epitope selection rational vaccine design. immune system differential distribution of mhc molecules...

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)

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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):

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1 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226

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MHC class I epitopes predicted (Tsol45):

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1 18 35 52 69 86 103 120 137 154 171 188 205 222

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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

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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

HLA 23KDa

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1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225 239

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Propred 23KDa

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1 19 37 55 73 91 109 127 145 163 181 199 217 235

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Propred I 23KDa

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23KDa consenso

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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

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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)

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1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225

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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

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

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

Epitope 1 in 32KDa

Epitope 1 in 32KDa and modified transferrin

Epitope 1 in modified Transferrin

Epitope 2 in 32KDa

Epitope 3 in 32KDa

Epitope 1 in Tsol18

Epitope 1 in Tsol18 and modified transferrin

Epitope 1 in modified transferrin

Epitope 2 in Tsol18

Epitope 1 in Tsol45

Epitope 2 in Tsol45

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

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GP50 epitopes detected by several webservers

GP50 modeled 3D structure

Epítopes GP50

TS14Highest signal: somewhere near GKIRTSLVEHCKGPKKK Second highest signal: somewhere near VANSTKKGIEYVHE Third highest signal: somewhere within EDPIGKQIAQLAKEWKEAM

Ts14 secondary structure prediction

Ts14 MHC I and MHC II consensus profile

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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

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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

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TsRS1 3D modeled structure

TsRS1 Ramachandran plot

TsRS1 epitope mapping

13-mers window skipping 3 aminoacids

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epitope selection rational vaccine design. immune system differential distribution of mhc molecules...

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