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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

Vaccine strains produce TCP in 1L growth volumes.A, Western immunoblot analysis of TcpA from CAH182 and CAH184 induced with 0.1% rhamnose (+R) in soy LB grown in large (1L) volumes. B, Pili produced by CAH182 (left) and CAH184 (right) in the above conditions as seen with transmission electron microscopy and negative staining.
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pone.0175170.g004: Vaccine strains produce TCP in 1L growth volumes.A, Western immunoblot analysis of TcpA from CAH182 and CAH184 induced with 0.1% rhamnose (+R) in soy LB grown in large (1L) volumes. B, Pili produced by CAH182 (left) and CAH184 (right) in the above conditions as seen with transmission electron microscopy and negative staining.

Mentions: A consideration for vaccine production is expression of the antigens in large culture volume in order to mass-produce the final vaccine formulation. To ensure that the rhamnose-inducible strains CAH182 and CAH184 continue to produce TCP in larger culture volumes, we grew 1-liter overnight cultures of each bacterial strain induced with 0.1% rhamnose at 37°C with agitation. Fig 4A (western immunoblot for TcpA) and 4B (TEM images) show that the vaccine strains CAH182 and CAH184, when grown in the presence of 0.1% rhamnose, produce TCP, even if grown in larger volumes of soy LB medium at 37°C, consistent with the strains potentially being amendable for mass vaccine production.


Production of putative enhanced oral cholera vaccine strains that express toxin-coregulated pilus
Vaccine strains produce TCP in 1L growth volumes.A, Western immunoblot analysis of TcpA from CAH182 and CAH184 induced with 0.1% rhamnose (+R) in soy LB grown in large (1L) volumes. B, Pili produced by CAH182 (left) and CAH184 (right) in the above conditions as seen with transmission electron microscopy and negative staining.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0175170.g004: Vaccine strains produce TCP in 1L growth volumes.A, Western immunoblot analysis of TcpA from CAH182 and CAH184 induced with 0.1% rhamnose (+R) in soy LB grown in large (1L) volumes. B, Pili produced by CAH182 (left) and CAH184 (right) in the above conditions as seen with transmission electron microscopy and negative staining.
Mentions: A consideration for vaccine production is expression of the antigens in large culture volume in order to mass-produce the final vaccine formulation. To ensure that the rhamnose-inducible strains CAH182 and CAH184 continue to produce TCP in larger culture volumes, we grew 1-liter overnight cultures of each bacterial strain induced with 0.1% rhamnose at 37°C with agitation. Fig 4A (western immunoblot for TcpA) and 4B (TEM images) show that the vaccine strains CAH182 and CAH184, when grown in the presence of 0.1% rhamnose, produce TCP, even if grown in larger volumes of soy LB medium at 37°C, consistent with the strains potentially being amendable for mass vaccine production.

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