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 > 1011 in humans How is this possible? ~ 30.000 genes in the humans genome! Immunoglobulin gene rearrangement

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

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

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

Gene Shuffling

Number of gene segments

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

Mechanism of gene rearrangement

RAG proteins (recombination-activating genes)

Addition of P and N nucleotides TdT: terminal deoxynucleotidyl transferase

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

CDR Regions CDR = complementarity determining region 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

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.

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

Example >BU02A02.1 GVQCEVHLLESGGGLVQPGGSLRLSCAASGFTF YSYAMSWVRQAPGKGLEWVSANSGSGGSTY YADSVRGRFTISRDNSKNTLYLQMNSLSAEDT AVYFCAKAPGYYYYYGMDVWGQGTTVTVSSG KNGHSRAFV 15 amino acids after end of CDR1

Somatic hypermutations

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

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

Cartoon by Eric Reits

Is the 11/23 rule always obeyed? Can D genes be inserted backwards? 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?

What did we find? Issue of next talk