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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

Pili produced by vaccine strains are stable after incubation in high heat or an acidic medium.A, Western immunoblot analysis for TcpA after heat-killing of the vaccine strains CAH182 and CAH184 at 56°C over two hours following overnight growth in soy LB with 0.1% rhamnose (+R) at 37°C. B, Transmission electron microscopy results of the negatively-stained pili after heat-killing for 60 minutes (CAH182, left and CAH184, right). C, Western immunoblot analysis of TcpA after treatment of the vaccine strains CAH182 and CAH184 with acid (pH 2.0) up to two hours at 37°C after overnight growth in soy LB at 37°C with 0.1% rhamnose (+R). D, Transmission electron microscopy results of negatively-stained pili after treatment with acid for 120 minutes (CAH182, left and CAH184, right).
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pone.0175170.g005: Pili produced by vaccine strains are stable after incubation in high heat or an acidic medium.A, Western immunoblot analysis for TcpA after heat-killing of the vaccine strains CAH182 and CAH184 at 56°C over two hours following overnight growth in soy LB with 0.1% rhamnose (+R) at 37°C. B, Transmission electron microscopy results of the negatively-stained pili after heat-killing for 60 minutes (CAH182, left and CAH184, right). C, Western immunoblot analysis of TcpA after treatment of the vaccine strains CAH182 and CAH184 with acid (pH 2.0) up to two hours at 37°C after overnight growth in soy LB at 37°C with 0.1% rhamnose (+R). D, Transmission electron microscopy results of negatively-stained pili after treatment with acid for 120 minutes (CAH182, left and CAH184, right).

Mentions: Heat-killed samples were then analyzed via SDS-PAGE and a western blot for TcpA indicated that TcpA protein was stable for up to one hour after heat-killing (Fig 5A). Intact pili were viewed via TEM in the 60 min killed samples (Fig 5B). However, at 120 min incubation at 56°C, TcpA levels were reduced by ~50% (Fig 5A) and few pili were visible by TEM (data not shown). Regardless, 60 min at 56°C was more than sufficient to ensure killing of the bacterial strains while retaining TCP.


Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus
Pili produced by vaccine strains are stable after incubation in high heat or an acidic medium.A, Western immunoblot analysis for TcpA after heat-killing of the vaccine strains CAH182 and CAH184 at 56°C over two hours following overnight growth in soy LB with 0.1% rhamnose (+R) at 37°C. B, Transmission electron microscopy results of the negatively-stained pili after heat-killing for 60 minutes (CAH182, left and CAH184, right). C, Western immunoblot analysis of TcpA after treatment of the vaccine strains CAH182 and CAH184 with acid (pH 2.0) up to two hours at 37°C after overnight growth in soy LB at 37°C with 0.1% rhamnose (+R). D, Transmission electron microscopy results of negatively-stained pili after treatment with acid for 120 minutes (CAH182, left and CAH184, right).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0175170.g005: Pili produced by vaccine strains are stable after incubation in high heat or an acidic medium.A, Western immunoblot analysis for TcpA after heat-killing of the vaccine strains CAH182 and CAH184 at 56°C over two hours following overnight growth in soy LB with 0.1% rhamnose (+R) at 37°C. B, Transmission electron microscopy results of the negatively-stained pili after heat-killing for 60 minutes (CAH182, left and CAH184, right). C, Western immunoblot analysis of TcpA after treatment of the vaccine strains CAH182 and CAH184 with acid (pH 2.0) up to two hours at 37°C after overnight growth in soy LB at 37°C with 0.1% rhamnose (+R). D, Transmission electron microscopy results of negatively-stained pili after treatment with acid for 120 minutes (CAH182, left and CAH184, right).
Mentions: Heat-killed samples were then analyzed via SDS-PAGE and a western blot for TcpA indicated that TcpA protein was stable for up to one hour after heat-killing (Fig 5A). Intact pili were viewed via TEM in the 60 min killed samples (Fig 5B). However, at 120 min incubation at 56°C, TcpA levels were reduced by ~50% (Fig 5A) and few pili were visible by TEM (data not shown). Regardless, 60 min at 56°C was more than sufficient to ensure killing of the bacterial strains while retaining TCP.

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