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1 INSILICO DESIGNING AND DEVELOPMENT OF VACCINE FOR V.CHOLERAE O139 IN
2 CHOLERA DISEASE
3
5 1PES Institute of Technology, Shimoga -580031, India
6 1Research work is carried out at DNA LABS INDIA, Hyderabad, India
7
8 1Corresponding Author Mobile, +91-9538020530; Email, sriranjini6@gmail.com
9
10 ABSTRACT
11 V.cholerae was first isolated by Italian anatomist Filipo Panici. V.cholerae is the etiological agent of cholera, a
12 major health concern in most of the developing countries. V cholerae carry strains the encode the cholera toxin.
13 These cholera toxins enters the Epithial cells and after crossing host line of defense it starts colonizing itself in
14 the small intestine. Cholera is usually a non contagious disease.
15 The main aim of this project is to design and develop a vaccine against cholera. Vibrio Cholerae is a bacterium
16 with 12,865 odd proteins causing cholera. Among these 1 protein sequence was selected having least identity
17 and least E- value. It was then screened by using SDSC workbench tool. Then antigenic determinants were
18 found by using different tools. The sequence with least identity was taken into consideration and then further
19 designed and used for docking studies. From this Docking analysis the epitope molecule LEALVEDL was
20 found to be the best vaccine candidate.
21
22 1. Introduction
23 Vibrio cholera also called as kommabacillus is a gram-negative comma shaped bacterium with a polar flagellum
24 that causes cholera in humans. V.cholera follows a fecal-oral infection path. It moves from contaminated food
25 and water and colonizes in the small intestine. V.cholera produces cholera toxin, the enterotoxin which acts on
26 the mucosal epithelium resulting in diarrhea.
27 There are two major biotypes of v.cholera i.e classical and ‘EI Tor’ which can be identified by
28 hemagglutination test. They occur both in marine and fresh water surfaces. It causes severe diarrheal disease in
29 humans by food and water. It is one of the most fatal illnesses known as diarrhea.
30 1.1 Clinical Symptoms:
31 Cholera symptoms include watery faces
32 With bits of mucus and mild fishy smell
33 Vomiting
34 Abdominal cramps
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35 Dehydration
36 Fever, it is rare, usually found in children.
37 The primary symptoms of cholera are profuse painless diarrhea and clear vomiting. These symptoms start
38 within 1 to 5 days after ingestion of bacteria. An untreated person may produce 10-15 lit. of diarrhea a day with
39 fatal results. If severe diarrhea and vomiting are not treated aggressively it may result in life threatening
40 dehydration and electrolytic imbalances.
41 1.2 Mode of transmission:
42
43 Transmission occurs through ingestion of contaminated water and food. Sudden large outbreaks are usually
44 caused by a contaminated water supply. Raw or undercooked seafood may be a source of infection in areas
45 where cholera is prevalent and sanitation is poor. Transmission due to direct person to person contact is rare.
46
47 2. Materials and Methods
48 2.1 Screening of Proteins
49
50 SDSC workbench: This bioinformatic tool is very essential in screening of proteins. By this screening the
51 protein with least identity was identified and its antigenic determinant was found.
52
53 The following protein ID with least identity was found are ;
54
55 Gene ID was found to be IF2_VIBC3 and its accession number is 172047700 with least identiy 20.62.
56
57 Least Identity – 21.89 , gene ID was found to be – 189046614 and the Accession number is
58 SECA_VIBC3
59
60 2.2 IMMUNOMED GROUP
61 This is mainly used to find out the antigenic determinants of the sequence that has least ident ity. From this tool
62 the Average antigenic propensity for protein sequence was found to be 1.0097
63
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64
65 Fig 1. Average Antigenic Propensity
n | Start Position | Sequence | End Position |
1 | 4 | ITVKALS | 10 |
2 | 13 | IGTPVDRLLEQ | 23 |
3 | 42 | KQKLLAHL | 49 |
4 | 84 | KNVQVEV | 90 |
5 | 92 | KKRTYVKR | 99 |
6 | 208 | QLEKVRE | 214 |
7 | 237 | TDYHVTT | 243 |
8 | 308 | DKTAVVAKADVVVGETIVVSE | 328 |
9 | 336 | KATEVIK | 342 |
10 | 352 | TINQVID | 358 |
11 | 395 | EVSRAPVVTIMGHVDH | 410 |
12 | 420 | RRTHVAS | 426 |
13 | 432 | ITQHIGAYHV | 441 |
14 | 469 | ATDIVVLVVAA | 479 |
15 | 484 | MPQTVEAIQHAKAAGVPLIVAVN | 506 |
16 | 549 | IDGLLEAILLQAEVLELKAVKQ | 570 |
17 | 572 | MASGVVIE | 579 |
18 | 586 | RGPVATVLVQS | 596 |
19 | 601 | KGDIVLCGQEYGR | 613 |
20 | 629 | GPSIPVEILGLSGVPA | 644 |
21 | 647 | DEATVVR | 653 |
22 | 670 | REVKLAR | 676 |
23 | 692 | GDVALNIVLKADVQGSVEAIADSLTK | 717 |
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24 | 719 | STDEVKVNIVGSGV | 732 |
25 | 737 | ETDAVLAAASNAIVVGF | 753 |
26 | 771 | DLRYYSIIYQLIDE | 784 |
27 | 798 | KQEIIGLAEVRDVFKSPKLGAIAGCMVTE | 826 |
28 | 833 | APIRVLRDNVVIY | 845 |
29 | 856 | KDDVAEV | 862 |
30 | 866 | YECGIGV | 872 |
31 | 876 | NDVRVGDQIEVFET | 889 |
66
67
68
69 2.3 MAPPP
70
Table 1
71 This tool is used to find out the type of MHC molecule (MHC 1 or 2) to which the epitope molecule binds.
72
73 Mappp results were found to be
74
Epitope | Position | MHC type | n- mer | Overall score | Cleavage Probability | MHC binding score | Group | |
0..897 | 898 | MTQITVKALSEEIGTPVDRL..VRVGDQIEVFETIEIQRTID | ||||||
QRKTRSTL | 66 | HLA_B8 | 8 | 0.8500 | 1.0000 | 0.7000 | n-term. trimmed | |
QRKTRSTL | 66 | HLA_B8 | 8 | 0.8500 | 1.0000 | 0.7000 | c-term. trimmed | |
QPRSDEEKL | 168 | HLA_B_0702 | 9 | 0.8429 | 1.0000 | 0.6857 | n-term. trimmed | |
QPRSDEEKL | 168 | H2_Ld | 9 | 0.8387 | 1.0000 | 0.6774 | n-term. trimmed | |
QPRSDEEKL | 168 | HLA_B_0702 | 9 | 0.8429 | 1.0000 | 0.6857 | n-term. trimmed | |
RRKAEEESR | 197 | HLA_B_2705 | 9 | 0.8514 | 1.0000 | 0.7027 | c-term. trimmed | |
PRGGKAGRK | 285 | HLA_B_2705 | 9 | 0.8514 | 1.0000 | 0.7027 | c-term. trimmed | |
KENELEEAI | 376 | H2_Kk | 9 | 0.8497 | 0.9994 | 0.7000 | trimmed twice | |
KENELEEAI | 376 | H2_Kk | 9 | 0.8498 | 0.9995 | 0.7000 | c-term. trimmed | |
ANPDNVKTEL | 512 | H2_Db | 10 | 0.9545 | 1.0000 | 0.9091 | n-term. |
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trimmed | ||||||||
GLLEAILLQA | 550 | HLA_A_0201 | 10 | 0.8528 | 0.9997 | 0.7059 | same length | |
GLLEAILLQA | 550 | HLA_A_0201 | 10 | 0.8529 | 1.0000 | 0.7059 | c-term. trimmed | |
LLQAEVLEL | 556 | HLA_A_0201 | 9 | 0.9028 | 1.0000 | 0.8056 | n-term. trimmed | |
ILLQAEVLEL | 555 | HLA_A_0201 | 10 | 0.9559 | 1.0000 | 0.9118 | n-term. trimmed | |
EVLELKAVK | 560 | HLA_A3 | 9 | 0.8422 | 0.9868 | 0.6977 | c-term. trimmed | |
AIADSLTKL | 710 | HLA_A_0201 | 9 | 0.8916 | 0.9776 | 0.8056 | same length | |
AIADSLTKL | 710 | HLA_A_0201 | 9 | 0.9028 | 1.0000 | 0.8056 | c-term. trimmed | |
DEVKVNIV | 721 | H2_Kk | 8 | 0.8667 | 1.0000 | 0.7333 | c-term. trimmed | |
RYYSIIYQLI | 773 | H2_Kd | 10 | 0.9107 | 1.0000 | 0.8214 | c-term. trimmed | |
KRNAPIRVL | 830 | HLA_B_2705 | 9 | 0.8649 | 1.0000 | 0.7297 | n-term. trimmed |
75 Table 2
76 2.4 DISCOVERY STUDIO 2.5
77 MINIMIZATION OF MHC MOLECULES
78 The antigenic determinants LEALVEDL was designed
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79
80 Fig 2. Epitope Molecule V.CHOLERAE
81 The minimization energy for this molecule is found to be 210.39238 kcal/mol.
82
83 The antigenic determinant GPVATVLVQSG was designed
84
85
86
87 2.5 Docking
88
89 This is mainly used to fit the epitope molecule into the MHC 1 molecule.
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91 Fig 4. Docked Molecule V.CHOLERAE
92 INFERENCE : The epitope molecule was docked with MHC I molecule successfully
93 C docker Energy was found to be 74.1119
94 C docker Interaction Energy was found to be 41.0479
95
96 3. DISCUSSION
97
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Thus from the above result it is found the antigenic determinant with least identity was ‘LEALVEDL’. The c
docker energy of this molecule was found to be 74.119 and C docker interaction energy was found to be
41.0479. By taking into consideration a vaccine is designed for cholera. The vaccine that is designed can be further sent for clinical trials and if it passes the FDA approval it can be used for curing this disease in future.
Support and help from the DNA Labs India, Hyderabad and Mr. Manas Ranjan Barik, is gratefully acknowledged.
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