International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 1

ISSN 2229-5518

1 INSILICO DESIGNING AND DEVELOPMENT OF VACCINE FOR V.CHOLERAE O139 IN

2 CHOLERA DISEASE

3

4 Dr. Ramani Iyengar1, Sriranjini A.S2, Jasmine2

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

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 2

ISSN 2229-5518

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

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 3

ISSN 2229-5518

64

65 Fig 1. Average Antigenic Propensity

There are 31 antigenic determinants in the sequence:

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

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 4

ISSN 2229-5518

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.

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 5

ISSN 2229-5518

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

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 6

ISSN 2229-5518

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

Fig 3. EpitopE Molecule 2, V-CHOLARAE

89 This is mainly used to fit the epitope molecule into the MHC 1 molecule.

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 7

ISSN 2229-5518

90

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

98

99

100

101

102

103

104

105

106

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.

Acknowledgements

Support and help from the DNA Labs India, Hyderabad and Mr. Manas Ranjan Barik, is gratefully acknowledged.

IJSER © 2012 http://www.ijser.org

International Journal of Scientific & Engineering Research Volume 3, Issue 7, June-2012 8

ISSN 2229-5518

107

108

1. REFERENCES

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

1. Archivist (1997), Cholera phage discovery. Arch Dis Child 76 (3): 274. DOI:
10.1136/adc.76.3.274.Bentivoglio M, Pacini, P (1995). Filippo Pacini: A determined observer. Brain
Research Bulletin 38 (2): 161–5. DOI: 10.1016/0361-9230(95)00083-Q. PMID 7583342.
3. Bertranpetit J, Calafell F (1996). Genetic and geographical variability in cystic fibrosis: evolutionary
considerations. Ciba Found. Symposium. 197: 97–114; discussion 114–8. PMID 8827370.
4. DiRita VJ, Parsot C, Jander G, Mekalanos JJ (June 1991). Regulatory cascade controls virulence in Vibrio
cholerae. Proc. Natl. Acad. Sci. U.S.A. 88 (12): 5403–7. DOI: 10.1073/pnas.88.12.5403. PMC 51881. PMID 2052618.
5. Faruque, Shah M.; Nair, Balakrish, ed (2008). Vibrio cholerae: Genomics and Molecular Biology. Caister
Academic Press. ISBN 978-1-904455-33-2.
6. Faruque, SM; Nair, GB (2002). Molecular ecology of toxigenic Vibrio cholerae. Microbiology and immunology 46 (2): 59–66. PMID 11939579.
7. Fraser, Claire M.; Heidelberg, John F.; Eisen, Jonathan A.; Nelson, William C.; Clayton, Rebecca A.; Gwinn, Michelle L.; Dodson, Robert J.; Haft, Daniel H. et al. (2000). DNA sequence of both chromosomes
of the cholera pathogen Vibrio cholerae. Nature 406 (6795): 477–83. DOI: 10.1038/35020000. PMID 10952301.
8. Harris JB, Khan AI, LaRocque RC, et al. (November 2005). Blood group, immunity, and risk o f infection with Vibrio cholerae in an area of endemicity. Infect. Immun. 73 (11): 7422–7. DOI:
10.1128/IAI.73.11.7422-7427.2005. PMC 1273892. PMID 16239542.Hartwell LH, Hood L, Goldberg ML, Reynolds AE, Silver LM, and Veres RC (2004). Genetics: From genes to genomes. Boston: Mc-Graw Hill.
pp. 551–552, 572–574.
10. Howard-Jones, N (1984). Robert Koch and the cholera vibrio: a centenary. BMJ 288 (6414): 379–81.
DOI:10.1136/bmj.288.6414.379. PMC 1444283. PMID 6419937.
11. Karaolis, David K. R.; Somara, Sita; Maneval, David R.; Johnson, Judith A.; Kaper, James B. (1999). A
bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Nature 399 (6734): 375–9. DOI: 10.1038/20715. PMID 10360577.
12. King AA, Ionides EL, J.Luckhurst, Bouma MJ (August 2008). Inapparent infections and cholera dynamics.
Nature 454 (7206): 877–80. DOI:10.1038/nature07084. PMID 18704085.
13. Lan R, Reeves PR (Jan 2002). Pandemic Spread of Cholera: Genetic Diversity and Relationships within the Seventh Pandemic Clone of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism (Free full text). Journal of Clinical Microbiology 40 (1): 172–181. DOI: 10.1128/JCM.40.1.172-181.2002. ISSN 0095-1137. PMC 120103. PMID 11773113.O'Neal C, Jobling M, Holmes R, Hol W (2005). Structural basis for the activation of cholera toxin by human ARF6-GTP. Science 309 (5737): 1093–6. DOI:
10.1126/science.1113398. PMID 16099990.
15. Ryan KJ, Ray CG (2004). Sherris Medical Microbiology (4th ed). McGraw Hill. pp. 376–7.
ISBN 0838585299.
16. Sack DA, Sack RB, Chaignat CL (August 2006). Getting serious about cholera. N. Engl. J. Med. 355 (7):
649–51. DOI: 10.1056/NEJMp068144. PMID 16914700.

17. Waldor, M. K.; Mekalanos, J. J. (1996). Lysogenic Conversion by a Filamentous Phage Encoding Cholera

Toxin. Science 272 (5270): 1910–4. DOI: 10.1126/science.272.5270.1910. PMID 8658163.

IJSER © 2012 http://www.ijser.org