Characterization of ShigETEC, a Novel Live Attenuated Combined Vaccine against Shigellae and ETEC

Abstract

Background: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) remain the two leading bacterial causes of diarrheal diseases worldwide. Attempts to develop preventive vaccines against Shigella and ETEC have not yet been successful. The major challenge for a broad Shigella vaccine is the serotype-specific immune response to the otherwise protective LPS O-antigen. ETEC vaccines mainly rely on the heat-labile enterotoxin (LT), while heat-stable toxin (ST) has also been shown to be an important virulence factor. Methods: We constructed a combined Shigella and ETEC vaccine (ShigETEC) based on a live attenuated Shigella strain rendered rough and non-invasive with heterologous expression of two ETEC antigens, LTB and a detoxified version of ST (STN12S). This new vaccine strain was characterized and tested for immunogenicity in relevant animal models. Results: Immunization with ShigETEC resulted in serotype independent protection in the mouse lung shigellosis model and induced high titer IgG and IgA antibodies against bacterial lysates, and anti-ETEC toxin antibodies with neutralizing capacity. Conclusions: ShigETEC is a promising oral vaccine candidate against Shigella and ETEC infections and currently in Phase 1 testing.

Keywords: ETEC; Shigella; cross-protection; heat-labile toxin; heat-stable toxin; non-invasive; rough; vaccine.

Conflict of interest statement

The authors declare potential conflict of interest as they are employees and/or shareholders of Eveliqure Biotechnologies GmbH.

Figures

Figure 1

Schematic illustration of the genetic construct carried by the invasion plasmid of ShigETEC. LTB is fused to ST via a GPGP (GlyPro) linker (blue). ST is detoxified by an N12S mutation (red). Expression is driven by the LTA promoter and halted by the LTB termination sequence. The fusion gene is expressed as a 3× tandem repeat. The construct also expresses infA as a separate gene with intrinsic promoter and terminator sequences.

Figure 2

(a) SDS-PAGE gel image of separated LPS from Shigella flexneri 2457T wild-type (WT) and Shigella flexneri 2457TΔrfbF mutant following staining with Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit. The lowest band represents the lipid A-core molecules, while the upper ladder-like pattern is the LPS molecule with various number of O-antigen repeating units. (b) Agglutination assay with Shigella flexneri 2457T wild-type (WT, top panel) and its isogenic ΔrfbF mutant (bottom panel) using rabbit anti-Shigella flexneri 1–6 serum. (c) HeLa cells were infected with the wild-type parental Shigella flexneri 2a 2457T or ShigETEC at a MOI of 80. Percentage of intracellular (invaded) bacteria relative to the inoculum was determined by CFU calculations after plating. Data are shown from two independent experiments.

Figure 3

Expression of detoxified ETEC antigens by ShigETEC. (a) ShigETEC whole cell lysates were tested for the expression of LTB-STN12S by binding to the LTB receptor, GM1 in ELISA. Bound LTB was detected by anti-CTB antibody. Expression level of LTB-STN12S was compared to serially diluted LTB (black bars). A rough, non-invasive Shigella mutant (ΔrfbFΔipaBC) lacking the LTB-STN12S fusion construct was used as negative control (blue bar). (b) Wild-type ST and its N12S mutant were generated recombinantly in E. coli. Supernatants (SN) of the cultures were used to stimulate T84 human epithelial cells and ST-induced cGMP production was measured by ELISA. Indicated amounts of synthetic ST were used as positive control. SN from bacteria carrying empty vector served as negative control. Triplicate measurements from two independent experiments were combined.

Figure 4

ShigETEC induces protection against lethal challenge with heterologous Shigella strains. Mice were vaccinated 3 times i.n. with 108 CFU ShigETEC (blue line) or buffer (grey line). Four weeks after the last vaccination, mice were challenged i.n. with lethal doses of (a) Shigella sonnei (9 × 106 CFU) or (b) Shigella flexneri 6 (1.2 × 107 CFU). Survival was monitored for 14 days. Data from two independent experiments with a total of 10 mice per group are shown.

Figure 5

Detection of serum IgG and mucosal IgA antibodies induced upon ShigETEC vaccination. (a) Mice were vaccinated 3 times i.n. with 108 CFU ShigETEC. Specific IgG antibody levels were evaluated against indicated antigens in serum obtained 4 weeks after the last vaccination using the indicated serum dilutions in ELISA. Symbols represent averages of duplicate measurements of sera from individual mice (43 mice per group) from three independent vaccination experiments. (b) Mice were vaccinated 3 times i.n. with 108 CFU ShigETEC and challenged with lethal doses of either S. sonnei or S. flexneri 6 four weeks after the last vaccination. Bronchoalveolar lavages (BAL) were taken two weeks after the challenge. Specific IgA antibody levels were evaluated against the indicated antigens using the indicated serum dilutions in ELISA. Symbols represent averages of duplicate measurement of BAL samples from individual mice from three independent vaccination experiments with 18 and 41 mice per group for mock and ShigETEC, respectively.

Figure 6

Toxin neutralizing capacity of mouse sera induced by ShigETEC vaccination. (a) Mice were vaccinated 3 times i.n. with 108 CFU ShigETEC. Serum at indicated dilutions was incubated with indicated amounts of LT, and LT-binding to GM1 coated plates was measured. Bound LT was detected with a polyclonal anti-cholera toxin antibody. (b) Sera from individual mice (3 times i.n. vaccinated with 108 CFU ShigETEC (blue symbols) or buffer (mock, black symbols) were pre-incubated with the 5 ng LT. LT induced cAMP release was measured in T84 human colon epithelial cells. (c) 5 ng synthetic ST was pre-incubated with pooled serum from mice vaccinated i.p. with LTB-STN12S (blue bar) or LTB-STWT (red bar) protein or vehicle (mock). ST-induced cGMP release was measured in T84 cells.