Download Materials and Methods - Welcome to the Biology Department

Presented by: Andrew Nelson, R.J. Dealy,
and Nick Bishop
Overview
• Intro
• Lactococcus lactis
• Materials and Methods
• Outline of what was done
• Interleukins (cytokines)
• Specific techniques used
• Quick summary of general
process
• Results and Discussion
• How and what results were
generated
• Relative Importance
• What was learned
Lactococcus lactis
• Gram positive, cocci, 0.5-1.5μm
• Used in the production of
fermented milk products
(buttermilk, cheese, etc…)
• Noninvasive (can’t multiply in vivo),
nonpathogenic
• Can serve as an antigen delivery
vehicle
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
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
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.
Materials and Methods
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, IL2 and IL-6
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)
Preparation for Immunization
• The bacterial strains which carried the
desired insert components were cultured,
washed and resuspended prior to
immunization
Mitomycin Pretreatment
Prior to immunization, cultured cells were
treated with mitomycin C. After treatment,
fewer than 1 in 104 cells remained viable.
Immunization
• Groups of lady mice, 6-8 weeks old, were
intranasally immunized with the modified
L. lactis
Pretend this is a mouse.
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
This was done for both TTFC-specific
antibodies and antilactococcal antibodies.
L. lactis
TTFC
Assay of IgA
• Fecal pellets from the subjects were
assyed for IgA concentration. IgA is the
main antibody (immunoglobulin) in
mucosal tissues
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….
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.
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 nonexpressor strain, control nonvaccinated group.
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
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.
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.