Limits...
Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus

View Article: PubMed Central - PubMed

ABSTRACT

The use of whole cell killed (WCK) oral cholera vaccines is an important strategy for cholera prevention in endemic areas. To overcome current vaccine limitations, we engineered strains of V. cholerae to be non-toxigenic and to express the protective protein colonization factor, toxin-coregulated pilus (TCP), under scale-up conditions potentially amenable to vaccine production. Two V. cholerae clinical strains were selected and their cholera toxin genes deleted. The tcp operon was placed under control of a rhamnose-inducible promoter. Production and stability of TCP were assessed under various conditions. The strains lack detectable cholera toxin production. The addition of 0.1% rhamnose to the growth medium induced robust production of TCP and TcpA antigen. The strains produced intact TCP in larger growth volumes (1 L), and pili appeared stable during heat-killing or acid treatment of the bacterial cultures. To date, no WCK cholera vaccines have included TCP. We have constructed putative strains of V. cholerae for use in a vaccine that produce high levels of stable TCP antigen, which has not previously been achieved.

No MeSH data available.


Related in: MedlinePlus

Vaccine strains do not produce cholera toxin.A, Cholera toxin production in the rhamnose-inducible tcp strains, and ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, wild-type C6706, RM3, which is the same ctx region deletion in the C6706 background, Bgd1 and Bgd5 strains. ngCTml-1OD600-1 ELISA measurements for three independent experiments presented as means with standard errors. A two-tailed standard t test yielded P values of <0.05 when CTX production of Bgd1 (*) and Bgd5 (*) were compared to all other strains. Bgd1 and Bgd5 were not significantly different from each other. B, Western immunoblot of cholera toxin B subunit (CTXB) in the listed strains, thereby confirming deletion of the genes encoding cholera toxin.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383245&req=5

pone.0175170.g002: Vaccine strains do not produce cholera toxin.A, Cholera toxin production in the rhamnose-inducible tcp strains, and ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, wild-type C6706, RM3, which is the same ctx region deletion in the C6706 background, Bgd1 and Bgd5 strains. ngCTml-1OD600-1 ELISA measurements for three independent experiments presented as means with standard errors. A two-tailed standard t test yielded P values of <0.05 when CTX production of Bgd1 (*) and Bgd5 (*) were compared to all other strains. Bgd1 and Bgd5 were not significantly different from each other. B, Western immunoblot of cholera toxin B subunit (CTXB) in the listed strains, thereby confirming deletion of the genes encoding cholera toxin.

Mentions: Two Vibrio cholerae O1 El Tor variant biotype clinical strains were selected as candidate strains to be included in an enhanced WCK cholera vaccine. While El Tor is the dominant biotype in current cholera incidences, classical biotype features (typically genetically classical cholera toxin genes) have emerged in the form of hybrid El Tor variant strains [18, 33–35]. These new pathogenic, clinically isolated variants have spread throughout Asian and African countries, appear to cause more severe disease and higher cases of fatalities [5], and are important to consider in future cholera vaccine development [36]. The selected strains were isolated from patients at Matlab Hospital in Bangladesh and were originally obtained from the International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B) [18]. These strains, Bgd1 and Bgd5, caused severe dehydration and acute watery diarrhea in the infected patients and produce high levels of cholera toxin (Fig 2A and 2B). Bgd1 and Bgd5 are Ogawa and Inaba serotypes, respectively (confirmed via western protein immunoblot in S1 Fig), classified by variations in the O-antigenic component of LPS [37]. Both strains contain a single copy of the cholera toxin genes (ctxA and ctxB) on the large chromosome only [18].


Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus
Vaccine strains do not produce cholera toxin.A, Cholera toxin production in the rhamnose-inducible tcp strains, and ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, wild-type C6706, RM3, which is the same ctx region deletion in the C6706 background, Bgd1 and Bgd5 strains. ngCTml-1OD600-1 ELISA measurements for three independent experiments presented as means with standard errors. A two-tailed standard t test yielded P values of <0.05 when CTX production of Bgd1 (*) and Bgd5 (*) were compared to all other strains. Bgd1 and Bgd5 were not significantly different from each other. B, Western immunoblot of cholera toxin B subunit (CTXB) in the listed strains, thereby confirming deletion of the genes encoding cholera toxin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383245&req=5

pone.0175170.g002: Vaccine strains do not produce cholera toxin.A, Cholera toxin production in the rhamnose-inducible tcp strains, and ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, wild-type C6706, RM3, which is the same ctx region deletion in the C6706 background, Bgd1 and Bgd5 strains. ngCTml-1OD600-1 ELISA measurements for three independent experiments presented as means with standard errors. A two-tailed standard t test yielded P values of <0.05 when CTX production of Bgd1 (*) and Bgd5 (*) were compared to all other strains. Bgd1 and Bgd5 were not significantly different from each other. B, Western immunoblot of cholera toxin B subunit (CTXB) in the listed strains, thereby confirming deletion of the genes encoding cholera toxin.
Mentions: Two Vibrio cholerae O1 El Tor variant biotype clinical strains were selected as candidate strains to be included in an enhanced WCK cholera vaccine. While El Tor is the dominant biotype in current cholera incidences, classical biotype features (typically genetically classical cholera toxin genes) have emerged in the form of hybrid El Tor variant strains [18, 33–35]. These new pathogenic, clinically isolated variants have spread throughout Asian and African countries, appear to cause more severe disease and higher cases of fatalities [5], and are important to consider in future cholera vaccine development [36]. The selected strains were isolated from patients at Matlab Hospital in Bangladesh and were originally obtained from the International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B) [18]. These strains, Bgd1 and Bgd5, caused severe dehydration and acute watery diarrhea in the infected patients and produce high levels of cholera toxin (Fig 2A and 2B). Bgd1 and Bgd5 are Ogawa and Inaba serotypes, respectively (confirmed via western protein immunoblot in S1 Fig), classified by variations in the O-antigenic component of LPS [37]. Both strains contain a single copy of the cholera toxin genes (ctxA and ctxB) on the large chromosome only [18].

View Article: PubMed Central - PubMed

ABSTRACT

The use of whole cell killed (WCK) oral cholera vaccines is an important strategy for cholera prevention in endemic areas. To overcome current vaccine limitations, we engineered strains of V. cholerae to be non-toxigenic and to express the protective protein colonization factor, toxin-coregulated pilus (TCP), under scale-up conditions potentially amenable to vaccine production. Two V. cholerae clinical strains were selected and their cholera toxin genes deleted. The tcp operon was placed under control of a rhamnose-inducible promoter. Production and stability of TCP were assessed under various conditions. The strains lack detectable cholera toxin production. The addition of 0.1% rhamnose to the growth medium induced robust production of TCP and TcpA antigen. The strains produced intact TCP in larger growth volumes (1 L), and pili appeared stable during heat-killing or acid treatment of the bacterial cultures. To date, no WCK cholera vaccines have included TCP. We have constructed putative strains of V. cholerae for use in a vaccine that produce high levels of stable TCP antigen, which has not previously been achieved.

No MeSH data available.


Related in: MedlinePlus