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B-Cell Epitopes

B-Cell Epitopes. Chapter 10. Claus Lundegaard. Antibodies. Antibodies. What are they?. Virtually any substance can elicit an antibody response. Clear extra cellular pathogens neutralizing antibodies Antibody repertoire > 10 11 in humans How is this possible?

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B-Cell Epitopes

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  1. B-Cell Epitopes • Chapter 10 Claus Lundegaard

  2. Antibodies

  3. Antibodies. What are they? • Virtually any substance can elicit an antibody response. • Clear extra cellular pathogens • neutralizing antibodies • Antibody repertoire • > 1011 in humans • How is this possible? • ~ 30.000 genes in the humans genome! • Immunoglobulin gene rearrangement

  4. Antibody Effect. Neutralizing Antibodies Virus or Toxin Neutralizing Antibodies Inhibit cellular infection Clear pathogen infection

  5. Paratope Antibody - Antigen interaction Antigen Fab (fragment antigen binding) Epitope Antibody

  6. B-Cells. How are they made? Stem Cell Precurser B-lymphocytes Gene rearrangements B-lymphocytes each displaying a unique B-cell receptor

  7. Gene Shuffling

  8. Number of gene segments

  9. The 12/23 rule of recombination recombination signal sequence (RSS) { Only combined 12 RSS to 23 RSS

  10. Mechanism of gene rearrangement

  11. RAG proteins (recombination-activating genes)

  12. Addition of P and N nucleotides TdT: terminal deoxynucleotidyl transferase

  13. Antibody variable regions, CDR’s (Complementarity-determining regions)

  14. CDR Regions Variable regions Alpha-carbon trace of the structure of the heavy chain and light chain variable regions of a typical antibody. The framework regions of both chains are shown in grey whilst the complementarity determining regions (CDRs) are coloured individually, i.e. Heavy chain CDR 1 = Light blue CDR 2 = Cerise CDR 3 = Yellow Light Chain CDR 1 = Red CDR 2 = Green CDR 3 = Blue CDR = complementarity determining region http://212.219.234.139/html/anti_alpha.html

  15. Identifying CDR regions • The Kabat definition is based on sequence variability and is the most commonly used • The Chothia definition is based on the location of the structural loop regions • The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software • The contact definition has been recently introduced by us and is based on an analysis of the available complex crystal structures.

  16. Identification of CDR’s (II) CDR-H1 Start Approx residue 26 (always 4 after a Cys) [Chothia / AbM defintion]; Kabat definition starts 5 residues later Residues before always Cys-XXX-XXX-XXX Residues after always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala Length 10 to 12 residues [AbM definition]; Chothia definition excludes the last 4 residues CDR-H2 Start always 15 residues after the end of Kabat / AbM definition) of CDR-H1 Residues before typically Leu-Glu-Trp-Ile-Gly, but a number of variations Residues after Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala Length Kabat definition 16 to 19 residues; AbM (and recent Chothia) definition ends 7 residues earlier CDR-H3 Start always 33 residues after end of CDR-H2 (always 2 after a Cys) Residues before always Cys-XXX-XXX (typically Cys-Ala-Arg) Residues after always Trp-Gly-XXX-Gly Length 3 to 25(!) residues

  17. Example • >BU02A02.1 • GVQCEVHLLESGGGLVQPGGSLRLSCAASGFTFYSYAMSWVRQAPGKGLEWVSANSGSGGSTYYADSVRGRFTISRDNSKNTLYLQMNSLSAEDTAVYFCAKAPGYYYYYGMDVWGQGTTVTVSSGKNGHSRAFV 15 amino acids after end of CDR1

  18. Somatic hypermutations

  19. B-Cell Activation (Proliferation depends on affinity) No Affinity No Affinity Low Affinity Somatic Hypermutations High Affinity Plasma cells Memory B-cells

  20. B-Cell Activation T Helper Cell TCR B Cell Class II MHC Bound Peptide

  21. Cartoon by Eric Reits

  22. Controversial issues • Is the 11/23 rule always obeyed? • Can you have multiple D genes? • Can D genes be inserted backwards? • I.e can both D and the inverted D genes be used? • Does V, D and J palindrom segments exits?

  23. What did we find? • Issue of next talk

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