Beneficial Uses of Viruses in Biotechnology

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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

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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

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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

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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

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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

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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

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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

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

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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
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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.
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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
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Vector Vaccines for HIV-1
Live AttenuatedViral Vectors
Adenovirus
Modified Vaccinia (MVA)
DNA from HIV is Cloned into Various Vectors
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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
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Vector Vaccines for HIV-1
Replicon Vaccines Virally encapsidated plasmid
vaccine
DNA from HIV is Cloned into Various Vectors
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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

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Principle
Cell transcribes DNA. Vaccine protein is
expressed on cell surface
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
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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

Multi drug resistant Pseudomonas