Development of an Acid-Resistant Salmonella Typhi Ty21a Attenuated Vector For Improved Oral Vaccine Delivery.

The licensed oral, live-attenuated bacterial vaccine for typhoid fever, Salmonella enterica serovar Typhi strain Ty21a, has also been utilized as a vaccine delivery platform for expression of diverse foreign antigens that stimulate protection against shigellosis, anthrax, plague, or human papilloma virus. However, Ty21a is acid-labile and, for effective oral immunization, stomach acidity has to be either neutralized with buffer or by-passed with Ty21a in an enteric-coated capsule (ECC). Several studies have shown that efficacy is reduced when Ty21a is administered in an ECC versus as a buffered liquid formulation, the former limiting exposure to GI tract lymphoid tissues. However, the ECC was selected as a more practical delivery format for both packaging/shipping and vaccine administration ease. We have sought to increase Ty21a acid-resistance to allow for removal from the ECC and immune enhancement. To improve Ty21a acid-resistance, glutamate-dependent acid resistance genes (GAD; responsible for Shigella spp. survival at very low pH) were cloned on a multi-copy plasmid (pGad) under a controllable arabinose-inducible promoter. pGad enhanced acid survival of Ty21a by 5 logs after 3 hours at pH 2.5, when cells were pre-grown in arabinose and under conditions that promote an acid-tolerance response (ATR). For genetically 100% stable expression, we inserted the gad genes into the Ty21a chromosome, using a method that allowed for subsequent removal of a selectable antibiotic resistance marker. Further, both bacterial growth curves and survival assays in cultured human monocytes/macrophages suggest that neither the genetic methods employed nor the resulting acid-resistance conferred by expression of the Gad proteins in Ty21a had any effect on the existing attenuation of this vaccine strain

Chromosomal integrant (Ty21a-Gad) can survive extreme pH when pre-grown for 24 hours under appropriate inducing conditions.

<p><b>A.</b> Selected bacteria were grown in TSB-MES pH 5.5 with or without glucose for 18 or 24 hours prior to acid challenge. Although Ty21a-Gad did not express much detectable Gad protein at 18 hours in the presence of glucose, Gad-expression was easily observed at 24 hours. Interestingly, Gad proteins were expressed even in the absence of glucose after 18 hours. In contrast to single copy Ty21a-Gad, the plasmid construct (Ty21a pGad) existed at ~ 10 copies per cell [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163511#pone.0163511.ref044" target="_blank">44</a>] and expressed a higher level of Gad proteins under all conditions. An antibody-binding, non-specific band was used as the loading control. <b>B</b>. Consistent with level of Gad protein expression, Ty21a-Gad acid survival was comparable to that of Ty21a after 18 hours of growth in TSB-MES pH 5.5 with glucose, but acid survival improved significantly (~3 logs) when Ty21a-Gad were grown for 24 hours prior to acid challenge.</p

Schematic diagram of GDAR system (adapted from Zhao et al., 2010).

<p>This system consists of paralogous GadA and GadB decarboxylases and an inner-membrane antiporter GadC. GadA and GadB are pyridoxal 5’-phosphate (PLP)- dependent enzymes that convert L-glutamic acid into GABA and CO₂, in a reaction that consumes a cytoplasmic proton. The inner membrane antiporter GadC transports GABA out of the cell in exchange for more glutamate.</p

Extreme acid survival of <i>S</i>. Typhi strains depends upon the pH of the pre-growth media.

<p><b>A.</b> Tested bacteria were pre-grown in either TSB-MES pH 4.5, TSB-MES pH 5.5, TSB-MOPS pH 6.5, commercial TSB (unbuffered pH 7.2), or TSB-MOPS pH 7.5 for 18 hours prior to the acid challenge, and survival at pH 2.5 for 3 hours was determined. Ty21a with pGad showed good survival when bacteria were pre-grown in mildly acidic pH media and in commercial unbuffered TSB, with the <i>p</i> value significance between Ty21apGad and Ty21a EV at pH 4.5, 5.5, 6.5 and 7.2 (unbuffered) are 0.0236, 0.0251, 0.0230 and 0.0468, respectively; however following pre-growth at pH 7.5, acid survival was decreased by ~ 3 logs (non-significant <i>p</i> value 0.1827). Our negative control Ty21a EV did not show any survival after pre-growth under all pH conditions. Although Ty2 showed good low pH-survival when pregrown at slightly acidic pH, it showed very poor survival when pre-grown at pH 7.5 or in commercial TSB at pH 7.2. Sf showed good survival under all pre-growth conditions, including pre-growth in TSB-MOS pH 7.5. <b>B.</b> The Gad protein expression of selected bacteria after 18 hours of growth in TSB-MES pH 4.5, TSB-MES pH 5.5, TSB-MOPS pH 6.5, unbuffered pH 7.2 TSB, and TSB-MOPS pH 7.5 was determined by Western blot with anti-Gad A/B antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163511#pone.0163511.ref035" target="_blank">35</a>]. Gad protein is expressed under all the pH conditions in Ty21a pGad, where Gad protein expression is under the control of an arabinose inducible promoter. However, in Sf where Gad protein expression is tightly regulated, Gad protein expression is only seen in TSB-MES pH 5.5, TSB-MOPS pH 6.5 and unbuffered pH 7.2 TSB. As expected, the negative control Ty2 grown in TSB-MES pH 5.5 did not express any Gad proteins. An antibody-binding, non-specific band was used as a loading control; qualitative differences in GadA/B expression were monitored.</p

Gad-induced acid resistance in Ty21a does not alter strain attenuation, as determined by entry into macrophages (A) and survival in macrophages (B).

<p>Entry into macrophages and survival in macrophages are key virulence traits of wildtype <i>S</i>. Typhi that have been lost due to mutation in Ty21a [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163511#pone.0163511.ref036" target="_blank">36</a>]. Macrophage entry and survival of strains expressing Gad proteins (Ty21a pGad and Ty21a-Gad), Ty21a and Ty2 were performed as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163511#pone.0163511.ref036" target="_blank">36</a>]. A. The percent of bacterial entry was calculated as the ratio of bacteria that entered into cultured human U937 macrophages, 3 hours after infection compared to the initial bacterial inoculum. Ty21a pGad (0.005%) and Ty21a-Gad (0.006%) showed low entry, comparable to Ty21a (0.008%), whereas Ty2 showed about ~5 fold better entry into macrophages (0.04%). B. The percent survival was calculated as the percentage of bacteria inside cultured human U937 macrophage cells 24 hours after infection, compared to bacteria internalized in macrophages at 4 hours. Ty21a pGad (37%) and Ty21a-Gad (49%) showed low survival, comparable to Ty21a (35%), whereas Ty2 showed robust replication within macrophages (341%). The percent survival of Ty21a pGad, Ty21a-Gad and Ty21a was significantly lower than that of Ty2 (<i>p</i> values were 0.0077, 0.0104and 0.0086 respectively; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163511#sec002" target="_blank">Methods</a> for statistical analyses).</p

Integration of Gad genes into the Ty21a chromosome using a suicide vector.

<p>We constructed the R6K-based suicide vectors, pMD-SV and pMD-SD, that require a trans-supply of pir-encoded pi protein for replication. Although these plasmids can replicate in strains that express pi protein such as RHO3, efficient plasmid suicide results upon transfer to Ty21a which does not encode pir. Strain S17λpir carrying a Kan^R pMD-SV, which contains homology to the Vi operon <i>vexA</i> gene, was conjugated (utilizing RP4 as the conjugal transfer mediator) with Ty21a to transfer the pMD-SV plasmid. Kanamycin-resistant Ty21a with pMD-SV integrated into the Vi operon were first selected. Next, the region containing the Kan^R gene between the FRT (flippase recognition target) sites was eliminated by expression of the flippase enzyme from replication temperature-sensitive plasmid pCP20, resulting in the <i>vexA</i> merodiploid strain MD290. The <i>gad</i> genes under control of an arabinose promoter were cloned into pMD-SD (pMD-SD-gad), integrated into <i>vexA</i> merodiploid MD290, and the region between FRT sites was eliminated to construct the Kan^S Ty21a-Gad (MD297).</p

Glucose in the pre-growth media is important for extreme acid survival of <i>S</i>. Typhi strains.

<p><b>A.</b> Bacteria were grown in commercial TSB supplemented with 0.25% glucose or TSB without added glucose for 18 hours and the pH of the cultures was determined. In the presence of glucose, the pH of Ty21a pGad and Ty21a pNW129 cultures dropped below 6.5 after 18 hours, whereas the pH of the Ty2 culture remained close to pH 7.2. The pH of the Sf culture dropped even further, to below pH 6. However, in the absence of glucose added to TSB, the pH drop was not observed with Ty21a strains, suggesting that glucose is responsible for the pH drop. The pH drop of Sf cultures was not dependent on glucose in the media. Data denoted by ** are significantly different (<i>p</i> < 0.05), but data marked * are not significant. <b>B.</b> Acid survival of bacteria was compared in the presence or absence of glucose in commercial TSB (pH 7.2). Acid survival ability of Ty21a pGad was greatly diminished (~ 3 logs after 3 hours) when the bacteria where grown in TSB without glucose. <b>C.</b> Acid survival of tested bacteria were compared in the presence and absence of glucose in TSB-MES pH 5.5. Acid survival ability of Ty21a pGad and Ty2 were significantly reduced (4 to 5 logs after 3 hours) when glucose was absent from the media. Even Ty21a acid survivability was reduced (~ 3 logs after 1 hour). However, Sf acid-survival was not affected by presence or absence of glucose in the media.</p

Extreme acid survival of bacteria depends on growth phase.

<p><b>A and B</b>. When tested bacteria were pre-grown to log phase in commercial TSB or TSB-MES pH 5.5, under aerobic conditions, Ty21a with pGad (Ty21a pGad), Ty21a with empty vector (Ty21aEV), or wildtype <i>S</i>. Typhi Ty2 did not show any substantive survival at pH 2.5 after 3 hours. In contrast, <i>S</i>. <i>flexneri</i> (Sf) showed ~0.01% survival in media at pH 2.5 after 3 hours. <b>C.</b> However, when bacteria were grown to stationary phase (i.e. for 18 hours) in regular TSB under aerobic conditions, prior to pH 2.5 acid challenge, Ty21a pGad showed ~5 log increase in acid survival (i.e. comparable to survival of Sf), compared to the negative control Ty21a EV after 3 hours. Surprisingly, under these conditions Ty2 did not show any survival. Importantly, the ~5 log increase in acid survival of Ty21a pGad was dependent upon media supplementation with arabinose. D. When tested bacteria were pre-grown to stationary phase in TSB-MES pH 5.5 under aerobic conditions, prior to the pH 2.5 acid challenge, Ty21a pGad, Ty2 and Sf showed good acid survival, whereas Ty2la pEV demonstrated ~5 logs less acid survival than that of the other strains. The <i>p</i> value significance between Ty21apGad and Ty21a EV at 1, 2 and 3 hours are 0.010, 0.018 and 0.025, respectively. <b>E.</b> When tested bacteria were pre-grown to stationary phase in regular TSB under anaerobic conditions, prior to the pH 2.5 acid challenge, <i>S</i>. <i>flexneri</i> showed solid acid survival. However, the Ty21a strains and Ty2 showed poor acid survival.</p