1. Epitope Selection Rational Vaccine design

2. Immune System Differential distribution of MHC molecules Cell activation affects the level of MHC expression The pattern of expression reflects the function of MHC molecules: Class I is involved in anti-viral immune responses Class 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+++ +++ Epithelial cells of thymus + +++ Neutrophils+++ - Hepatocytes + - Kidney + - Brain + - Erythrocytes --

3. 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

4. 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

5. 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

6. Antigen Presentation Pathway – MHCI Intracellular antigens Viruses

7. Antigen Presentation Pathway – MHCII Extracellular antigens Bacteria and Parasites

8. Antigen Presentation Pathways

9. 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

10. 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.

11. Diagnostic Epitopes and Potential Vaccine Epitopes Design for T.solium

12. Epitope identification for vaccines Tsol18 Tsol45 OA4 (22.5KDa) Ts23 OA2 (32.5KDa) Ts32

13. 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.

14. Tsol18: Aminoacid sequence: MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFAL SATEIGVFWDAGEMVGHGVEEIKVKVEKAIHPYK IWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIM VFGSQRFATTLPKKQIKHKKVRRS Glycosilation and Phosphorilation site: Glycosilation: pos. 57-60 Asn glycosilation Phosphorilation: pos. 94-96 Protein kinase C Pos. 5-8 and 21-24 Casein kinase II phosphorilation site

15. 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)

16. Secondary structure prediction: Predicted by GOR 4. (Tsol18) MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFALSATEIGVFWDAGEMVGHGVEEIKVKVE KAIHPYK cccceeeehhhhhhhcccccccccceecceeeccccccceeeeeeccccceccchhhhhhhhh hhccccc IWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIMVFGSQRFATTLPKKQIKHKKVRRS eeeeeeccccchhhhhhhcccceeeeeccccccceeeecccccccccchhhhccceeeec 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%

17. MHC class I epitopes predicted: Predicted by ProPred I (Tsol18)

18. 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

19. Tsol18 modeled 3D structure

20. Tsol45 : Aminoacid sequence: MASQFHLILLLTSILAGNHKATSREVGREQPLHSLFLWGPPFSTKIGLSWRGAF SEDGDKVLTLKAALTSDPNNTKTTYQILGYGRATLKGLTPNTSYIVTATANLSG NTILVLRKHIHTPLDDTNPMENYFHWGPVTNQSIQVSWDQLDPEDARSMIVTLT AEMASNPSVERSESAIPSVGRITVDGLMPDTLYIATLTVLENGRQFLTSTRDIRT LKTGHGGVTVVTTSGSGIASAILGLLFTCTVLVLA Glycosilation sites:

21. Phosphorilation tsol45

22. Tsol45 Hidrophobicity: Predicted by SOSUI (ref 6) Average of hydrophobicity : 0.006324 Conclusion: 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)

23. MHC class II epitopes predicted (Tsol45):

24. MHC class I epitopes predicted (Tsol45):

25. Secondary structure prediction: Predicted by GOR 4. (Tsol45) MVCRFALIFLVAVVLASGDRTFGDDIFVPYLRCFALSATEIGVFWDAGEMVGHGVEEIKVKVEKAIHPYK cccceeeehhhhhhhcccccccccceecceeeccccccceeeeeeccccceccchhhhhhhhhhhccccc IWNATVSANNGKVIIRDLKAKTIYRVDVDGYRNEIMVFGSQRFATTLPKKQIKHKKVRRS eeeeeeccccchhhhhhhcccceeeeeccccccceeeecccccccccchhhhccceeeec 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%

26. 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

27. Consensus 3D-Modeling by threading, Rosetta and Molecular Dynamics refinement: (Tsol45). MHC I,II consensus best epitopes.

28. Fusion Epitope

29. Tsol18 selected fusion epitope

30. Tsol45 selected fusion epitope

31. OA4 (22.5KDa) Ts23 modeled 3D structure

32. Glycosilation sites on 23KDa

33. Phosphorilation sites on 23 KDa

34. 23KDa MHC II profile

35. 23KDa Phosphorilation sites

36. 23KDa best inmunogenic epitopes

37. 23KDa electronic density: inmunogenic epitopes

38. Stability of the epitope with the higest Peak in Epitope prediction (red Epitope) 23KDa (100ps)

39. Potential Energy change in MD stability

40. Red epitope (23KDa) (after 1500ps MD)

41. Accesibilidad 23KDa

43. HLA 23KDa

44. Propred 23KDa

45. Propred I 23KDa

46. 23KDa consenso

48. Epitopes inmunogenicos 23KDa (ninguno es expuesto a superficie)

49. OA2 (32.5KDa) Ts32 modeled 3D structure

50. Glycosilation sites for 32 KDa

52. Phosphorilation sites for 32 KDa

53. 32KDa MHC II profile

54. MHC I and MHC II 32KDa MCH II MHC I

55. Signal sequence peptide: MSFQLYLILLVTSVLA Phosphorilation sites

56. N-Glycosilation has no effect on immunogenicity

57. O-glycosilation ???

58. Conseso 32KDa (SVM HLA Propred Propred I)

59. 32KD modeled 3D structure: Green(threading method) Red(refinement with 113ps MD in vacum)

61. Best immunogenic epitopes for 32KDa

62. Epitope 1

63. Epitope 2

64. Epitope 3

65. Electronic density of 32KDa: Best immunogenic epitopes

68. Accesibility of aminoacid residues in 32KDa protein

70. Western blot (32KDa vs. Tsol45)

71. Comparison of 32KDa and Tsol45 32KDa(green), Tsol45(white)

72. Alignment of 32KDa and Tsol45 Alignment of sequences aminoacids

73. Alignment of 32KDa and Tsol45 Alignment of sequences aminoacids

74. Alignment of 32KDa and Tsol45 Alignment of sequences aminoacids

75. 32KDa and Tsol45 The highest similar Block Pattern is not an Epitope in both proteins

78. Alignment of the best epitopes of Tsol45 and 32KDa Alignment

79. Western blot (23KDa – Tsol45)

80. Local alignment Tsol45 vs. 23KDa

81. Local alignment 23KDa vs. Tsol45

82. MHC II MHC I 23KDa / Tsol45

83. Multiepitopic fusion protein cysticercosis vaccine Tsol18 Tsol45 23KDa OA2 32KDa OA4

84. Multiepitopic fusion protein cysticercosis vaccine

87. Epitope 1 in 32KDa

88. Epitope 1 in 32KDa and modified transferrin

89. Epitope 1 in modified Transferrin

90. Epitope 2 in 32KDa

91. Epitope 2 in 32KDa and modified transferrin

92. Epitope 2 in modified Transferrin

93. Epitope 3 in 32KDa

94. Epitope 3 in 32KDa and modified transferrin

95. Epitope 3 in modified Transferrin

96. Epitope 1 in Tsol18

97. Epitope 1 in Tsol18 and modified transferrin

98. Epitope 1 in modified transferrin

99. Epitope 2 in Tsol18

100. Epitope 2 in Tsol18 and modified transferrin

101. Epitope 2 in modified transferrin

102. Epitope 1 in Tsol45

103. Epitope 1 in Tsol45 and modified transferrin

104. Epitope 1 in modified transferrin

105. Epitope 2 in Tsol45

106. Epitope 2 in Tsol45 and modified transferrin

107. Epitope 2 in modified transferrin

108. Diagnostic Epitopes GP50 Ts14 Ts18 TsRS1

109. GP50 protein Gp50 is an important protein candidate to differentiate the two stages in human T. solium cysticercosis: Active disease Calcified cyst stage

110. GP50 MHC II profile

111. GP50 epitopes detected by several webservers

112. GP50 modeled 3D structure

113. Epítopes GP50

115. TS14 Highest signal: somewhere nearGKIRTSLVEHCKGPKKK Second highest signal: somewhere near VANSTKKGIEYVHE Third highest signal: somewhere within EDPIGKQIAQLAKEWKEAM

116. Ts14 secondary structure prediction

118. Ts14 MHC I and MHC II consensus profile

119. Ts14 (epitope maping) 13-mers window skipping 3 aminoacids

120. Ts18 MHC II epitope profiles for different alleles

121. Ts18 MHC I and MHC II consensus profile

122. Ts18 epitope mapping 13-mers window skipping 3 aminoacids

123. Ts18 modeled 3D structure

124. Ts18 var1 3D structure

125. Ts18 Ramachandran Plot for the 3D modeled structure

126. TsRS1 MHC I and MHC II consensus profile

127. TsRS1 3D modeled structure

128. TsRS1 Ramachandran plot

129. TsRS1 epitope mapping 13-mers window skipping 3 aminoacids