epitope selection rational vaccine design. immune system differential distribution of mhc molecules...
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Epitope Selection
Rational Vaccine design
Immune SystemDifferential distribution of MHC molecules
Cell activation affects the level of MHC expressionThe pattern of expression reflects the function of MHC molecules:
Class I is involved in anti-viral immune responsesClass II involved in activation of other cells of the immune system
Tissue MHC class I MHC class II
T cells +++ +/-B cells +++ +++Macrophages +++ ++Other APC +++ +++
Epithelialcells of thymus + +++
Neutrophils +++ -Hepatocytes + -Kidney + -Brain + -Erythrocytes - -
Distinct Cells in Immune System• Lymphocytes (B cells, T cells)
- Determining specificity of immunity
• Monocyte/macrophage, dendritic cells, natural killer cells and other members of myeloid cells
- Antigen presentation
- Mediation of immunologic functions
• Specialized epithelial and stromal cells
- Providing anatomic environment
T Lymphocytes
• Helper (CD4+) and Cytotoxic (CD8+) T cells• Help B cells develop into antibody-producing
cells (HTL) • Directly killing of target cells (CTL)• Enhance the capacity of monocytes and
macrophage• Secretion of cytokines
Major Histocompatibility Complex (MHC)
• Transfer of information about proteins within a cell to the cell surface
• MHC I are expressed on the great majority of cells and recognized by CD8+ T cells
• MHC II are expressed on B cells, macrophages, dendritic cells and recognized by CD4+ T cells
• Responsible for graft rejection• Found on chromosome 6 in human and 17 in
mouse
Antigen Presentation Pathway – MHCI
• Intracellular antigens
• Viruses
Antigen Presentation Pathway – MHCII
• Extracellular antigens
• Bacteria and Parasites
Antigen Presentation Pathways
Select proteins with specific function(s)
• Assignment by homology• Sequence -> Structure prediction• Structure -> Function prediction• Sequence -> Function prediction (ProtFun)
– Secondary structure– Signal peptide– Trans membrane– Phosphorylation– Glycosylation
Peptides Binding to MHC Molecules
• MHC I molecules bind short peptides, usually between 8 and 10 residues.
• The typical length of a class I ligand comprises 9 amino acids.
• Class II ligands consist of 12 to 25 amino acids.
• A core of nine amino acids is essential for peptide/MHC binding.
Diagnostic Epitopes and Potential Vaccine Epitopes Design for
T.solium
Epitope identification for vaccines
Tsol18Tsol45
OA4 (22.5KDa) Ts23OA2 (32.5KDa) Ts32
Rationale
• The same parameters are use for vaccine development and diagnostic test, but we need to introduce the structural information and epitope distribution on the surface of the protein in vaccine development.
• The use of MD (molecular dynamic) is useful in epitope stability prediction.
Tsol18:
Aminoacid sequence:MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFALSATEIGVFWDAGEMVGHGVEEIKVKVEKAIHPYKIWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIMVFGSQRFATTLPKKQIKHKKVRRS
Glycosilation and Phosphorilation site:
Glycosilation: pos. 57-60 Asn glycosilationPhosphorilation: pos. 94-96 Protein kinase C Pos. 5-8 and 21-24 Casein kinase II phosphorilation site
Tsol18
• Hidrophobicity:• Predicted by SOSUI • Average of hydrophobicity : 0.070769• Conclusion: Tsol18 is a soluble protein• Number of transmembrane helices:• Predicted by HMMTOP server (refs. 2, 3)• No transmembrane helices detected• Cell localization:• Predicted by TMHMM and TMPred• Tsol 18 is a extra cellular protein (secreted)
Secondary structure prediction:Predicted by GOR 4. (Tsol18)
MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFALSATEIGVFWDAGEMVGHGVEEIKVKVEKAIHPYK
cccceeeehhhhhhhcccccccccceecceeeccccccceeeeeeccccceccchhhhhhhhhhhccccc
IWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIMVFGSQRFATTLPKKQIKHKKVRRSeeeeeeccccchhhhhhhcccceeeeeccccccceeeecccccccccchhhhccceeeec
• Sequence length : 130• GOR4 :• Alpha helix (Hh) : 29 is 22.31%• 310 helix (Gg) : 0 is 0.00%• Pi helix (Ii) : 0 is 0.00%• Beta bridge (Bb) : 0 is 0.00%• Extended strand (Ee) : 35 is 26.92%• Beta turn (Tt) : 0 is 0.00%• Bend region (Ss) : 0 is 0.00%• Random coil (Cc) : 66 is 50.77%• Ambigous states (?) : 0 is 0.00%• Other states : 0 is 0.00%
MHC class I epitopes predicted:Predicted by ProPred I (Tsol18)
0
5
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35
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45
50
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109
Series1
Tsol18
Sequence with maximum similarity according to BLAST:
• No significantly homologous sequence available (E-value =2.9 for the best hit)
Templates for modeling:
• No suitable templates for modeling were found
Tsol18 modeled 3D structure
• Tsol45:
Aminoacid sequence:
MASQFHLILLLTSILAGNHKATSREVGREQPLHSLFLWGPPFSTKIGLSWRGAFSEDGDKVLTLKAALTSDPNNTKTTYQILGYGRATLKGLTPNTSYIVTATANLSGNTILVLRKHIHTPLDDTNPMENYFHWGPVTNQSIQVSWDQLDPEDARSMIVTLTAEMASNPSVERSESAIPSVGRITVDGLMPDTLYIATLTVLENGRQFLTSTRDIRTLKTGHGGVTVVTTSGSGIASAILGLLFTCTVLVLA
Glycosilation sites:
Phosphorilation tsol45
Tsol45
Hidrophobicity:Predicted by SOSUI (ref 6)
Average of hydrophobicity : 0.006324Conclusion: Tsol45 is a soluble protein
Number of transmembrane helices:Predicted by HMMTOP server (refs. 2, 3)• No transmembrane helices detected
Cell localization:• Predicted by TMHMM and TMPred• Tsol 45 is a extra cellular protein (secreted)
MHC class II epitopes predicted (Tsol45):
0
10
20
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40
50
60
1 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226
Series1
MHC class I epitopes predicted (Tsol45):
0
10
20
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60
1 18 35 52 69 86 103 120 137 154 171 188 205 222
Series1
Secondary structure prediction:Predicted by GOR 4. (Tsol45)
MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFALSATEIGVFWDAGEMVGHGVEEIKVKVEKAIHPYKcccceeeehhhhhhhcccccccccceecceeeccccccceeeeeeccccceccchhhhhhhhhhhcccccIWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIMVFGSQRFATTLPKKQIKHKKVRRSeeeeeeccccchhhhhhhcccceeeeeccccccceeeecccccccccchhhhccceeeec
Sequence length : 130GOR4 :Alpha helix (Hh) : 29 is 22.31%310 helix (Gg) : 0 is 0.00%Pi helix (Ii) : 0 is 0.00%Beta bridge (Bb) : 0 is 0.00%Extended strand (Ee) : 35 is 26.92%Beta turn (Tt) : 0 is 0.00%Bend region (Ss) : 0 is 0.00%Random coil (Cc) : 66 is 50.77%Ambigous states (?) : 0 is 0.00%Other states : 0 is 0.00%
Tsol45
Sequence with maximum similarity according to BLAST:
• 45W antigen ToW6 Taenia ovis (Evalue=2e-81)• Tsa9 Taenia saginata (Evalue=2e-63)• Glucoprotein EG95-QH-3 Echinococus
(Evalue=4e-08)
Templates for modeling:• No suitable templates for modeling were found
Consensus 3D-Modeling by threading, Rosetta and Molecular Dynamics refinement: (Tsol45). MHC I,II
consensus best epitopes.
Fusion Epitope
Tsol18 selected fusion epitope
Tsol45 selected fusion epitope
OA4 (22.5KDa) Ts23modeled 3D structure
Glycosilation sites on 23KDa
Phosphorilation sites on 23 KDa
23KDa MHC II profile
0
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35
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45
50
1 17 33 49 65 81 97 113 129 145 161 177 193 209 225 241
23KDa
Phosphorilation sites
23KDa best inmunogenic epitopes
23KDa electronic density: inmunogenic epitopes
Stability of the epitope with the higest Peak in Epitope prediction
(red Epitope) 23KDa (100ps)
Potential Energy change in MD stability
Red epitope (23KDa) (after 1500ps MD)
Accesibilidad 23KDa
Accesibilidad 23KDa
HLA 23KDa
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225 239
Series1
Propred 23KDa
0
5
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35
40
45
1 19 37 55 73 91 109 127 145 163 181 199 217 235
Series1
Propred I 23KDa
0
1
2
3
4
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6
7
8
9
10
1 21 41 61 81 101 121 141 161 181 201 221 241
Series1
23KDa consenso
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 20 39 58 77 96 115 134 153 172 191 210 229 248
Series1
Epitopes inmunogenicos 23KDa (ninguno es expuesto a superficie)
OA2 (32.5KDa) Ts32 modeled 3D structure
Glycosilation sites for 32 KDa
Glycosilation sites for 32KDa
Phosphorilation sites for 32 KDa
32KDa MHC II profile
0
10
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60
1 17 33 49 65 81 97 113 129 145 161 177 193 209 225
Series1
MHC I and MHC II 32KDa
MCH II
MHC I
Signal sequence peptide: MSFQLYLILLVTSVLA
Phosphorilation sites
N-Glycosilation has no effect on immunogenicity
O-glycosilation ???
Conseso 32KDa (SVM HLA Propred Propred I)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225
Series1
32KD modeled 3D structure: Green(threading method)
Red(refinement with 113ps MD in vacum)
Proteina 32KDa Epítopes pronosticados
EKDKATTEPPIGQYFHWGEVDFQSVSLSWETEKLHSLLDAEITIKAVLTPGPGPERSTIAYLSDGEVTLDGLIPNSSYAVTAAVVRGKTTILELKKNIQTKVVDKSLIEKFFHWGPITAKSISLRWDLEGQDDLLDGVIQMTAVRNSDPTSKTTTSEPFSKGKATLDGLVSNSLYAVTVTGLVEGYKYFDFTENIKTLDTGHGKGGVARTGGFGITSVVVGLLFTCMALVLD XXX Sitios de glicosilación que no afectan a los epítopes inmunogénicos XXX Sitios de glicosilación que SI afectan a los epítopes inmunogénicos Epítopes escogidos: ITIKAVLTP ‘Loop puro’ dentro de la proteína total VVRGKTTILE ‘Loop y ¼ de hoja beta’ dentro de la proteína total IQMTAVRNSDPTSK ‘Loop y ¾ de hoja beta’ dentro de la proteína total FGITSVVVGLLFTCMA ‘Alfa hélice – loop – beta’ dentro de la proteína total Potenciales epítopes de fusión:
1) IQMTAVRNSDPTSKITIKAVLTP 2) ITIKAVLTPIQMTAVRNSDPTSK escogido 3) IQMTAVRNSDPTSKVVRGKTTILE 4) VVRGKTTILEIQMTAVRNSDPTSK escogido 5) FGITSVVVGLLFTCMAITIKAVLTP 6) ITIKAVLTPFGITSVVVGLLFTCMA 7) FGITSVVVGLLFTCMAVVRGKTTILE 8) VVRGKTTILEFGITSVVVGLLFTCMA
Posteriormente se tuvo problemas con el cuarto epitope, se fuciono con el tercer epitope de ts18var1 y se obtuvieron las siguientes combinaciones: FGITSVVVGLLFTCMAQEAKGEIRASLAEYCRGLKNKT QEAKGEIRASLAEYCRGLKNKT FGITSVVVGLLFTCMA escogida
Best immunogenic epitopes for 32KDa
Epitope 1
Epitope 2
Epitope 3
Electronic density of 32KDa: Best immunogenic epitopes
Electronic density of 32KDa: Best immunogenic epitopes
Electronic density of 32KDa: Best immunogenic epitopes
Accesibility of aminoacid residues in 32KDa protein
Western blot (32KDa vs. Tsol45)
Comparison of 32KDa and Tsol45 32KDa(green), Tsol45(white)
Alignment of 32KDa and Tsol45
Alignment of sequences aminoacids
Alignment of 32KDa and Tsol45
Alignment of sequences aminoacids
Alignment of 32KDa and Tsol45
Alignment of sequences aminoacids
32KDa and Tsol45
The highest similar Block Pattern is not an Epitope in both proteins
Alignment of the best epitopes of Tsol45 and 32KDa
Alignment
Western blot (23KDa – Tsol45)
Local alignment Tsol45 vs. 23KDa
Local alignment 23KDa vs. Tsol45
MHC II MHC I23KDa / Tsol45
Multiepitopic fusion protein cysticercosis vaccine
Tsol18
Tsol45
23KDa OA2
32KDa OA4
Multiepitopic fusion protein cysticercosis vaccine
Multiepitopic fusion protein cysticercosis vaccine
Multiepitopic fusion protein cysticercosis vaccine
Epitope 1 in 32KDa
Epitope 1 in 32KDa and modified transferrin
Epitope 1 in modified Transferrin
Epitope 2 in 32KDa
Epitope 2 in 32KDa and modified transferrin
Epitope 2 in modified Transferrin
Epitope 3 in 32KDa
Epitope 3 in 32KDa and modified transferrin
Epitope 3 in modified Transferrin
Epitope 1 in Tsol18
Epitope 1 in Tsol18 and modified transferrin
Epitope 1 in modified transferrin
Epitope 2 in Tsol18
Epitope 2 in Tsol18 and modified transferrin
Epitope 2 in modified transferrin
Epitope 1 in Tsol45
Epitope 1 in Tsol45 and modified transferrin
Epitope 1 in modified transferrin
Epitope 2 in Tsol45
Epitope 2 in Tsol45 and modified transferrin
Epitope 2 in modified transferrin
Diagnostic Epitopes
GP50Ts14Ts18
TsRS1
GP50 protein
• Gp50 is an important protein candidate to differentiate the two stages in human T. solium cysticercosis:
• Active disease
• Calcified cyst stage
GP50 MHC II profile
0
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1 25 49 73 97 121 145 169 193 217 241 265
Series1
GP50 epitopes detected by several webservers
GP50 modeled 3D structure
Epítopes GP50
Epítopes GP50
TS14Highest signal: somewhere near GKIRTSLVEHCKGPKKK Second highest signal: somewhere near VANSTKKGIEYVHE Third highest signal: somewhere within EDPIGKQIAQLAKEWKEAM
Ts14 secondary structure prediction
Ts14 secondary structure prediction
Ts14 MHC I and MHC II consensus profile
0
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1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65
Series1
Ts14 (epitope maping) 13-mers window skipping 3 aminoacids
Ts18 MHC II epitope profiles for different alleles
Ts18 MHC I and MHC II consensus profile
0
5
10
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45
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73
1 3 5 7 9 11 13 15 17 19
A65
0
0.0
0.4
0.8
1.2
1.6
1 3 5 7 9 11 13 15 17 19
A65
0
0.0
0.4
0.8
1.2
1.6
1 3 5 7 9 11 13 15 17 19
A65
0
0.0
0.4
0.8
1.2
1.6
1 3 5 7 9 11 13 15 17 19A
650
0.0
0.4
0.8
1.2
1.6
1 3 5 7 9 11 13 15 17 19
A65
0
0.0
0.4
0.8
1.2
1.6
1 3 5 7 9 11 13 15 17 19
A65
0
0.0
0.4
0.8
1.2
1.6
Ts18 epitope mapping
13-mers window skipping 3 aminoacids
Ts18 modeled 3D structure
Ts18 var1 3D structure
Ts18 Ramachandran Plot for the 3D modeled structure
TsRS1 MHC I and MHC II consensus profile
0
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1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69
TsRS1 3D modeled structure
TsRS1 Ramachandran plot
TsRS1 epitope mapping
13-mers window skipping 3 aminoacids
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14 16 18
A65
0
0.0
0.4
0.8
1.2
1.6