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

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.

Fig 1. The Shanchol vaccine does not contain TcpA protein.

A, Western immunoblot of TcpA protein present in ΔtcpA, wild-type N16961 (El Tor), clinical El Tor variant strains Bgd1 (Ogawa) and Bgd5 (Inaba), and rhamnose-inducible tcp strains in the Bgd1 and Bgd5 background with cholera toxin genes deleted (referred to as CAH182 and CAH184, respectively; +R, with 0.1% rhamnose) as compared to the Shanchol vaccine in ELISA units (EU). B, Western immunoblot of LPS present in these strains compared to Shanchol, probed with anti-LPS antiserum 72.1 (detects Ogawa and Inaba LPS).

Fig 2. 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.

Fig 3. 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.

Fig 4. 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.

Fig 5. 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).