Historical definition of Antigen

Historical definition of Antigen An antigen is a foreign substance that elicits the production of antibodies that specifically binds to the antigen.

Historical definition of Antigen An antigen is a foreign substance that elicits the production of antibodies that specifically binds to the antigen.

Any substance able to provoke an immune response in the human body

Antibody structure (1)

Antibody structure (2)

Antibody structure (3)

B-cell receptor biology

Antibody structure (4)

Antigen structure (1)

Hen Egg White Lysozyme – 1VED

Antigen structure (1)

Hen Egg White Lysozyme – 1VED

Antigen structure (2) Sequential Epitope

Conformational Epitope

Hen Egg White Lysozyme – 1VED

Antigen structure (3)

Hen Egg White Lysozyme – 1VED

Antigen structure (4)

Hen Egg White Lysozyme – 1VED

Identification of epitope in the target protein sequence (1) sequence scanning by overlapping peptides for sequential epitopes

Antigen structure (5)

Hen Egg White Lysozyme – 1VED

Identification of epitope in the target protein sequence (2) scanning of coupled peptide for conformational epitopes

Can we identify B-cell epitopes by using bioinformatic approaches?

Hen Egg White Lysozyme – Protein sequence analysis

Reverse Vaccinology (protective antibodies)

Reverse Vaccinology (protective antibodies)

Rappuoli R. Curr Opin Microbiol 2000

QUESTION TIME (1)

MHC: the Key to how T-cells see the “Universe”

CD4+ T-Cell

HLA/peptide/TCR complex

CD8+ T-Cell Proteasome

Ribosomes

Pathogens Exogenous particles etc

APC

HLA-class II HLA-class I

TCR

T-cell receptor feature

Different evolution of the diversity in the proteins involved in the specific T-cell response Vertebrate only No intraspecie gene variability

TCR

Multiple gene fragments for variable portions present in the gene Rearrangment of the variable gene portion at somatic level Large repertoire (1018 different combinations) in each individual for the antigen recognition

Antigenic peptide Strongly conserved among different phyla

(MHC) HLA

Large intraspecie/population variability Gene duplication Codominance of the genes for increasing the possibility of the antigen recognition (from 6 to 14 different proteins for individual in human)

Different evolution of the diversity in the proteins involved in the specific T-cell response

TCR

Antigenic peptide

(MHC) HLA

Enough large repertoire for antigen recognition..... i.e. There will be always one or more TCRs capable to recognise a HLA-peptide complex and start the immune if response Determine a protein portion might be recognised as an antigen.... i.e. the binding of a peptide to a MHC

MHC/peptide complex peptide

MHC molecule

cell membrane

The Major Histocompatibility Complex (Human Leukocyte Antigen, in Human) The Key to How T Cells See the “Universe”

The discovery Agglutination of the leucocytes of twins with the aid of leuco-agglutination sera Monozygotic twins Leuco-agglutination sera Au Ch Ce Le Bo Te Ro P.Va + + + + + J.Va + + + + +

De -

Ds + +

Rb -

Vc -

Ba + +

Pu + +

Lx + +

E.Ro H.Ro

+ +

+ +

-

-

+ +

-

+ +

-

+ +

+ +

-

+ +

+ +

+ +

D.Te B.Te

+ +

+ +

+ +

+ +

+ +

-

-

-

+ +

-

+ +

+ +

-

-

-

-

+

+

+

-

+ +

+ -

-

+ +

+

+ +

Dizygotic twins L.Go + + V.Ro + +

Dausset and Brecy, Nature, 1958 180: 1430

The discovery (2) • Leuco-agglutination sera formed after blood transfusion • Monospecific leuco-agglutination antibodies formed during pregnancy • Family studies showing Mendelian segregation • Importance in trasplantation and tests of the human transplant failures • Development of microtoxicity tests (Terasaky) • Computer facility for analysing multiple 2x2 c2 tables • Development of the Mixed Lymphocyte Reaction tests • Studies on cousin marriage families (identified thanks to the Roman Catholic church archives) • Identification of the cross-over between the different “Leukocytes groups” identified • Idea that it was one genetic system and not different groups (like blood groups) • 10 WORKSHOPS in less than 30 years!!! • No patent requests and full sharing of reagents betwen groups and setting of a repository

The discovery (3) The molecular genetic of HLA HLA complex

Chromosome 6

6p21.3

0 Kb

Class I

Class III

Class II 4000 Kb

Map of the Human MHC from the Human Genome Project 3,838,986 bp 224 genes on chromosome 6

The MHC sequencing consortium Nature 401, 1999

http://webace.sanger.ac.uk/cgi-bin/ace/pic/6ace?name=MHC&class=Map&click=400-1

Detailed Map of HLA region

Map of the HLA Complex - Microsatellites

HLA-DRB1

Class I MHC heavy (a) chain genes: HLA-A, HLA-B & HLA-C

HLA Class I MHC region

HLA CLASS I

CD8 T cell

MHC I

Cytoplasm

Nucleated cell

EACH LOCUS ENCODES EITHER AN ALPHA CHAIN GENE OR A BETA CHAIN GENE Genes: TAP and proteosome components HLA class II peptide loading

HLA Class II MHC region

HLA CLASS II

CD4 T cell

MHC II Intravesicular

Antigen presenting cell

Extracellular

Differential distribution of MHC molecules Tissue

MHC class I

MHC class II

T cells B cells Macrophages Other APC

+++ +++ +++ +++

+/+++ ++ +++

Epithelial cells of thymus

+

+++

+++ + + + -

-

Neutrophils Hepatocytes Kidney Brain Erythrocytes

Genes: TNF, C2, C4, factor B, etc.

HLA Class III MHC region

WHAT TYPES OF GENES ARE ENCODED BY THE MHC? 

The MHC basically contains 3 types of genes:

Type of gene Class I MHC

MHC region Class I region

Function Peptide presentation to CD8 T cells

Class II MHC

Class II region

Peptide presentation to CD4 T cells

Class III MHC

Class III region

Multiple: do not involve antigen presentation examples: genes for C4, C3, factor B (Bf)

HLA characteristics - Polygenic: many genes: - HLA-A, -B, -C; HLA-DR, -DP, -DQ

HLA (genetic products)

HLA characteristics - Polygenic: many genes: - HLA-A, -B, -C; HLA-DR, -DP, -DQ - Codominance: - both paternal and maternal genes are expressed

Diversity of MHC molecules in the individual DP  a

DQ  a

DR 1 a

B C

A Polygeny

DP  a

DQ  a

DR 1 a

B C

DP  a

DQ  a

DR 1 a

B C

A Variant alleles polymorphism

HAPLOTYPE 1

HAPLOTYPE 2

A Additional set of variant alleles on second chromosome

MHC molecules are CODOMINANTLY expressed Two of each of the six types of MHC molecule are expressed Genes in the MHC are tightly LINKED and usually inherited in a group

The combination of alleles on a chromosome is an MHC HAPLOTYPE

HLA characteristics - Polygenic: many genes: - HLA-A, -B, -C; HLA-DR, -DP, -DQ - Codominance: - both paternal and maternal genes are expressed - Polyallelic: many gene alleles at each locus (e.g., HLA-B) - HLA-B8, HLA-B15, HLA-B27 - Polymorphic: the HLA molecules are highly polymorphic (variations in primary amino acid sequence) as a consequence of polyallelism

A. HLA-A

D. HLA-DP

B. HLA-B

E. HLA-DQ

C. HLA-C

F. HLA-DRB1

Map of the genes in the HLA Database

http://www.anthonynolan.org.uk/HIG/data.html April 2003 update

Number of HLA antigens/alleles identified over the years

http://www.anthonynolan.org.uk/HIG/data.html April 2003 update

HLA antigens (serological variants) identified HLA-A A1 A2 A3 A11 A23(9) A24(9) A25(10) A26(10) A29(19) A30(19) A31(19) A32(19) A33(19) A34(10) A36 A43 A66 A68(28) A69(28) A74(19)

HLA-B B7 B8 B13 B14 B15 B18 B27 B35 B37 B38(16) B39(16) B40 B41 B42 B44(12) B45(12) B46 B47 B48 B49(21) B50(21) B51(5) B52(5) B53 B54(22) B55(22) B56(22) B57(17) B58(17) B59 B67 B73 B78

HLA-C Cw1 Cw2 Cw3 Cw4 Cw5 Cw6 Cw7 Cw8 Cw9 Cw10

HLA-DR DR1 DR15(2) DR16(2) DR3 DR4 DR11(5) DR12(5) DR13(6) DR14(6) DR7 DR8 DR9 DR10 DR52 DR53 DR51

HLA-DQ DQ5(1) DQ6(1) DQ2 DQ7(3) DQ8(3) DQ9(3) DQ4

HLA-DP DPw1 DPw2 DPw3 DPw4 DPw5 DPw6

Number of HLA alleles in the HLA/IMGT database Class I A

B

C

E

F

G

H

J

K

L

274

519

133

6

1

15

0

0

0

0

DRA

DRB

DQA1

DQB1

DPA1

DPB1

DMA

DMB

DOA

DOB

3

404

24

53

20

103

4

6

8

8

DRB1

DRB2

DRB3

DRB4

DRB5

DRB6

DRB7

DRB8

DRB9

TOT

329

1

38

12

17

3

2

1

1

404

Class II

HLA-DR

Other non-HLA genes MICA

MICB

MICC

MICD

MICE

TAP1

TAP2

LMP2

LMP7

55

0

0

0

0

6

4

0

0

http://www.anthonynolan.org.uk/HIG/data.html April 2003 update

Nomenclature for factors of HLA alleles Nomenclature

Indicates

HLA HLA-DRB1

the HLA region and prefix for an HLA gene a particular HLA locus, i.e. DRB1

HLA-DRB1*13

a group of alleles encoding the DR13 specificity

HLA-DRB1*1301

a specific HLA allele

HLA-DRB1*1301N

a null allele

HLA-DRB1*130102

an allele which differ by a synonymous mutation

HLA-DRB1*13010102

an allele which contaning a mutation outside the coding region HLA-DRB1*13010102N a null allele contaning a mutation outside the coding region HLA-DRB1*13010103L an allele contaning a mutation outside the coding region which leads to a lower level of expression By default: HLA-DRB1*13010101

How diverse are HLA molecules in the population? IF

• each individual had 6 types of MHC • the alleles of each MHC type were randomly distributed in the population • any of the about 2000 alleles could be present with any other allele

~2 x 1032 unique combinations In reality MHC alleles are NOT randomly distributed in the population

Alleles segregate with lineage and race

Frequency (%) Group of alleles

CAU

AFR

ASI

HLA-A1

15.18

5.72

4.48

HLA- A2

28.65

18.88

24.63

HLA- A3

13.38

8.44

2.64

HLA- A28

4.46

9.92

1.76

HLA- A36

0.02

1.88

0.01

x

x

x

Secondary Hot-spots of recombination

Primary Hot-spots of recombination

x

HLA ancestral haplotypes Based on: - the full HLA class I and II typing, - microsatellite analysis on class III region (in particular the area of the TNF genes),

53 ancestral haplotype in the HLA genomic region has been identified. Each one include the HLA-B and –C and TNF variants They could/couldn’t include the HLA-A and/or –DR and DQ alleles Nomenclature are based on HLA-B serotype The most frequents are present in >1-3% population i.e. Ancestral Haplotype 8.1: HLA-A1; -Cw7, -B8, TNFA2, DRB1*0301 and DQB1*0201

HLA-DR haplotypes

expressed genes

pseudogenes

Inheritance of MHC haplotypes

Parents DP-1,2 DQ-3,4 DR-5,6 B-7,8 C-9,10 A-11,12

DP

DP

DQ DR

DQ DR

BC

BC

DP-9,8 DQ-7,6 DR-5,4 B-3,2 C-1,8 A-9,10

DP-1,8 DQ-3,6 DR-5,4 B-7,2 C-9,8 A-11,10

A

A

Children

X DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9

DP-2,8 DQ-4,6 DR-6,4 B-8,2 C-10,8 A-12,10 DP-2,9 DQ-4,7 DR-6,5 B-8,3 C-10,10 A-12,9

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

DP

DQ DR

BC

A

Haplotype

HLA identical (share both haplotypes)

Haplotype

HLA haploidentical (“half”-identical; share one haplotype)

Haplotype

HLA nonidentical no shared haplotypes)

Which is the functional role of the polymorphisms of HLA molecules?

RIBBON DIAGRAM: PEPTIDE WITHIN THE GROOVE FOR CLASS I MHC

SPACE-FILLING DIAGRAM: PEPTIDE WITHIN THE GROOVE FOR CLASS I MHC

Class I MHC

Class I MHC

Class I MHC

Interaction between HLA class I and peptide

Pocket F

Pocket B HLA classe I

RIBBON DIAGRAM: PEPTIDE HANGS OUT OF THE GROOVE (CLASS II MHC)

SPACE-FILLING DIAGRAM: PEPTIDE HANGS OUT OF THE GROOVE (CLASS II MHC)

Interaction between HLA class II and peptide

P4

P6

P1 HLA classe II

P9

Class II MHC

Class II MHC

Class II MHC

HLA polymorphisms (1) +0.7

A. HLA-DP2(E69) -0.7

+0.7

B. HLA-DP2K69 -0.7

Berretta et al 2003.

HLA polymorphisms (2) HLA-DP2 P4 affinities

HLA-DP2k69 P4 affinities

Y W

Y W

V T S

V T S

R Q

R Q P N M L K I H

P N M L K I H G F E

G

C

C

F E D

D (WT) A 0.01

0.1

A (WT)

1

10

100 0.01

0.1

1

10

100

IC50 M

IC50 M

Position P4 of HLA-DP2 preferentially bind polar, aromatic or positive charged residues.

Posizione P4 of HLA-DP2k69 bind preferentially only aromatic residues. Berretta et al 2003.

HLA supertypes HLA alleles sharing the peptide binding motif “perform” the same “work” and are grouped in a supertype Nine HLA-class I supertypes have been identified: A1, A2, A3, A24, B7, B27, B44, B58 and B62

Nine HLA-DR supertypes have been identified: 1, 3, 4, 7, 8, 11, 13, 15, 51 Four HLA-DP supertypes have been identified: 2, 3, 4, 9

Non-self

Individual peptides derived from a pathogen Simplistic model for HLA molecules encoded by a single chr.: 1st set of 3 ellipses: potential epitopes presented to CD8 T cells 2nd set of 4 ellipses: potential epitopes presented to CD4 T cells

Being polygenic & polyallelic: many peptides can be presented to CD4 T cells and CD8 T cells

For the human population: ~all peptides can be presented to CD4 T cells and CD8 T cells Non-self

Potential presentation of any pathogen that contains peptides or proteins!

QUESTION TIME (2)

Identification of T-cell microbial antigens by reverse immunogenetic

HLA molecules are peptide binding molecules

Pocket B

HLA class I

Pocket F P1

P4 P6

HLA class II

P9

HLA-A*0201

ANCHOR AMINOACIDS

HLA-A*1101

ANCHOR AMINOACIDS

Adapted from Janeway et al., Immunobiology (2001)

HLA-DRB*0404

SECONDARY PRIMARY ANCHOR ANCHOR AMINOACIDS AMINOACIDS

PEPTIDE CORE

Adapted from Janeway et al., Immunobiology (2001)

Peptide binding motif analysis (1) (qualitative) HLA class II

HLA class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I Class I

X[LM]XXXXXXX[VL] X[LM]XXXXX[VL] X[LM]XXXXXX[VL] X[L]XXXXXXX[LV] X[L]XXXXXX[LV] X[L]XXXXX[LV] X[L]XXXXXXX[L] X[L]XXXXXX[L] X[L]XXXXX[L] X[VLIMQ]XXXXXX[L] X[VLIMQ]XXXXXXX[L] X[VLIMQ]XXXXX[L]

A*0201 A*0201 A*0201 A*0202 A*0202 A*0202 A*0204 A*0204 A*0204 A*0205 A*0205 A*0205

Class II Class II Class II Class II Class II Class II Class II Class II Class II Class II Class II Class II Class II

[IL]XX[N]X[AS]XX[IL] [W]XX[VK]X[DS]XX[N] [FY]XX[A]X[NT]XX[Q] [A]XX[A]XXXX[T] [V]XX[M]X[K]XXX XXX[Y]XXXXX [I]XX[I]XX[V]XX [L]XX[F]XX[I]XX [V]X[A]XXXXX[V] [IL]XX[L]X[R]XX[Y] [V]XX[V]X[K]XX[F] [MV]XXX[MY]XX[MV]X [FL]XXX[FL]XX[IA]X

DRB3*0301 DRB1*0407 DRB1*0407 DRB1*1302 DRB1*1302 DRB1*1302 DRB1*1501 DRB1*1501 DRB1*1201 DRB1*1301 DRB1*1301 DPB1*0201 DPB1*0201

“Peptide binding motif analysis” (2) (quantitative)‫‏‬

Amicosante M. Sarcoidosis Dif. Lung Dis. 2008

“Peptide binding motif analysis” (3)‫‏‬

1

3

2

Seghrouchni F., Berretta F., Amicosante M.; Curr. Pharmacogen. 2005

Pathogen genome

Pathogen immunome

Pathogen proteome

Portion of pathogen derived pepetides recognised by HLA molecules

Subject’s pathogen immunome (i.e. Pathogen epitopes recognised by the combination of the subject’s HLA molecules)‫‏‬