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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

TcpA and pilus production in vaccine strains.A, Western immunoblot of TcpA production in rhamnose-inducible tcp, ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, Bgd1 and Bgd5 strains under AKI-inducing conditions as compared to growth in soy LB (traditional lysogeny broth amended to replace tryptone with papain-digested soybean meal to avoid prion risk from animal material) with or without addition of 0.1% rhamnose. B, Transmission electron microscopy of phosphotungstic acid-negatively stained pili produced by CAH182 (left) and CAH184 (right) after growth in soy LB with 0.1% rhamnose. C, CTX-Kmϕ phage transduction via TCP in wild-type N16961, Bgd1 and Bgd5 in AKI growth conditions and vaccine strains CAH182 and CAH184 with (+R) and without (-R) 0.1% rhamnose grown in soy LB. Data from three independent experiments +/- standard errors. A two-tailed standard t test yielded no significant differences among strains.
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pone.0175170.g003: TcpA and pilus production in vaccine strains.A, Western immunoblot of TcpA production in rhamnose-inducible tcp, ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, Bgd1 and Bgd5 strains under AKI-inducing conditions as compared to growth in soy LB (traditional lysogeny broth amended to replace tryptone with papain-digested soybean meal to avoid prion risk from animal material) with or without addition of 0.1% rhamnose. B, Transmission electron microscopy of phosphotungstic acid-negatively stained pili produced by CAH182 (left) and CAH184 (right) after growth in soy LB with 0.1% rhamnose. C, CTX-Kmϕ phage transduction via TCP in wild-type N16961, Bgd1 and Bgd5 in AKI growth conditions and vaccine strains CAH182 and CAH184 with (+R) and without (-R) 0.1% rhamnose grown in soy LB. Data from three independent experiments +/- standard errors. A two-tailed standard t test yielded no significant differences among strains.

Mentions: To verify production of TCP that is dependent upon rhamnose addition, the final vaccine strains CAH182 and CAH184 (parental strains are Bgd1 and Bgd5, respectively) were grown in an animal-free medium (soy-based LB broth) at 37°C overnight with or without the addition of 0.1% rhamnose (vol/vol). Whole cell extracts (WCE) were assayed via western immunoblot for the presence of TcpA, the major pilin that forms TCP, using anti-TcpA anti-serum. Both CAH182 and CAH184 produced stable TcpA protein only when grown in the presence of rhamnose (Fig 3A). Parental strains Bgd1 and Bgd5, along with control V. cholerae El Tor strain N16961, only produced TcpA when grown in AKI-inducing conditions as previously described [27] and did not produce TcpA in soy LB with or without the inclusion of rhamnose (Fig 3A).


Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus
TcpA and pilus production in vaccine strains.A, Western immunoblot of TcpA production in rhamnose-inducible tcp, ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, Bgd1 and Bgd5 strains under AKI-inducing conditions as compared to growth in soy LB (traditional lysogeny broth amended to replace tryptone with papain-digested soybean meal to avoid prion risk from animal material) with or without addition of 0.1% rhamnose. B, Transmission electron microscopy of phosphotungstic acid-negatively stained pili produced by CAH182 (left) and CAH184 (right) after growth in soy LB with 0.1% rhamnose. C, CTX-Kmϕ phage transduction via TCP in wild-type N16961, Bgd1 and Bgd5 in AKI growth conditions and vaccine strains CAH182 and CAH184 with (+R) and without (-R) 0.1% rhamnose grown in soy LB. Data from three independent experiments +/- standard errors. A two-tailed standard t test yielded no significant differences among strains.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383245&req=5

pone.0175170.g003: TcpA and pilus production in vaccine strains.A, Western immunoblot of TcpA production in rhamnose-inducible tcp, ctx knockout strains CAH182 and CAH184 as compared to wild-type N16961, Bgd1 and Bgd5 strains under AKI-inducing conditions as compared to growth in soy LB (traditional lysogeny broth amended to replace tryptone with papain-digested soybean meal to avoid prion risk from animal material) with or without addition of 0.1% rhamnose. B, Transmission electron microscopy of phosphotungstic acid-negatively stained pili produced by CAH182 (left) and CAH184 (right) after growth in soy LB with 0.1% rhamnose. C, CTX-Kmϕ phage transduction via TCP in wild-type N16961, Bgd1 and Bgd5 in AKI growth conditions and vaccine strains CAH182 and CAH184 with (+R) and without (-R) 0.1% rhamnose grown in soy LB. Data from three independent experiments +/- standard errors. A two-tailed standard t test yielded no significant differences among strains.
Mentions: To verify production of TCP that is dependent upon rhamnose addition, the final vaccine strains CAH182 and CAH184 (parental strains are Bgd1 and Bgd5, respectively) were grown in an animal-free medium (soy-based LB broth) at 37°C overnight with or without the addition of 0.1% rhamnose (vol/vol). Whole cell extracts (WCE) were assayed via western immunoblot for the presence of TcpA, the major pilin that forms TCP, using anti-TcpA anti-serum. Both CAH182 and CAH184 produced stable TcpA protein only when grown in the presence of rhamnose (Fig 3A). Parental strains Bgd1 and Bgd5, along with control V. cholerae El Tor strain N16961, only produced TcpA when grown in AKI-inducing conditions as previously described [27] and did not produce TcpA in soy LB with or without the inclusion of rhamnose (Fig 3A).

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