Presented by: Andrew Nelson, R.J. Dealy,

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• Presented by Andrew Nelson, R.J. Dealy,
• and Nick Bishop

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Overview

• Intro
• Lactococcus lactis
• Interleukins (cytokines)
• Quick summary of general process
• Relative Importance

• Materials and Methods
• Outline of what was done
• Specific techniques used
• Results and Discussion
• How and what results were generated
• What was learned

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Lactococcus lactis

• Gram positive, cocci, 0.5-1.5µm
• Used in the production of fermented milk products
  (buttermilk, cheese, etc)
• Noninvasive (cant multiply in vivo),
  nonpathogenic
• Can serve as an antigen delivery vehicle

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Interleukins (cytokines)

• Cytokines (secreted signaling molecules)
• Immune system depends on them
• Can be useful immune response modulators for
  vaccines
• Administration of IL-2 and IL-6 has been shown to
  increase antibody responses to antigens

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What was done

• Constitutive expression strains were engineered
  to accumulate a test antigen (TTFC) and murine
  interleukins (cytokines) IL-2 and IL-6
• Mice were intranasally immunized with variations
  of these expression strains (as well as controls)
• Some recombinants were treated (killed) with
  mitomycin C prior to immunization
• Both systemic and mucosa anti-TTFC antibody
  responses were measured

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Relative Importance

• Treatment of disease
• At this point, virtually all recombinant delivery
  systems had been derived by infectious agents
  (Salmonella spp., Mycobacterium bovis, etc)
• Cytokine delivery could enable tailored vaccines
  against particular pathogens.

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Materials and Methods
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Recombination of DNA

• PCR amplification using Vent polymerase
• DNA-modifying enzymes and restriction
  endonucleases used under standard conditions
• L. lactis transformed by electroporation of cells
  grown in presence of glycine
• Recombinant strains expressing TTFC, IL-2 and
  IL-6

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Immunoblotting

• Cell walls were digested using a buffer solution
  and separated from the cells by centrifugation
• Proteins extracted using electrophoresis and
  electroblotted
• Transfer of TTFC and murine cytokines detected by
  immunoblotting (western blot)

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Preparation for Immunization

• The bacterial strains which carried the desired
  insert components were cultured, washed and
  resuspended prior to immunization

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Mitomycin Pretreatment

• Prior to immunization, cultured cells were
  treated with mitomycin C. After treatment, fewer
  than 1 in 104 cells remained viable.

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Immunization

• Groups of lady mice, 6-8 weeks old, were
  intranasally immunized with the modified L. lactis

Pretend this is a mouse.
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Detection of Antibodies

• The presence of antibodies was detected using
  enzyme-linked immunosorbent assay (ELISA). This
  method was developed using antibodies which react
  with serum antigens or antibodies and signal
  their presence

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• This was done for both TTFC-specific antibodies
  and antilactococcal antibodies.

L. lactis
TTFC
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Assay of IgA

• Fecal pellets from the subjects were assyed for
  IgA concentration. IgA is the main antibody
  (immunoglobulin) in mucosal tissues

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Results

• Feces of mice examined to determine if cytokines
  would influence IgA in mucosal tissues
• IgA levels in mice inoculated with different
  strains of recombinant L. lactis showed the same
  results as the control groups
• Therefore, IgA levels in gastrointestinal tract
  were unaffected.
• But, blood serum antibody levels were affected.

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• Mean anti-TTFC IgG levels
• Increased levels in
• - TTFC IL-2
• - TTFC IL-6

Recombinant bacterial strains from left to
right TTFC, TTFC IL-2, TTFC IL-6, control
non-expressor strain, control nonvaccinated group.
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Mean Anti-TTFC IgA Levels Increased levels
in - TTFC IL-6
Recombinant bacterial strains from left to
right TTFC, TTFC IL-2, TTFC IL-6, control
non-expressor strain, control nonvaccinated group.
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• Anti-TTFC antibody levels
• a IL-2
• b IL-6
• Inoculated mice with viable strains and mitomycin
  C killed strains of L. lactis
• TTFC antigen delivery does not require bacterial
  viability
• IL-2 and IL-6 cytokine delivery does require
  bacterial viability

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Results

• So...
• By demonstrating an increase in levels of IgA and
  IgG antibodies in mice inoculated with TTFC
  IL-2 and TTFC IL-6, it has been shown that
• The viable recombinant lactococci were successful
  in delivering cytokines to the immune system.

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Conclusions

• L. lactis can be used an effective method for
  delivering cytokines to the immune system via
  intranasal inoculation.
• This strategy may potentially be applied as a
  method of vaccination for a particular pathogen
  of interest.