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

<div><p>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 <i>V</i>. <i>cholerae</i> 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 <i>V</i>. <i>cholerae</i> clinical strains were selected and their cholera toxin genes deleted. The <i>tcp</i> 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 <i>V</i>. <i>cholerae</i> for use in a vaccine that produce high levels of stable TCP antigen, which has not previously been achieved.</p></div

Model for intestinal colonization dynamics of <i>V</i>. <i>cholerae</i>.

<p><i>V</i>. <i>cholerae</i> may be ingested as free-living cells (i), as forming microcolonies (ii), or as part of a biofilm (iii) (A). Cells in the lumen will first come in contact with the mucus layer (B). The bacterium must reach the intestinal epithelium by penetrating through the viscous mucus layer covering it (C). Once the bacterium reaches the intestinal epithelium, we hypothesize that noncommitted (reversible) attachment occurs, mediated by adhesins such as GbpA or Mam7 (D). Subsequently, specific attachment adhesins might be produced that would allow <i>V</i>. <i>cholerae</i> to bind in a committed fashion (E), the cells multiply (F), and, once a certain concentration of cells has been reached, the toxin coregulated pilus is produced, allowing for microcolony formation and toxin production (G).</p

Proteolysis of ToxR during late stationary phase at alkaline pH.

<p><b>(A)</b> ToxR immunoblot of O395 wild-type or Δ<i>toxR</i> grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). <b>(B)</b> ToxR immunoblots of O395 wild-type grown at different time points in LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). <b>(C)</b> ToxR immunoblots of O395 wild-type grown overnight in LB starting pH 7.0 unbuffered at 37°C, pelleted, and resuspended in phosphate buffered saline (PBS) at pH 7.0, pH 8.3, or pH 9.3 for 12 hours.</p

Viability and morphology of <i>V</i>. <i>cholerae</i> mutants after 48 hours at alkaline pH.

<p>Fluorescent (F) and differential interference contrast (DIC) images of O395 Δ<i>toxR</i>, Δ<i>rseP</i>, Δ<i>rseP</i>Δ<i>toxR</i>, Δ<i>rpoE</i> or Δ<i>rpoE</i>Δ<i>toxR</i>, <i>toxR248</i>, <i>toxR248</i>Δ<i>rseP</i>, <i>toxR248</i>Δ<i>rpoE</i>, and <i>toxR-phoA</i> grown for 48 hours in LB starting pH 9.3 (unbuffered). The cells were observed after treatment with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. Viable and culturable cells appear green and elongated; viable but dormant cells appear green and round; dead cells appear red and round.</p