MSc Defense, 2004

En Route To Peptide Based Vaccination

Elucidation Of Two HLA-B*1501 Structures And Establishment Of ANovel Mass Spectrometry MHC Disulfide Bond Configuration Assay

Gustav Roder, Dec. 2004

Project Corner StonesToday’s Agenda

MHC-I in Cellular ImmunologyCytotoxic T Cell Response

Apoptosis – Cell Death

MHC-I Protective Immunology

MHC-I in Cellular ImmunologyAntigen Processing

Immunology, Biochemistry and BioinformaticsMaking Vaccine Peptide Candidates

Cloning Expression Purification Active HCs

MHC-I Heavy Chain Manufacturing

Generation of Binding Data

Prediction of Peptide Binders

HC Refolding ELISA KD

KDQBC ANN Novel Peptides

Some Cooperation Results

Sylvester-Hvid C, Nielsen M, Lamberth K, Roder G, Justesen S, Lundegaard C, Worning P, Thomadsen H, Lund O, Brunak S, Buus S.

2004. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation. Tissue Antigens 63:395-400

Lund O, Nielsen M, Kesmir C, Petersen AG, Lundegaard C, Worning P, Sylvester-Hvid C, Lamberth K, Roder G, Justesen S, Buus S, Brunak S. 2004. Definition of supertypes for HLA molecules using clustering of specificitymatrices. Immunogenetics 55:797-810

Making the MHC-I Heavy ChainsGenetic Manipulation – An Overview

Making the MHC-I Heavy ChainsGenetic Manipulation – The Construct

Making the MHC-I Heavy ChainsGenetic Manipulation – QuikChange Mutagenesis

Making the MHC-I Heavy ChainsProduction – Expression and Purification

Purification by IMAC

Making the MHC-I Heavy ChainsProduction – Expression and Purification

Ferre et al.Protein Sci. 2003, 12:551

Melander et al.J.Chromatogr. 1984, 317:67

Purification by HIC

Fractions tested in RIA or ELISA

Making the MHC-I Heavy ChainsProduction – Dose Response by ELISA

Testing of Peptide Binding Capability

HC Dose-Response in Quantitative ELISA

Sylvester-Hvid, C. et al. 2002. Establishment of a quantitative ELISA capable of determining peptide - MHC class I interaction. Tissue Antigens 59:251-258

2m-B*5801Why?

Potential Advantages :

Previously :

Sylvester-Hvid, C. et al. 1999. A Single-Chain Fusion Molecule Consisting of Peptide,Major Histocompatibility Gene Complex Class I Heavy Chain and b2-Microglobulin Can Fold Partially Correctly, but Binds Peptide Inefficiently. Tissue Antigens 59:251-258

On-site 2m mediated refolding of HC

No need for 2m production

2m-B*5801Making the Construct

2m-B*5801Expression and Purification

2m PCR HC-Vector PCR HC-Vector PCR

B2m-B*5801-HB Colony PCR

69oC 69oC (58-68)oC

2m-B*5801Expression and Purification

Fermentation IMAC Purification IEX Purification

2m-B*5801Testing the Functionality

Does 2m-B*5801 fold correctly and is it independent of external 2m

3M 2m1M pep

20nM 2m-B*5801HB2nM B*5801HB

Dependent on peptide and external 2m

MHC DSB MS AssayWhy?

Active? Active?

Why do we need ‘just another’ assay

MHC DSB MS AssayThe Basic Principle

MHC DSB MS AssayTrypsin Digestion Optimization

Assay Conditions: •trypsin/substrate ratio 1.5/100•2M urea, 25mM Tris-HCl, pH8.0•2hrs at 37C

% H

C L

oss

MHC DSB MS AssayHLA-A*0206-HB

MHC DSB MS AssaySumming Up

MHC DSB MS AssayFunctional DSB Isomer

MHC DSB MS AssayFunctional DSB Isomer

MHC DSB MS AssaySumming Up

MHC DSB MS AssayConclusion

Gorman, J. J. et al. 2002. Protein disulfide bond determination by mass spectrometry. Mass Spectrom. Rev. 21:183-216

• This assay is consistent with peptide binding data from RIA

• This assay does NOT need any addition of 2m or peptide

• This assay gets results faster than does ELISA and RIA

• This assay is capable of doing finger print analysis

HLA-B*1501 Crystal StructuresMaking the MHC Complexes

Denatured MHC Heavy ChainFolded ß2-microglobulin Peptide

Mixing Pot

Size Exclusion Chromatography

Concentration > 3 mg/mL

HLA-B*1501 Crystal StructuresMaking the Crystals

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Hanging Drops

MHC-I Complex

HLA-B*1501 Crystal StructuresFrom Diffraction to Model

Model Building

HLA-B*1501 Crystal StructuresProcess Flowchart

HLA-B*1501 Crystal StructuresOverall Structure

HLA-B*1501 Crystal StructuresPeptide Conformations

ILGPPGSVY

LEKARGSTY

Epstein-Barr virusnuclear protein 3EBNA3A (406-414)

Human ubiquitinConjugating enz.(83-91)

HLA-B*1501 Crystal StructuresILGPPGSVY Binding Pockets

Matsumura, M. et al. 1992. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science. 257:927-934

A pocket

Acknowlegdements

Department Of Medical Microbiology And Immunology

Department Of Medicinal Chemistry

Søren Buus

Kasper LamberthLise-Lotte NielsenSune JustesenJeanette NielsenAnne SchmiegelowChristian LeisnerChristina Sylvester-HvidHenrik FerreKirstine Grandahl

…

And the rest!

Michael Gajhede

Thomas BlicherOle KristensenBritt Johannessen

Department Of Medical Anatomy

Mogens Cläesson

Thank You!