Lecture 33 - Research Analysis II - Vaccines

Question 1

What was the rationale for these experiments?

Answer 1

• Not much is know about how empirical vaccines induce such long-lived immunity
• Want to investigate this, so it can be applied to rational design of vaccines
• It was known that the Yellow Fever vaccine activates multiple TLRs in DCs to promote robust immunity
  
  • → co-administer vaccine with TLR agonists

Question 2

Describe the nanoparticles that were used in the experiment

Answer 2

• Synthetic polymer
• WIll degrade
• Contains TLR agonists or Ag
  
  • TLR agonists:
    
    • Synthetic
    • **MPL **& R837
  • Ag:
    
    • ​OVA
    • Influenza HA
    • *Bacillus anthracis *ag
• Same size as virus

Question 3

What were the adjuvants used in this experiment?

Answer 3

• MBL
  
  • ​TLR4 ligand
  • LPS-derived
• R837
  
  • ​TLR7 ligand
• R848​
  
  • TLR7 & TLR8 ligand

Question 4

What was the antigen used in these experiments?

Answer 4

• Influenza HA
• Protective antigen Bacillus anthracis
• Ovalbumin (OVA)

Question 5

Describe the requirements for delivery of the antigen and adjuvants

Answer 5

• The adjuvants had to be administered separately from the antigen for a robust immune response
• When the adjuvants and Ag were administered together, there was much less Ab produced

⇒ big implications for the field of immunology

Question 6

How does Alum function?

Answer 6

Activates the inflammasome

Question 7

Describe the effect of adjuvant nanoparticles on the magnitude of the Ab response

Answer 7

• Synergistic response when vaccine was administered with both TLR ligands
• Using just one or the other TLR ligands had a much lower Ab titre
• Measured with an ELISA assay
• Not additive, but synergistic response

Question 8

How do we test Ab affinity?

Answer 8

Surface plasmon resonance assay (SPR)

Question 9

How did the group test for Ab function?

Answer 9

Viral neutralisation assay

• If the virus is blocked by the Ab, it won’t be able to infect cells
• Presence of plaques: virus was able to infect

Question 10

Describe the SPR assay

Answer 10

• Measures rates of binding and disociation of Ab from the Ag
• Read out:
  
  • High affinity:
    
    • ​Fast ON
    • Slow OFF
  • and vice versa
• Determines the K_d

Question 11

Describe how the role of DCs was investigated

Answer 11

1st experiment

• Design
  
  • ​ELISA assay
  • DCs stimulated with nanoparticles containing various combinations of adjuvants
• Results
  
  • ​Pro-inflammatory cytokines are produced by the DCs in response to adjuvants in the nanoparticles

2nd experiment

• Design:
  
  • CD11c-DTR transgenic mice
    
    • DTR: diphtheria toxin receptor
  • When DT is administered, all DCs are depleted from the mouse
  • Immunise with vaccine
• Results:
  
  • Low Ab titres in DC depleted mice, compared to WT mice
• Conclusion
  
  • Immunity from vaccine is dependent on DCs

Question 12

Which adaptor proteins are involved in TLR signalling?

Answer 12

• MyD88
• TRIF

Question 13

Describe how the role of TLRs in the vaccine was investigated

Answer 13

• Design
  
  • ​MyD88 and TRIF KO mice (either one or the other)
  • Immunisation
  • Looks at Ab responses (ELISA assay)
• Results
  
  • ​No immunity (low Ab titre) in *MyD88-/- *KO mice
  • No immunity in *Trif-/- *mice
• Conclusion
  
  • ​TLRs are essential in generation of immunity

Question 14

What are μMT mice?

Answer 14

No B cells

Lack the mu heavy chain

Question 15

Describe how the role of B cells was investigated

Answer 15

• Design
  
  • μMT mice
  • Adoptive transfer of Trif-/- and MyD88-/- B cells
    
    • One or the other, or both
  • Immunise
  • Measure response (ELISA assay)
• Results
  
  • V. low Ab titre in B cells w/o:
    
    • TRIF
    • MyD88
    • TRIF & Myd88 on different B cells
• Conclusion
  
  • TLR signalling in B cells is crucial for generation of immunity
  • Both signalling pathways must be present in a single B cell to mount a response
    
    • →synergy

Question 16

Describe how the role of CD4 T cells was investigated

Answer 16

First Experiment

• Design
  
  • ​Immunisation with vaccine with various adjuvants present
  • Look at the IFN-γ CD4 T cell responses with FACS
  • Look at Ab titres
• Results
  
  • Many *antigen-specific *IFN-γ+ CD4+ T cells when both adjuvants were present
  • Few cells when only one of the adjuvants was present
• Conclusion
  
  • ​Need both adjuvants for a robust CD4 T cell response & high Ab titre

Second experiment

• Looking at the Ab titre in absence of CD4 T cells
• Design
  
  • ​CD4 T cell depleted mice
  • Immunisation
  • Measure Ab titres with ELISA assay
• Results
  
  • ​Low Ab titre in CD4 T cell depleted mice
• Conclusion
  
  • ​CD4 T cells are required for the synergistic enhancement of Ab responses

Question 17

Which cells are required for immunisation with this vaccine?

Answer 17

• DCs
• B cells
• CD4 T cells

Question 18

Outline the chemokines involved in the development of B cell responses

Answer 18

• CXCR5
• CCR4

Question 19

Outline the various consequences of B cell activation

Answer 19

• Extrafollicular response
  
  • ​Short lived plasma cells
  • Low affinity IgM and IgG secretion
  • BLIMP
• Follicular response
  
  • ​Long lived memory cell formation
  • Isotype switching and affinity maturation
  • High affinity IgG secretion
  • Bcl6

Question 20

What was the effect of the adjuvants on GC formation?

How was this investigated

Answer 20

• Design
  
  • Confocal microscopy
  • Vaccinate
  • Look at n° of GCs in lymphoid organs
• Results:
  
  • Dual-combination vaccine results in generation of many GCs that are very long lived
    
    • → high affinity Ab produced, long lived titres

Question 21

Describe how programming of B cell memory responses was investigated

Answer 21

• Design
  
  • ​Systems biology approach:
    
    • Immunise mice
    • ​Look at expression of genes uniquely associated with:
      
      • Plasma cells
      • GCs
      • Memory B cells
    • such as **Bcl6 **and BLIMP
• Results
  
  • With dual immunisation, there is expression of Bcl6
  • With immunisation with single adjuvant, expression of BLIMP
• Conclusions
  
  • ​Dual immunisation programmes memory responses in B cells

Question 22

Which TFs are important for programming of B cell responses to either short lived plasma cell or memory B cell?

Answer 22

• Bcl-6
• BLIMP

Question 23

How was protective immunity in mice demonstrated?

Answer 23

• Design
  
  • ​Some mice immunised, some untreated
  • Mice challenged with H1N1, which is normally lethal
• Results
  
  • ​Untreated mice don’t survive
  • Vaccine w/o adjuvant: die
  • With dual combination vaccine: mice are protected
• Conclusion
  
  • ​The vaccine immunises the mice and leads to survival when challenged with H1N1

Question 24

Describe the experiments with vaccination in macaques

Answer 24

• Design
  
  • Vaccination of Rhesus macques with various adjuvant combinations
  • Infection with influenza
  • Measure Ab titres
• Results
  
  • Highest Ab titre in monkeys that received dual vaccination
• Conclusion
  
  • ​Vaccination with dual adjuvant induces protective immunity in non-human primates

Question 25

Describe how the effect of adjuvant on Ab response was measured

Answer 25

Magnitude

• ELISA assay
• Compare Ab titre in response across the groups
• Results:
  
  • ​Dual adjuvant administration induce high titres of Ab
  • Adjuvants co-administered singly had poor response
  • Synergistic effect of dual administration of adjuvants

Quality

• Surface plasmon resonance (SPR) assay
• Compare affinity of Abs produced in response across the groups
• Results
  
  • ​Dual adjuvant administration generated Abs of highest affinity