CONVENTIONAL VACCINE DEVELOPMENT

PROBLEM Lethal germ Dead mouse

LIVE VACCINES Related but harmless germ gives protection against lethal pathogen. Examples are the original pox vaccine and some TB vaccines

BENEFITS AND DRAWBACKS Pros Relatively safe Strong response Long memory Cons Expensive to produce and store Antigens not identical to the real pathogen

Organisms with disabled virulent properties. Examples include yellow fever, measles, rubella, and mumps. LIVE ATTENUATED VACCINES

BENEFITS AND DRAWBACKS Pros Strong response against native antigens Long memory Cons Risk of reversion/ conversion Production and storage costs

Previously virulent microorganisms which have been killed with chemicals or heat. Examples are flu, cholera, bubonic plague, polio and hepatitis A KILLED VACCINES

BENEFITS AND DRAWBACKS Pros Generally very safe Response against native antigens Cons No CTL Shorter memory

TOXOIDS Toxoids are inactivated toxins, which is used where the toxin cause illness or death, but the producing organism otherwise will be eliminated. Examples of toxoid-based vaccines include tetanus and diphtheria.

BENEFITS AND DRAWBACKS Pros Relatively safe Easy to produce Cons No CTL Shorter memory

SUBUNIT VACCINES

Johri et al. Nature Reviews Microbiology 4, 932 942 (December 2006) doi:10.1038/ nrmicro1552

ANTIGEN DISCOVERY

Eluted from Ab trapping Johri et al. Nature Reviews Microbiology 4, 932 942 (December 2006) doi:10.1038/ nrmicro1552

CTL Peptide Scan

Avian Flu vaccine development by Reverse genetics techniques from the National Institute of Allergy and Infectious Diseases

Cleavage sites and binding affinities Collaborators approaches

Bioinformatics is data driven Most methods needs a large number of training examples Best if made the same way Validation can be done by different methods Good collaborators are invaluable

in vitro cleavage measurements www.benbest.com/ lifeext/aging.html

Peptide Digest Workflow Purification of Peptides by RP-HPLC: Synthesized peptides are polished to >98% purity (1h Gradient, 25 cm x 4 mm C18 column), lyophilized and used for digestion Digestion: 100 ul Volume - 15 ul/ Timepoint MSMS-Analysis very fast analysis of peptide digests can be performed in one day multiple time points possible (Instrument time: 90 min/timepoint) Data Processing: Waters Protein Expression System (1-2h processing) Excel-Macros (Manual, 30 min/timepoint)

Peptide polishing: very high purity of peptide substrates required, but some peptides ordered at >80% purity are quite dirty -> time consuming polishing of peptides (4-8h) Protocol has been established for routine purification high hydrophobicity of some peptides leads to solubility /purification problems

MHC Binding

RMAS Assay: classical way to measure peptide binding - However not quantitative (no determination of the affinity) TAP difficient cell line At 37 C At 26 C Add peptide

RMAS Assay: classical way to measure peptide binding - However not quantitative (no determination of the affinity) TAP difficient cell line At 37 C At 26 C Add peptide Measure T cell activation

Experimental description of peptide-mhc binding How to examine HLA specificity? What the HLA has bound in vivo Elution and sequencing of natural ligands Simpel motif ~ low sensitivity predictions What the HLA will, or will not, bind in vitro Determine the binding strength of any peptide Extended motif ~ higher sensitivity predictions

How to determine peptide affinity Law of mass action Binding [R] + [L] k off k on [RL] K D = k off (S -1 )/k on (M -1 S -1 ) 100% 50% Saturation assay Binding [MHC] + [P] Binding hot peptide k off k on [P*MHC] Inhibition assay Peptide [M] Peptide Log [M] Cold Peptide Log [M] K D = (10-15 -10-6 M) Log IC 50

How to do radioactive biochemical inhibition binding assays Obtain purified HLA Or recombinant heavy chain & b2m Obtain indicator peptide Perform dose titration of any inhibitory peptide Separate free from bound peptide Calculate binding and IC 50

A spun column binding assay b2m peptide G50 MHC

A spun column binding assay b2m peptide G50 Non binding test peptid Binding test peptid MHC

The radioactive biochemical binding assay PROS Truly quantitative Can address affinities in the low nm level Reproducible CONS Radioactive Not a standard method Waste problem

The Quantitative ELISA Capable of Determining Peptide-MHC Class I Interaction Made possible by our recent development of highly active recombinant MHC class I heavy chains functional equivalents of empty molecules Pros: L.O.Pedersen et al.,, EJI. 2001, 31: 2986 Reasonably simple, sensitive and quantitative Does not depend on labeled peptide It is easily adaptable to other laboratories Disseminated protocol and standard reagents

Strategy for the assay Step I: Folding of MHC class I molecules in solution

Strategy for the assay Step I: Folding of MHC class I molecules in solution Incubation

Strategy for the assay Step I: Folding of MHC class I molecules in solution Incubation Step II: Detection of de novo folded MHC class I molecules by ELISA

Strategy for the assay Step I: Folding of MHC class I molecules in solution Incubation Step II: Detection of de novo folded MHC class I molecules by ELISA Development Sensitivity below 0.1 nm or 5 x 10-15 M MHC class I complex!

ELISA driven assay nm complex detected nm peptide offered Concentrations of complexes generated are plotted as a function of the concentration of peptide offered Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251

ELISA driven assay Results are expressed as: nm complex detected nm peptide offered B MAX : Amount of detected complex including 95% confidence interval Concentrations of complexes generated are plotted as a function of the concentration of peptide offered Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251

ELISA driven assay Results are expressed as: nm complex detected nm peptide offered B MAX : Amount of detected complex including 95% confidence interval K D : Peptide affinity including 95% confidence interval Concentrations of complexes generated are plotted as a function of the concentration of peptide offered Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251

ELISA driven assay Results are expressed as: nm complex detected nm peptide offered B MAX : Amount of detected complex including 95% confidence interval K D : Peptide affinity including 95% confidence interval Automated, 384 format Concentrations of complexes generated are plotted as a function of the concentration of peptide offered Sylvester-Hvid, C. et al., Tissue Antigens (2002) 59:251

AlphaScreen Pros Homologous proximity assay. No washing => fast development Donor bead Acceptor bead Cons Expensive reagents Specialized reader O 2 <200 nm

AlphaScreen Biotin Streptavidin anti-mouse IgG W6/32 mouse anti- HLA

AlphaScreen 480 nm O 2 560 nm Biotin Streptavidin anti-mouse IgG W6/32 mouse anti- HLA

AlphaScreen

TAP Binding

TAP Binding TAP is very hard to purify for in vitro assays (membrane bound) Most used assay is the radioactive RIA assay IC 50 from RIA assays are dependent of the affinity of the competing peptide