This article was downloaded by: [Northern Alberta Inst of Technology] On: 02 January 2015, At: 13:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioengineered Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbie20
The hows and whys of constructing a native recombinant cholera vaccine a
Mina Boustanshenas & Bita Bakhshi
a
a
Department of Bacteriology; Faculty of Medical Sciences; Tarbiat Modares University; Tehran, Iran Published online: 16 Sep 2013.
Click for updates To cite this article: Mina Boustanshenas & Bita Bakhshi (2014) The hows and whys of constructing a native recombinant cholera vaccine, Bioengineered, 5:1, 53-55, DOI: 10.4161/bioe.26420 To link to this article: http://dx.doi.org/10.4161/bioe.26420
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Article Addendum
Article Addendum
Bioengineered 5:1, 53–55; January/February 2014; © 2014 Landes Bioscience
Mina Boustanshenas and Bita Bakhshi*
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
Department of Bacteriology; Faculty of Medical Sciences; Tarbiat Modares University; Tehran, Iran
E
mergence of different ctxB genotypes within virulent Vibrio cholerae populations accentuates the need to develop a vaccine that has the potential to protect against all cholera toxin genotypes. Oral administration of rCTB—alone and in combination with 2 dominant domestic killed whole cells of V. cholerae (O1 Ogawa El Tor and O1 Inaba El Tor) plus one standard V. cholerae (O1 Ogawa classic ATCC 14035)—has shown satisfactory protection as a potent vaccine candidate against toxigenic V. cholerae.
Keywords: recombinant CTB, cholera toxin, native vaccine, rabbit, Vibrio cholerae *Correspondence to: Bita Bakhshi; Email: [email protected] Submitted: 06/29/2013 Revised: 09/04/2013 Accepted: 09/09/2013 Published Online: 09/16/2013 http://dx.doi.org/10.4161/bioe.26420 Addendum to: Boustanshenas M, Bakhshi B, Ghorbani M. Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model. J Appl Microbiol 2013; 114:509-15; PMID:23082752; http://dx.doi.org/10.1111/ jam.12043
At the beginning of the 21st century, it is still important to treat cholera in epidemic or endemic areas of developing countries.1,2 Production of cholera toxin (CT) and its ability to adhere to the epithelial cells and colonize in the small intestine of the host are the most important virulence factors of epidemic Vibrio cholerae strains.3,4 The CT is composed of two subunits: CTA is an enzymatic subunit that can activate adenylate cycles and increase the secretion of chloride and bicarbonate into intestinal lumen, and CTB is a pentameric nontoxic antigen with the ability to bind to GM1 ganglioside receptors on host epithelial cells, resulting in the entrance of CTA Vaccination is a feasible and powerful strategy for prevention and control of cholera outbreaks. Different types of cholera vaccines have been developed, including killed whole cell V. cholerae with or without adjuvants. So far, the developed cholera vaccines have had poor immunogenicity due to the mucosal nature of infection caused by V. cholerae.
A potent immunogenic cholera vaccine still remains a target.5,6 Two major strategies have been used to develop a cholera vaccine with long-term immunity: live-attenuated V. cholerae strains lacking the ability to produce CT5,7,8 and killed whole cell toxogenic V. cholerae strains in combination with CTB.7-9 Several live-attenuated vaccine candidates have been developed including CVD103-HgR, CVD111, CVD101, CVD103, Peru-14, and Peru-15, which have been used in the clinical trials.5,6,8,10,11 These are single dose vaccines, which can elicit a high titer of serum vibriocidal antibodies because they can actively colonize the host intestinal lumen.6,1214 The major defect of the engineered live vaccine strains is the probability of acquiring the enterotoxin gene through horizontal gene transfer. This, in turn, can transform the strains carrying toxins into virulent strains which could be troublesome, especially in epidemic areas.15,16 Vaccinations with oral killed whole cell plus CTB require multiple doses for a long-term immunity outcome. It can also be reasonably safe and has many advantages, including (1) prevention of bacterial colonization in intestinal lumen because of oral administration of vaccine, (2) improvement of adverse effects, and (3) low cost and ease of use.6,9,17 There have been many efforts to produce rCTB in prokaryotic and eukaryotic systems with no satisfactory results. Recently, we introduced a potent expression system consisting of BL21 (DE3) plus pAE_ctxB construct as an expression vector and have compared
www.landesbioscience.com Bioengineered 53
©2013 Landes Bioscience. Do not distribute
The hows and whys of constructing a native recombinant cholera vaccine
References 1.
2.
3.
4.
5.
6.
7.
Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. Lancet 2004; 363:223-33; PMID:14738797; http:// dx.doi.org/10.1016/S0140-6736(03)15328-7 Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981; 292:413-7; PMID:7019725; http://dx.doi. org/10.1038/292413a0 Parsot C, Mekalanos JJ. Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. Proc Natl Acad Sci U S A 1990; 87:9898902; PMID:2124707; http://dx.doi.org/10.1073/ pnas.87.24.9898 Gennaro ML, Greenaway PJ, Broadbent DA. The expression of biologically active cholera toxin in Escherichia coli. Nucleic Acids Res 1982; 10:488390; PMID:6290992; http://dx.doi.org/10.1093/ nar/10.16.4883 Kenner JR, Coster TS, Taylor DN, Trofa AF, BarreraOro M, Hyman T, Adams JM, Beattie DT, Killeen KP, Spriggs DR, et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126-9; PMID:7561195; http://dx.doi.org/10.1093/infdis/172.4.1126 Taylor DN, Killeen KP, Hack DC, Kenner JR, Coster TS, Beattie DT, Ezzell J, Hyman T, Trofa A, Sjogren MH, et al. Development of a live, oral, attenuated vaccine against El Tor cholera. J Infect Dis 1994; 170:1518-23; PMID:7995992; http://dx.doi. org/10.1093/infdis/170.6.1518 Fontana MR, Monaci E, Yanqing L, Guoming Q, Duan G, Rappuoli R, Pizza M. IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin. Vaccine 2000; 19:75-85; PMID:10924789; http:// dx.doi.org/10.1016/S0264-410X(00)00137-7
however, have not been satisfactory, and the expressed rCTB reported were too low. CTB has been described as a potent mucosal adjuvant for an effective oral immunization antigen mixed with killed whole-cell Vibrio cholerae.23,24 We have evaluated the biological and stability of our rCTB using GM1-ELISA assay. The result has shown that rCTB has the ability to bind to its receptor. In addition, rCTB was shown to stimulate the immune response using a rabbit model.14 Our previous results showed that two different ctxB genotypes were present among V. cholerae isolated from recent outbreaks in Iran.25 The circulation of inhabitant strains in a region can underline the significance of developing a potent local vaccine composed of our domestic strains with their own cholera toxin antigenic specifications for the control and prevention of cholera outbreaks in this region. Our investigation of the level of anti-CTB IgG antibodies in a rabbit immunized with rCTB in combination
with 2 different killed domestic toxigenic V. cholerae strains (O1 Ogawa El Tor and O1 Inaba El Tor) plus one standard V. cholerae strain (O1 Ogawa classic ATCC 14035) showed significant production of vibriocidal antibodies. Furthermore, the immunized rabbits were challenged with the live toxigenic V. cholerae O1 serotype Ogawa biotype classic ATCC 14035 strains, and the fluid accumulation index was calculated by ileal loop assay. The results showed that rCTB alone and in combination with killed whole cell strains can produce protective immunity against V. cholerae and can be used as a potent vaccine candidate.26 We will attempt to assess the immunological responses against a native vaccine composed of oral killed whole cell plus CTB in combination with an additional mucosal adjuvant to improve its potential for stimulating mucosal immunity.
8.
15. Kimsey HH, Waldor MK. CTXphi immunity: application in the development of cholera vaccines. Proc Natl Acad Sci U S A 1998; 95:7035-9; PMID:9618534; http://dx.doi.org/10.1073/ pnas.95.12.7035 16. Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996; 272:1910-4; PMID:8658163; http://dx.doi. org/10.1126/science.272.5270.1910 17. Simerska P, Moyle PM, Olive C, Toth I. Oral vaccine delivery--new strategies and technologies. Curr Drug Deliv 2009; 6:347-58; PMID:19534712; http:// dx.doi.org/10.2174/156720109789000537 18. Boustanshenas M, Bakhshi B, Ghorbani M, Norouzian D. Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae. Indian J Med Microbiol 2013; 31:10-4; PMID:23508422; http://dx.doi. org/10.4103/0255-0857.108705 19. Arêas APM, Oliveira MLS, Ramos CRR, SbrogioAlmeida ME, Raw I, Ho PL. Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli. Protein Expr Purif 2002; 25:481-7; PMID:12182829; http://dx.doi.org/10.1016/S1046-5928(02)00026-8 20. L’hoir C, Renard A, Martial JA. Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit. Gene 1990; 89:47-52; PMID:2197181; http://dx.doi. org/10.1016/0378-1119(90)90204-5 21. Slos P, Dutot P, Reymund J, Kleinpeter P, Prozzi D, Kieny MP, Delcour J, Mercenier A, Hols P. Production of cholera toxin B subunit in Lactobacillus. FEMS Microbiol Lett 1998; 169:29-36; PMID:9851032; ht t p : //d x .doi.or g /10.1111/j.1574 - 69 6 8 .19 98 . tb13295.x
9.
10.
11.
12.
13.
14.
Rijpkema SGT, Bik EM, Jansen WH, Gielen H, Versluis LF, Stouthamer AH, Guinée PA, Mooi FR. Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model. Infect Immun 1992; 60:2188-93; PMID:1587587 Ryan ET, Calderwood SB. Cholera vaccines. Clin Infect Dis 2000; 31:561-5; PMID:10987721; http:// dx.doi.org/10.1086/313951 Gotuzzo E, Butron B, Seas C, Penny M, Ruiz R, Losonsky G, Lanata CF, Wasserman SS, Salazar E, Kaper JB, et al. Safety, immunogenicity, and excretion pattern of single-dose live oral cholera vaccine CVD 103-HgR in Peruvian adults of high and low socioeconomic levels. Infect Immun 1993; 61:3994-7; PMID:8359923 Kenner JR, Coster TS, Taylor DN, Trofa AF, BarreraOro M, Hyman T, Adams JM, Beattie DT, Killeen KP, Spriggs DR, et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126-9; PMID:7561195; http://dx.doi.org/10.1093/infdis/172.4.1126 Butterton JR, Boyko SA, Calderwood SB. Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains. Vaccine 1993; 11:1327-35; PMID:8296486; http://dx.doi.org/10.1016/0264-410X(93)90103-5 Svennerholm AM, Sack DA, Holmgren J, Bardhan PK. Intestinal antibody responses after immunisation with cholera B subunit. Lancet 1982; 1:3058; PMID:6120313; http://dx.doi.org/10.1016/ S0140-6736(82)91568-9 Boustanshenas M, Bakhshi B, Ghorbani M. Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model. J Appl Microbiol 2013; 114:509-15; PMID:23082752; http://dx.doi. org/10.1111/jam.12043
54 Bioengineered
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Volume 5 Issue 1
©2013 Landes Bioscience. Do not distribute
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
it with other expression systems.14,18 Our previous study demonstrated that expression of rCTB in pAE_ctxB construct is more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct.18 The yield of recombinant CTB produced within this construct in E.coli BL21 (DE3) was about 700 mg l-1. The ctxB sequence in pAE_ ctxB construct was designed according to codon usage of E. coli for high expression yield.19 Replacement of certain codons (avoid hairpin structures), deletion of signal peptide from ctxB sequence (avoid the secretion of recombinant CTB in culture medium) and addition of 6His-tag sequence at the N terminus of recombinant CTB protein (facilitate the purification of recombinant protein through Ni2+ charged column chromatography)19 were all performed on the ctxB gene. Other investigators have used E. coli,20 Lactobacillus, Bacillus brevis,21,22 and V. cholerae strains (lacking the CTA gene) for the production of rCTB. The results,
24. Clemens JD, Sack DA, Harris JR, Chakraborty J, Neogy PK, Stanton B, Huda N, Khan MU, Kay BA, Khan MR, et al. Cross-protection by B subunitwhole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis 1988; 158:372-7; PMID:3042876; http:// dx.doi.org/10.1093/infdis/158.2.372 25. Aliabad NH, Bakhshi B, Pourshafie MR, Sharifnia A, Ghorbani M. Molecular diversity of CTX prophage in Vibrio cholerae. Lett Appl Microbiol 2012; 55:27-32; PMID:22502605; http://dx.doi. org/10.1111/j.1472-765X.2012.03253.x
26. Dashtbani-Roozbehani A, Bakhshi B, Katouli M, Pourshafie MR. Comparative sequence analysis of recA gene among Vibrio cholerae isolates from Iran with globally reported sequences. Lett Appl Microbiol 2011; 53:313-23; PMID:21707677; http://dx.doi. org/10.1111/j.1472-765X.2011.03108.x
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
©2013 Landes Bioscience. Do not distribute
22. Goto N, Maeyama J, Yasuda Y, Isaka M, Matano K, Kozuka S, Taniguchi T, Miura Y, Ohkuma K, Tochikubo K. Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant. Vaccine 2000; 18:216471; PMID:10715532; http://dx.doi.org/10.1016/ S0264-410X(99)00337-0 23. Clemens JD, Sack DA, Harris JR, Chakraborty J, Khan MR, Stanton BF, Kay BA, Khan MU, Yunus M, Atkinson W, et al. Field trial of oral cholera vaccines in Bangladesh. Lancet 1986; 2:124-7; PMID:2873397; http://dx.doi.org/10.1016/S0140-6736(86)91944-6
www.landesbioscience.com Bioengineered 55
Bioengineered Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbie20
The hows and whys of constructing a native recombinant cholera vaccine a
Mina Boustanshenas & Bita Bakhshi
a
a
Department of Bacteriology; Faculty of Medical Sciences; Tarbiat Modares University; Tehran, Iran Published online: 16 Sep 2013.
Click for updates To cite this article: Mina Boustanshenas & Bita Bakhshi (2014) The hows and whys of constructing a native recombinant cholera vaccine, Bioengineered, 5:1, 53-55, DOI: 10.4161/bioe.26420 To link to this article: http://dx.doi.org/10.4161/bioe.26420
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Article Addendum
Article Addendum
Bioengineered 5:1, 53–55; January/February 2014; © 2014 Landes Bioscience
Mina Boustanshenas and Bita Bakhshi*
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
Department of Bacteriology; Faculty of Medical Sciences; Tarbiat Modares University; Tehran, Iran
E
mergence of different ctxB genotypes within virulent Vibrio cholerae populations accentuates the need to develop a vaccine that has the potential to protect against all cholera toxin genotypes. Oral administration of rCTB—alone and in combination with 2 dominant domestic killed whole cells of V. cholerae (O1 Ogawa El Tor and O1 Inaba El Tor) plus one standard V. cholerae (O1 Ogawa classic ATCC 14035)—has shown satisfactory protection as a potent vaccine candidate against toxigenic V. cholerae.
Keywords: recombinant CTB, cholera toxin, native vaccine, rabbit, Vibrio cholerae *Correspondence to: Bita Bakhshi; Email: [email protected] Submitted: 06/29/2013 Revised: 09/04/2013 Accepted: 09/09/2013 Published Online: 09/16/2013 http://dx.doi.org/10.4161/bioe.26420 Addendum to: Boustanshenas M, Bakhshi B, Ghorbani M. Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model. J Appl Microbiol 2013; 114:509-15; PMID:23082752; http://dx.doi.org/10.1111/ jam.12043
At the beginning of the 21st century, it is still important to treat cholera in epidemic or endemic areas of developing countries.1,2 Production of cholera toxin (CT) and its ability to adhere to the epithelial cells and colonize in the small intestine of the host are the most important virulence factors of epidemic Vibrio cholerae strains.3,4 The CT is composed of two subunits: CTA is an enzymatic subunit that can activate adenylate cycles and increase the secretion of chloride and bicarbonate into intestinal lumen, and CTB is a pentameric nontoxic antigen with the ability to bind to GM1 ganglioside receptors on host epithelial cells, resulting in the entrance of CTA Vaccination is a feasible and powerful strategy for prevention and control of cholera outbreaks. Different types of cholera vaccines have been developed, including killed whole cell V. cholerae with or without adjuvants. So far, the developed cholera vaccines have had poor immunogenicity due to the mucosal nature of infection caused by V. cholerae.
A potent immunogenic cholera vaccine still remains a target.5,6 Two major strategies have been used to develop a cholera vaccine with long-term immunity: live-attenuated V. cholerae strains lacking the ability to produce CT5,7,8 and killed whole cell toxogenic V. cholerae strains in combination with CTB.7-9 Several live-attenuated vaccine candidates have been developed including CVD103-HgR, CVD111, CVD101, CVD103, Peru-14, and Peru-15, which have been used in the clinical trials.5,6,8,10,11 These are single dose vaccines, which can elicit a high titer of serum vibriocidal antibodies because they can actively colonize the host intestinal lumen.6,1214 The major defect of the engineered live vaccine strains is the probability of acquiring the enterotoxin gene through horizontal gene transfer. This, in turn, can transform the strains carrying toxins into virulent strains which could be troublesome, especially in epidemic areas.15,16 Vaccinations with oral killed whole cell plus CTB require multiple doses for a long-term immunity outcome. It can also be reasonably safe and has many advantages, including (1) prevention of bacterial colonization in intestinal lumen because of oral administration of vaccine, (2) improvement of adverse effects, and (3) low cost and ease of use.6,9,17 There have been many efforts to produce rCTB in prokaryotic and eukaryotic systems with no satisfactory results. Recently, we introduced a potent expression system consisting of BL21 (DE3) plus pAE_ctxB construct as an expression vector and have compared
www.landesbioscience.com Bioengineered 53
©2013 Landes Bioscience. Do not distribute
The hows and whys of constructing a native recombinant cholera vaccine
References 1.
2.
3.
4.
5.
6.
7.
Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. Lancet 2004; 363:223-33; PMID:14738797; http:// dx.doi.org/10.1016/S0140-6736(03)15328-7 Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981; 292:413-7; PMID:7019725; http://dx.doi. org/10.1038/292413a0 Parsot C, Mekalanos JJ. Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. Proc Natl Acad Sci U S A 1990; 87:9898902; PMID:2124707; http://dx.doi.org/10.1073/ pnas.87.24.9898 Gennaro ML, Greenaway PJ, Broadbent DA. The expression of biologically active cholera toxin in Escherichia coli. Nucleic Acids Res 1982; 10:488390; PMID:6290992; http://dx.doi.org/10.1093/ nar/10.16.4883 Kenner JR, Coster TS, Taylor DN, Trofa AF, BarreraOro M, Hyman T, Adams JM, Beattie DT, Killeen KP, Spriggs DR, et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126-9; PMID:7561195; http://dx.doi.org/10.1093/infdis/172.4.1126 Taylor DN, Killeen KP, Hack DC, Kenner JR, Coster TS, Beattie DT, Ezzell J, Hyman T, Trofa A, Sjogren MH, et al. Development of a live, oral, attenuated vaccine against El Tor cholera. J Infect Dis 1994; 170:1518-23; PMID:7995992; http://dx.doi. org/10.1093/infdis/170.6.1518 Fontana MR, Monaci E, Yanqing L, Guoming Q, Duan G, Rappuoli R, Pizza M. IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin. Vaccine 2000; 19:75-85; PMID:10924789; http:// dx.doi.org/10.1016/S0264-410X(00)00137-7
however, have not been satisfactory, and the expressed rCTB reported were too low. CTB has been described as a potent mucosal adjuvant for an effective oral immunization antigen mixed with killed whole-cell Vibrio cholerae.23,24 We have evaluated the biological and stability of our rCTB using GM1-ELISA assay. The result has shown that rCTB has the ability to bind to its receptor. In addition, rCTB was shown to stimulate the immune response using a rabbit model.14 Our previous results showed that two different ctxB genotypes were present among V. cholerae isolated from recent outbreaks in Iran.25 The circulation of inhabitant strains in a region can underline the significance of developing a potent local vaccine composed of our domestic strains with their own cholera toxin antigenic specifications for the control and prevention of cholera outbreaks in this region. Our investigation of the level of anti-CTB IgG antibodies in a rabbit immunized with rCTB in combination
with 2 different killed domestic toxigenic V. cholerae strains (O1 Ogawa El Tor and O1 Inaba El Tor) plus one standard V. cholerae strain (O1 Ogawa classic ATCC 14035) showed significant production of vibriocidal antibodies. Furthermore, the immunized rabbits were challenged with the live toxigenic V. cholerae O1 serotype Ogawa biotype classic ATCC 14035 strains, and the fluid accumulation index was calculated by ileal loop assay. The results showed that rCTB alone and in combination with killed whole cell strains can produce protective immunity against V. cholerae and can be used as a potent vaccine candidate.26 We will attempt to assess the immunological responses against a native vaccine composed of oral killed whole cell plus CTB in combination with an additional mucosal adjuvant to improve its potential for stimulating mucosal immunity.
8.
15. Kimsey HH, Waldor MK. CTXphi immunity: application in the development of cholera vaccines. Proc Natl Acad Sci U S A 1998; 95:7035-9; PMID:9618534; http://dx.doi.org/10.1073/ pnas.95.12.7035 16. Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996; 272:1910-4; PMID:8658163; http://dx.doi. org/10.1126/science.272.5270.1910 17. Simerska P, Moyle PM, Olive C, Toth I. Oral vaccine delivery--new strategies and technologies. Curr Drug Deliv 2009; 6:347-58; PMID:19534712; http:// dx.doi.org/10.2174/156720109789000537 18. Boustanshenas M, Bakhshi B, Ghorbani M, Norouzian D. Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae. Indian J Med Microbiol 2013; 31:10-4; PMID:23508422; http://dx.doi. org/10.4103/0255-0857.108705 19. Arêas APM, Oliveira MLS, Ramos CRR, SbrogioAlmeida ME, Raw I, Ho PL. Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli. Protein Expr Purif 2002; 25:481-7; PMID:12182829; http://dx.doi.org/10.1016/S1046-5928(02)00026-8 20. L’hoir C, Renard A, Martial JA. Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit. Gene 1990; 89:47-52; PMID:2197181; http://dx.doi. org/10.1016/0378-1119(90)90204-5 21. Slos P, Dutot P, Reymund J, Kleinpeter P, Prozzi D, Kieny MP, Delcour J, Mercenier A, Hols P. Production of cholera toxin B subunit in Lactobacillus. FEMS Microbiol Lett 1998; 169:29-36; PMID:9851032; ht t p : //d x .doi.or g /10.1111/j.1574 - 69 6 8 .19 98 . tb13295.x
9.
10.
11.
12.
13.
14.
Rijpkema SGT, Bik EM, Jansen WH, Gielen H, Versluis LF, Stouthamer AH, Guinée PA, Mooi FR. Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model. Infect Immun 1992; 60:2188-93; PMID:1587587 Ryan ET, Calderwood SB. Cholera vaccines. Clin Infect Dis 2000; 31:561-5; PMID:10987721; http:// dx.doi.org/10.1086/313951 Gotuzzo E, Butron B, Seas C, Penny M, Ruiz R, Losonsky G, Lanata CF, Wasserman SS, Salazar E, Kaper JB, et al. Safety, immunogenicity, and excretion pattern of single-dose live oral cholera vaccine CVD 103-HgR in Peruvian adults of high and low socioeconomic levels. Infect Immun 1993; 61:3994-7; PMID:8359923 Kenner JR, Coster TS, Taylor DN, Trofa AF, BarreraOro M, Hyman T, Adams JM, Beattie DT, Killeen KP, Spriggs DR, et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126-9; PMID:7561195; http://dx.doi.org/10.1093/infdis/172.4.1126 Butterton JR, Boyko SA, Calderwood SB. Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains. Vaccine 1993; 11:1327-35; PMID:8296486; http://dx.doi.org/10.1016/0264-410X(93)90103-5 Svennerholm AM, Sack DA, Holmgren J, Bardhan PK. Intestinal antibody responses after immunisation with cholera B subunit. Lancet 1982; 1:3058; PMID:6120313; http://dx.doi.org/10.1016/ S0140-6736(82)91568-9 Boustanshenas M, Bakhshi B, Ghorbani M. Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model. J Appl Microbiol 2013; 114:509-15; PMID:23082752; http://dx.doi. org/10.1111/jam.12043
54 Bioengineered
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Volume 5 Issue 1
©2013 Landes Bioscience. Do not distribute
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
it with other expression systems.14,18 Our previous study demonstrated that expression of rCTB in pAE_ctxB construct is more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct.18 The yield of recombinant CTB produced within this construct in E.coli BL21 (DE3) was about 700 mg l-1. The ctxB sequence in pAE_ ctxB construct was designed according to codon usage of E. coli for high expression yield.19 Replacement of certain codons (avoid hairpin structures), deletion of signal peptide from ctxB sequence (avoid the secretion of recombinant CTB in culture medium) and addition of 6His-tag sequence at the N terminus of recombinant CTB protein (facilitate the purification of recombinant protein through Ni2+ charged column chromatography)19 were all performed on the ctxB gene. Other investigators have used E. coli,20 Lactobacillus, Bacillus brevis,21,22 and V. cholerae strains (lacking the CTA gene) for the production of rCTB. The results,
24. Clemens JD, Sack DA, Harris JR, Chakraborty J, Neogy PK, Stanton B, Huda N, Khan MU, Kay BA, Khan MR, et al. Cross-protection by B subunitwhole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis 1988; 158:372-7; PMID:3042876; http:// dx.doi.org/10.1093/infdis/158.2.372 25. Aliabad NH, Bakhshi B, Pourshafie MR, Sharifnia A, Ghorbani M. Molecular diversity of CTX prophage in Vibrio cholerae. Lett Appl Microbiol 2012; 55:27-32; PMID:22502605; http://dx.doi. org/10.1111/j.1472-765X.2012.03253.x
26. Dashtbani-Roozbehani A, Bakhshi B, Katouli M, Pourshafie MR. Comparative sequence analysis of recA gene among Vibrio cholerae isolates from Iran with globally reported sequences. Lett Appl Microbiol 2011; 53:313-23; PMID:21707677; http://dx.doi. org/10.1111/j.1472-765X.2011.03108.x
Downloaded by [Northern Alberta Inst of Technology] at 13:17 02 January 2015
©2013 Landes Bioscience. Do not distribute
22. Goto N, Maeyama J, Yasuda Y, Isaka M, Matano K, Kozuka S, Taniguchi T, Miura Y, Ohkuma K, Tochikubo K. Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant. Vaccine 2000; 18:216471; PMID:10715532; http://dx.doi.org/10.1016/ S0264-410X(99)00337-0 23. Clemens JD, Sack DA, Harris JR, Chakraborty J, Khan MR, Stanton BF, Kay BA, Khan MU, Yunus M, Atkinson W, et al. Field trial of oral cholera vaccines in Bangladesh. Lancet 1986; 2:124-7; PMID:2873397; http://dx.doi.org/10.1016/S0140-6736(86)91944-6
www.landesbioscience.com Bioengineered 55
