Lecture 32 - Vaccination II

Question 1

Compare **empirical **and rational approaches to vaccine development

List vaccines that have been developed in these ways

Answer 1

Empirical

• “Trial & error” methodologies
• Based on knowledged gained by direct observation of an experiment
• Many traditional vaccines

Rational

• Based on logic and deduction
• Using data from experiements to guide and inform
• HPV (Gardasil; VLP), HBsAg subunit vaccine

Question 2

What is the imporance of Dendritic cells in vaccine design?

Answer 2

A good vaccine strategy needs to license / activate DCs

• DCs play a crucial role in shaping the immune response
  
  • Become activated by sensing ‘Danger’
  • Intracellular transduction pathway
  • Transcription of genes
• Activation of DCs
  
  • ​Recognise PAMPs / DAMPs through PRRs
    
    • TLRs
    • C-type lectin receptors
    • RLRs
    • NLRs
• Release of pro-inflammatory cytokines that regulate:
  
  • ​Adaptive immune response generation
  • Proliferation
  • Inflammation
  • Apoptosis
  • Immune regulation

Question 3

What is therapeutic vaccination?

Answer 3

Vaccination after establishment of infection / disease

eg. Improvement of T cell immunity to tumours

Question 4

How has basic research in the field of immunology impacted modern therapeutics?

Answer 4

• Understanding mechanisms of immune induction
  
  • ​Role of innate immunity
  • Requirements of lymphocyte activation
  • Induction of immune memory
• Understanding cell mediated immunity
  
  • ​Clearance of intracellular infection
  • Recognition of tumour Ags by CD8 T cells
• Therapeutic vaccination
  
  • ​eg in tumour immunotherapy

Question 5

What can techniques such as ELISA & MHC-peptide tetramer staining tell us?

Answer 5

• Can tell us about the responses elicited by vaccines
• Whether or not the vaccine is effective

Question 6

How can vaccine efficacy be measured?

Answer 6

• Ab responses
  
  • ​ELISA
    
    • ​Titre (magnitude of response)
    • Isotype
      
      • IgG: memory formation
      • IgA: mucosal immunity
  • Surface plasmon resonance technology
    
    • ​Binding affinity
• Cell mediated responses
  
  • ​Intracellular cytokine staining assays
  • pMHC tetramers
    
    • ​Detection of specific T cells (ie specific for vaccine Ag)
    • Look at the proliferation of these specific T cells
  • Dye-base in vivo and in vitro killing assays
    
    • Ability of T cells to kill infected cells
  • Bead arrays & ELISA
    
    • ​Overall cytokine levels in blood & tissue

Question 7

What do molecular biology techniques allow in vaccine development?

Answer 7

• DNA sequencing
  
  • ​Sequence pathogen genomes
  • Identify important genes for virulence
• Determine antigenic determinants of pathogens
• Mutation of pathogen
  
  • ​Rational attenuation for production of vaccine strains

Question 8

Which things must be considered for rational design of a vaccine?

Answer 8

• Type of vaccine
  
  • Attenuated, inactivated, subunit, DNA?
  • eg might need to use subunit vaccine, as the pathogen cannot be attenuated
• Type of response required
  
  • ​Cell mediated or humoral?
  • Based on the type of infection caused by the pathogen (intra- or extracellular)
• Target
  
  • ​The pathogen itself?
  • Product? (toxin, secretory factor)
• Safety profile & risk
  
  • ​Toxicity, AEs, tolerance

Question 9

How were pathogens attenuated in the past?

Answer 9

• Passage of the pathogen through cells or other organisms
• The pathogen must adapt to this new environment:
  
  • Different selective pressures
  • Mutation of the pathogen
• In so doing, the pathogen loses its ability to infect humans
• Disadvantages:
  
  • Do not know which mutations have occurred
  • If there aren’t enough mutations, pathogen can revert to virulence

Question 10

Describe the process of rational attenuation of pathogens

Answer 10

Example approach by reverse genetics:

1. ​Clone genome of pathogen
2. Identify genes:
   
   • ​Virulence
   • Replication
   • Antigenic proteins
3. Mutate or delete virulence gene, whilst leaving others in tact
   
   • Mutate by selective mutagenesis
   • ​Deletion is safer: can’t revert to virulence
     
     • But can the virus still replicate?
4. Pathogen can no longer cause harm, but can infect and induce an immune response

Question 11

Describe vector vaccines

Which vectors are used?

What are the advantages and disadvantages of this method?

Answer 11

Bacteria or viruses

• Approach
  
  • ​Attenuated pathogen
  • Engineered to express Ags from another organism
• Example vehicles:
  
  • ​Adenoviruses
  • Attenuated Salmonella
  • Poxviruses
    
    • Vaccinia
    • Canarypox
  • Genomes are quite large, and thus can accommodate additional genes without compromise
• Advantages:
  
  • ​Mimics natural infection
  • High levels of expressed Ag
  • Mucosal administration
  • Elicits both T & B cell responses
  • Antigens are properly folded
    
    • ​Important for conformational Ags
    • Maintain proper 2° and 3° structure
• Disadvantages
  
  • ​Can be dangerous in the immunocompromised
  • The vector can be the target of an immune response with repeated use

Question 12

List the various types of subunit vaccines

Answer 12

• Recombinant proteins
• Peptide vaccines
• VLPs: virus-like particles

Question 13

How are recombinant proteins made?

What are the disadvantages of these vaccines?

Answer 13

• ​Generation:
  
  • Using recombinant DNA technology
    
    • ​Plasmid encoding the Ag is introduced into an organism
    • Yeast
    • Bacteria
    • Insect cells
  • Organism produces the Ag
• Disadvantages
  
  • ​Low immunogenicity
  • Requires adjuvants
  • Need to identify the protective Ag
  • Can be difficult to manufacture (purification of recomb. proteins)

Question 14

Describe the features of peptide vaccines

How are they manufactured?

What are the advantages and disadvantages?

Answer 14

• Composition:
  
  • Peptide represents epitope of:
    
    • B cell
    • CD8 T cell
    • ​CD4 T cell
  • Adjuvant
  • ​TLR agonists
  • ISCOMs
• Manufacture
  
  • ​Synthetically generated
• Advantages
  
  • ​Very safe
  • Very defined composition
  • No risk of reversion
• Disadvantages
  
  • Not very immunogenic
  • ​B cell determinants are often not native-like
    
    • Often linear sequences: thus limited to immune responses against non-conformational antigens
  • T cell determinants are MHC restricted
    
    • eg only HLA-A2
    • The vaccine would thus only work in individuals with this haplotype

Question 15

What are ISCOMs?

Answer 15

Immune Stimulating COMplexes

• Used in peptide vaccines
• Like an adjuvant
• Helps the take up of the peptides by DCs

Question 16

What are **polytope **vaccines?

What are the advantages and disadvantages?

Answer 16

Solves problem of MHC restriction of peptide vaccines

• Genetic engineering to generate a peptide that has multiple epitopes
• These epitopes are:
  
  • ​Different epitopes of the same organism
    
    • Bind different HLA molecules
  • Epitopes from different organisms
• Advantages​
  
  • ​Provides a range of epitopes from a single organism that are presented by different MHC molecules
• Disadvantages
  
  • ​Appropriate processing of T cell epitopes
  • Competition for MHC binding
    
    • One epitope may be preferentially presented over the others
  • Some T cell responses can predominate

Question 17

Describe the features of VLP subunit vaccines

Answer 17

• MOA:
  
  • Capsid or envelope of some viruses self assemble in the absence of the genome
  • VLP binds and enters cell
  • Capsid subunits induce immune response
    
    • ​Both cellular and humoral
• eg. Gardasil HPV vaccine

Question 18

Describe the Gardasil vaccine

Answer 18

• VLP subunit vaccine
• **L1 **(major capsid protein) of HPV self-assembles
  
  • ​L1 of HPV types 6, 11 & 18

Question 19

What is Pam₂Cys?

Describe its use in vaccines

Answer 19

• PAMP (lipopeptide) recognised by TLR2
• Stimulates the formation of TLR2/6 heterodimers
• Triggers formatino of an endosome
• (Whatever is bound to Pam₂Cys will be taken up into the cell)
• Engineered onto subunit vaccines to increase delivery to DCs
• DC captures whatever is bound to Pam₂Cys, intiating a signalling cascade
  
  • ​MyD88
  • Activation of TFs
  • Transcription of genes
  • Activation of DCs
    
    • ​Pro-inflammatory molecule expression
    • Co-stimulatory molecule expression

Question 20

What is TLR2?

List the important features

What is its role in vaccines?

Answer 20

• Features:
  
  • PRR expressed on many immune cells
    
    • ​DCs, macrophages, PMNs, monocytes, lymphocytes
  • Recognises bacterial-derived lipopeptides
    
    • ​eg. Pam₂Cys
  • Present in the endosome
• Role in vaccines:
  
  • ​Subunit vaccines can be made immunogenic if they express Pam₂Cys
  • Pam₂Cys ligates TLR2
  • Transduction pathway leading to the activation of NFKB
  • NFKB turns on pro-inflammatory & activating genes in DC
  • Activation of DCs
    
    • ​Expression of co-stimulatory molecules, migration to draining LNs
  • Induction of a robust immune response

Question 21

Describe DNA vaccines:

• Contents
• Generation
• Mechanism

Answer 21

• Contents:
  
  • ​Plasmid encoding:
    
    • Eukaryotic promoter
    • Gene for pathogen Ag
    • Poly-A tail
    • Bacterial origin or replication (Ori)
      
      • Allows replication of the plasmid in bacteria
    • Selective marker for growth (antibiotic resistance)
    • Unmethylated CpG motifs
  • Saline solution
• Generation
  
  • ​Generated in bacteria
  • Replicated in bacteria
    
    • ​Hence requirement of Ori
  • Harvested
• Mechanism
  
  • ​Injection of plasmid into muscle / coating on gold beads and ‘gene gun’ administration
  • Transfection (DNA enters host cells)
  • DNA moves to nucleus
  • Transcription of DNA into mRNA by RNApol
  • mRNA translated into protein in the cytosol
    
    • Transfected cell processes and presents protein, or
    • Release of protein, capture and presentation by APC

Question 22

Describe how DNA vaccines can be administered

Answer 22

1. Injection into muscle
2. Gene gun
   
   • ​​Plasmid coated onto gold beads
   • Air highly pressurised → penetrates skin
   • Plasmid taken up by skin DCs

Question 23

What are the advantages and disadvantages of DNA vaccines?

Answer 23

• Advantages
  
  • ​Cheap to generate DNA
  • Easy to manufacture
  • Long lasting response
  • Can deliver a combination of plasmids
  • ​Various Ags
    
    • Immunomodulatory molecules
    • Unmethylated CpG motifs: TLR9 agonist
• Disadvantages
  
  • ​Oncogene activation
    
    • Can’t control where the DNA will insert
  • Low uptake into cells
  • Hypersensitivity against Ag or DNA
  • Limited administration
    
    • Only injection into muscle / gene gun
  • Efficacy in small animal models doesn’t translate
  • Large doses required

Question 24

What is the main advantage of administration of DNA vaccines by gene guns?

Answer 24

Delivers DNA efficiently to skin DCs

Question 25

Describe **immunomodulation **of DNA vaccines

Answer 25

Used to shape the immune response

• ​Plasmids encoding pro-inflammatory cytokines
  
  • ​GM-CSF
  • IFN-gamma
  • IL-12
  • IL-15
• Co-administration of mAb/cytokine
  
  • ​mAb: anti-CTLA4 (Ipilimumab)
  • Enhances the immune response

Question 26

How can DNA vaccines be improved?

Answer 26

1. Immunomodulation
   
   • Immune response is enhanced against antigen
   • Co-administration of Ipilimumab / cytokine
   • Plasmid encoded cytokines
2. Viral / bacterial vectors
   
   • ​Faster replication and expression of antigen

Question 27

What is one significant benefit of subunit vaccines over whole pathogen vaccines?

Answer 27

• Whole pathogen vaccines pose a risk to the individual; could infect & cause harm
• Subunits can not cause harm

Question 28

Why is use of adjuvants a problem?

Answer 28

• There are very many adjuvants available that are approved for use in humans
• Alum is one of the only ones
  
  • Very good induction of Ab responses
  • Poor induction of cell-mediated responses

Question 29

What is systems biology?

List some of the techniques that are used

How can it be used in vaccine design?

Answer 29

• Systems biology:
  
  • “Computational and mathematical modelling of complex biological systems”
  • Use of:
    
    • ​Gene array chips
      
      • ​See which genes are ON or OFF
      • Snapshot of the genetic landscape of a cell or tissue
    • NextGen Sequencing
      
      • ​Sequence entire genome of pathogen or host
    • RNA-seq
      
      • ​Sequencing of RNA
      • Look at the mRNA that is produced
      • Indicates which genes are being utilised
    • Bioformatics tools
      
      • ​Supercomputers
      • Need to be able to interpret all this information
        
        • ​Pathway analysis
        • Differential expression of genes
        • Signalling pathways
• In vaccine design:
  
  • Really powerful tool
  • Comprises a range of techniques to help us understand the effect of a vaccine on the body as a whole:
    
    • ​Genomic level
    • Phenotypic level
    • Mechanism of protection
  • **eg **Yellow fever vaccine

Question 30

Describe how systems biology was used to investigate the Yellow Fever vaccine

Answer 30

Yellow fever vaccine:

• ​Live-attenuated vaccine
• Been around for 65 years
• ‘Travel vaccine’
• Very effective vaccine
  
  • ​Hence, also used as a vaccine delivery vector
• Molecular mechanisms of inducing immunity are unknown

Querec et al. 2009 trial

• Aim: Identification of the molecular signatures evoked by the vaccine
• Design:
  
  • ​Administration of the vaccine to naïve individuals
  • Two separate trials
  • At two time points, bleed the individuals to acquire peripheral blood lymphocytes
  • Perform gene array and **transcription profiling **on these samples
  • Analysed which genes were switched on and off
  • Indentify the genes that were commonly induced after vaccination
  • These genes were then associated with a specific type of immunity
    
    • ​eg anti-viral / parasitic response
      
      • ​Gene network analysis:
        
        • ​Look at the overal impact of the genes
• Conclusions
  
  • ​Genes induced after vaccination:
    
    • Help regulate the innate immune response
    • have IRF7/9 binding sites
      
      • ​ie Interferon binding sites