Possible Limitations of Traditional Vaccine Production

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Possible Limitations of Traditional Vaccine
Production

• Not all infectious agents can be grown in culture
• Animal/human cell culture expensive if needed
• Yield of viruses from cultures can be low
• Safety precautions for culture of live agents
• Insufficient killing/attenuation of agents
• Reversion of attenuated agents
• Traditional vaccines dont work for all agents
• Traditional vaccines may have shorter shelf-lives

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New Strategies

• Delete virulence genes
• Reversion nearly impossible
• Use live nonpathogenic carriers for immunization
• Clone antigenic determinants into alternative
  host systems for production
• Address autoimmune system response/problems
• Kill infected cells that wouldnt naturally die

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Human Diseases for Which Recombinant Vaccines Are
Currently Being Developed
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Animal Virus Structure
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Subunit Vaccine Development

• Clone HSV glycoprotein gD gene
• Transfect into CHO cells
• Isolate secreted glycoprotein
• Purify and use as vaccine
• Protects mice from HSV

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Envelope HSV-1 gD Protein

• Envelope protein
• transmembrane domain
• Clone gene
• Truncate
• Make soluble gene product as vaccine
• Less effective than membrane bound version but
  works well enough to be effective

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Foot-and-Mouth Subunit Vaccine

• Clone gene for capsid VP1 protein
• Express in E. coli as fusion protein
• Purify protein and use as vaccine
• Worked
• Fusion protein regulations make second generation
  nonfusion vaccine necessary

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Antigenic Envelope proteins

• Exterior and interior domains, plus transmembrane
  domain
• Exterior domain primarily involved in activating
  immune response
• Antigens commonly have multiple
  determinants/epitopes
• Short peptides corresponding to epitopes may be
  sufficient

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Peptide Vaccines

• Peptides generally not good antigens by
  themselves
• Improved antigenicity when attached to larger
  carrier protein
• Highly antigenic protein such as hepatitis B core
  protein works very well as a carrier
• Up to 500X better than peptide alone

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Peptide Limitations

• Epitopes must be continuous run of amino acids
• Peptide must assume same conformation as it would
  as part of complete protein
• Sometimes single epitopes do no elicit sufficient
  immunological response
• Posttranslational modifications???
• Response to carrier proteins upon repeated use?

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Yeast Retroposon Ty p1 Protein

• Another potentially useful carrier
• Studied using Plasmodium falciparum (malaria)
• Sometimes subunit vaccines with different
  carriers used in concert provide best immunity

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DNA Vaccines

• Genetic immunization
• Gold projectiles coated with influenza A gene
  biolistically delivered into mouse ears
• No response to vector
• Only expressed gene
• Can therefore be reused repeatedly

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Shigella-based Delivery Systems

• Shigella can enter cells without phagocytosis
• Directs plasmids to cytoplasm (with inserted
  genes)
• Provides immunity to expressed antigens

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Microparticle DNA Delivery

• DNA adheres to cationic surface
• Can be injected into tissue and DNA slowly
  released over several days
• Gives prolonged though transient expression by
  cells
• Provides up to 250-fold better effect as compared
  to naked DNA

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Advantages of Genetic Immunization Over
Conventional Vaccines
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Rhesus Monkey Immunization Against Simian
Immunodeficiency Virus

• DNA constructs for expression of SIV proteins
  injected at 0 and 8 weeks
• Booster of recombinant vaccinia virus expressing
  same proteins at 24 weeks
• Immunity to SIV infection through mucosal tissues
  at 7 months post booster

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Cholera Toxin

• B subunit for binding to membrane receptor site
• A2 peptide acts as connector between B and A1
  subunits
• A1 peptide is the enzymatically active component
  which modifies target G protein, locking
  adenylate cyclase in on position in intestinal
  mucosal cells

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Deletion-attenuated Toxin Gene

• Clone toxin gene
• Cut out internal
• segment
• Ligate using linker
• Conjugate into wt
• cell
• Recombination
• Chromosomal gene
• now attenuated by
• deletion

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Deletion-attenuated Salmonella Strains

• Salmonella strains cause many diseases
• Enteric fever, infant death, typhoid fever, food
  poisoning
• Attenuated strains constructed by deletion of
  biosynthetic, regulatory and/or virulence genes
  (100-fold reduction in virulence)
• Many double deletions used to make reversion less
  likely
• Useful for human and animal inoculation

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Deleted Genes and Their Function in the
Development of Attenuated Strains of Salmonella
spp.
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Attenuated Leishmania

• Protozoan parasite
• Traditionally attenuated strains persist in host
  (without symptoms) for extended periods
• Traditional attenuated strains have undesirable
  property of reversion
• Delete DHFR/Thymidylate synthase
• This strain persists only for a few days in host
  but provides immunity

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Attenuated L. major Immunity

• Inoculate with attenuated L. major
• Challenge with wt L. major
• Few/small lesions in previously inoculated mice
• even very sensitive host strains

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Attenuated Herpes Simplex Virus

• Subunit vaccines ineffective
• Live replication defective virus induce immune
  response
• Double deletion in genome prevents replication
  and makes reversion very unlikely
• Strain induces immunity and reduces viral
  shedding/latent infection

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Vector Vaccines

• Use one organism to express antigens from another
  pathogenic organism
• Vaccinia virus a good candidate vector
• Broad host range
• Well-characterized and benign
• But spreads like
• Large ds DNA genome (187 kb)
• Replicates and expressed in cytoplasm
• brings own DNAP, RNAP, etc.
• New genes introduced by in vivo recombination

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Recombinant Vaccinia Virus

• Plasmid with antigen expression construct
• Vaccinia controls
• Transform into viral infected cells
• Select for double recombinants
• Targeted for TK gene so cells are TK-
• Use recombinant virus for expression in host
  animals

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TK Selection Issues

• TK- spontaneous mutants not that uncommon
• Background in selection protocols can be high
• Develop better site

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Improved Vaccinia Vector

• Insert NeoR gene for kanamycin analogue G-418
  resistance
• Many proteins successfully expressed using this
  system

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Attenuating Vaccinia

• Vaccinia virus could have deleterious effects on
  certain patients (immunosuppressed or deficient)
• Virus persists in host because it inhibits
  interferon-mediated antiviral pathway
• Makes competitive inhibitor of kinase that knocks
  out eIF-2a (eukaryotic translational initiation
  factor 2a)

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Interferon-Mediated Antiviral Activity and
Vaccinia
Vaccinia Protein
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Use of K3L Mutant Vaccinia

• K3L mutant vaccinia persist in host for less time
  and at lower levels than wt virus

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Vaccines Against Bacteria

• Not all bacterial diseases treatable by
  antibiotics
• Resistance is proliferating to many antibiotics
• Antibiotics usually require refrigeration
• Patients dont always complete antibiotic regimen

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Tuberculosis

• 2 billion persons currently infected
• 2-3 million deaths/yr
• Antibiotics work but resistant strains becoming
  more common
• Attenuated BCG strain of Mycobacterium bovis
  developed in early 1900s used for immunization
• But infects immunocompromised individuals
• Gives false positive for standard tuberculosis
  test

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Subunit Vaccines to Tuberculosis

• Isolate proteins secreted into culture medium
• Identify which induce protection
• Construct subunit vaccine(s) based on promising
  antigens

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Improving BCG Strain

• E. coli/Mycobacterium shuttle vector
• Introduce identified antigen gene into expression
  module
• Vaccine was more potent than traditional one

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Bacteria as Antigen Delivery Systems