. Vaccine protein is expressed on cell surface Principle: Mammalian expression control elements l DNA l DNA Antigen gene Phage broken down. Vaccine-encoding DNA released Vaccine expression cassette cloned into bacteriophage l DNA Immune response Grow l phage in E. coli & purify Antigen – presenting cells engulf l particles Inoculate - injection / oral

  19. 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 Multi drug resistant Pseudomonas