b cell epitopes - cbs · b cell epitopes tuesday, 9 june 2009. technical university of denmark -...

Technical University of Denmark - DTUDepartment of systems biology

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B cell Epitopes

Tuesday, 9 June 2009


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Antibody Effect

Virus or Toxin Antibodies



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Antibodies



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Antibody - Antigen interactionAntigen

Antibody



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Antibody - Antigen interaction

Fab

Antigen

Antibody



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Fab

Antigen

Epitope

Antibody



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Fab

Antigen

Paratope

Antibody



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Structural Epitopes



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Discontinuous Epitopes



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Discontinuous Paratopes



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Variable regionsAlpha-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 chainCDR 1 = Light blue

CDR 2 = CeriseCDR 3 = Yellow

Light ChainCDR 1 = Red

CDR 2 = GreenCDR 3 = Blue

CDR RegionsCDR = complementarity determining region

http://212.219.234.139/html/anti_alpha.html





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B-Cells

B-lymphocytes each displaying a unique B-cell receptor

Stem Cell

Precurser B-lymphocytes

Gene rearrangements


Figure 4-2


Figure 4-3


Figure 4-4


Figure 4-5


Figure 4-6



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P-nucleotide introduction



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N-nucleotide introduction



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Somatic Hypermutations

© 2001 by Garland Publishing



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B-Cell ActivationNo AffinityLow AffinityNo Affinity

High Affinity

Somatic Hypermutations

Memory B-cells

Plasma cells



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B-Cell Activation

B Cell

T Helper Cell

Class II MHC

Bound Peptide

TCR



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Cartoon by Eric Reits



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Monoclonal Abs

•Hybridomas

•Phage Display



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Hybridomas

http://www.britannica.com/


http://www.britannica.com

http://www.britannica.com


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Phage Display



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Phage Display

http://www.bio.anl.gov/highthroughputbiology/images/phage_display1.jpg





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Identification of germ-line genes

Why?

As bookkeeping and reporting tool

For detection of potentially important residues in binding affinity.



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Joinsolver



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Vbase



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Vquest



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VDJsolver



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Questions to be addressed

•Can multiple D genes be inserted?

•Violation of 12/23 rule

•Can D genes be inserted backwards?

•Is there a D gene preference?

•Is there a reading frame preference for D genes?

•If yes, is it part of the gene rearrangement?



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Data sets

•6329 clonally unrelated rearrangements.

•1968 un-mutated functional

•3707 mutated functional

•274 un-mutated non-functional

•380 mutated non-functional



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SequencesSequencesSequences Permutated sequencesPermutated sequencesPermutated sequences

Distance from heptamer to gene end

No. ofseq

No. withP % with P No. of

seqNo. with

P % with P p-value

VH geneVH geneVH geneVH geneVH geneVH geneVH geneVH gene

1 1448 474 32.7 1635 103 6.3 <10-5

2 1027 48 4.7 1068 65 6.1 0.091

3 762 53 7.0 612 36 5.9 0.245

JH geneJH geneJH geneJH geneJH geneJH geneJH geneJH gene

1 324 60 18.5 350 23 6.6 <10-5

2 184 2 1.0 209 3 1.4 0.560

3 219 8 3.7 250 14 5.6 0.220

5’ end of D gene 5’ end of D gene 5’ end of D gene 5’ end of D gene 5’ end of D gene 5’ end of D gene 5’ end of D gene 5’ end of D gene

1 519 128 24.7 619 54 8.7 <10-5

2 343 31 9.0 347 26 7.5 0.275

3 474 25 5.3 454 17 3.7 0.168

3’ end of D gene3’ end of D gene3’ end of D gene3’ end of D gene3’ end of D gene3’ end of D gene3’ end of D gene3’ end of D gene

1 616 86 14.0 684 58 8.5 0.001

2 266 30 11.3 276 24 8.7 0.195

3 460 5 1.1 485 9 1.9 0.241

P nucleotides



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How many types of D genes?

•Conventional D genes

• Identified in 81% of sequences unmutated sequences, 64% of mutated sequences

•Inverted D genes

•Long inverted D genes can not be excluded

•Two or more D genes



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D gene usage27 conventional D genes, 34 known alleles



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D-gene usage and JH gene

•JH proximal D genes more often recombined to JH4 than JH6 and JH distal D genes more often to JH6



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Inverted (palindrom) D genes



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Inverted (palindrom) D genes

Inverted D genes are not used!(or used extremely infrequent)



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Multiple D genes

•65 sequences with two D genes

•Average length of shortest D genes: 11.6bp

•Average length of longest D genes: 18.8bp

•Average length of D genes in permuted sequences: 11.3bp

•Average length of D genes in normal sequences: 17.8bp

• => multiple D genes are not present!!!

Longest-D Shortest-DV-gene J-gene



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D gene reading frames

• The recombination mechanism utilizes each D gene reading frame at same frequency

Reading Frame StopStopStop HydrophilicHydrophilicHydrophilic HydrophobicHydrophobicHydrophobic

Gene P NP P NP P NP

D2-2*01 RIL**YQLLC (1) 6.5 34.7 GYCSSTSCYA (2) 61.2 32.6 DIVVVPAAM (3) 32.2 32.6

D2-2*02 RIL**YQLLY (1) 11.3 46.7 GYCSSTSCYT (2) 55.0 20.0 DIVVVPAAI (3) 33.8 33.3

D2-2*03 WIL**YQLLC (1) 0.0 50.0 GYCSSTSCYA (2) 66.7 50.0 DIVVVPAAM (3) 33.3 0.0

D2-8*01 RILY*WCMLY (1) 2.4 42.9 GYCTNGVCYT (2) 68.3 28.6 DIVLMVYAI (3) 29.3 28.6

D2-8*02 RILYWWCMLY (1) 0.0 0.0 GYCTGGVCYT (2) 88.9 0.0 DIVLVVYAI (3) 11.1 100

D2-15*01 RIL*WW*LLL (1) 1.5 32.5 GYCSGGSCYS (2) 70.8 37.5 DIVVVVAAT (3) 27.7 30.0

D2-21*01 SILWW*LLF (1) 8.3 50.0 AYCGGDCYS (2) 58.3 25.0 HIVVVIAI (3) 33.3 25.0

D2-21*02 SILWW*LLF (1) 0.0 54.5 AYCGGDCYS (2) 78.0 18.2 HIVVVTAI (3) 22.0 27.3

Total - 10.8 33.6 - 62.2 32.4 - 26.9 34.0



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VDJsolver performanceUnmutated sequences

Mutated sequences

#: p<0.01§: P<0.001


b cell epitopes - cbs · b cell epitopes tuesday, 9 june 2009. technical university of denmark -...

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