Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity

Abstract

Live, attenuated Shigella vaccine candidates, such as Shigella sonnei strain WRSS1, Shigella flexneri 2a strain SC602, and Shigella dysenteriae 1 strain WRSd1, are attenuated principally by the loss of the VirG(IcsA) protein. These candidates have proven to be safe and immunogenic in volunteer trials and in one study, efficacious against shigellosis. One drawback of these candidate vaccines has been the reactogenic symptoms of fever and diarrhea experienced by the volunteers, that increased in a dose-dependent manner. New, second-generation virG(icsA)-based S. sonnei vaccine candidates, WRSs2 and WRSs3, are expected to be less reactogenic while retaining the ability to generate protective levels of immunogenicity seen with WRSS1. Besides the loss of VirG(IcsA), WRSs2 and WRSs3 also lack plasmid-encoded enterotoxin ShET2-1 and its paralog ShET2-2. WRSs3 further lacks MsbB2 that reduces the endotoxicity of the lipid A portion of the bacterial LPS. Studies in cell cultures and in gnotobiotic piglets demonstrate that WRSs2 and WRSs3 have the potential to cause less diarrhea due to loss of ShET2-1 and ShET2-2 as well as alleviate febrile symptoms by loss of MsbB2. In guinea pigs, WRSs2 and WRSs3 were as safe, immunogenic and efficacious as WRSS1.

Fig. 1

Steps involved in the construction of S. sonnei vaccine candidates WRSs2 and WRSs3. The parental strain Moseley, which is tetracycline resistant, was also the source for WRSS1 [47]. WRSs2 and WRSs3 were constructed by successive deletions of senA, senB, virG(icsA) and msbB2 using the lambda red recombineering technique. Tetracycline resistance was eliminated by selection on fusaric acid plates.

Fig. 2

Secretion of ShET2-1 and ShET2-2 in Shigella culture supernatants. Cultures of Moseley, 2457T and 2457TΔipaB with FLAG-tagged ShET2-1 and FLAG-tagged ShET2-2 were grown to log phase and treated with or without Congo Red for TTSS-mediated secretion. Lanes 1–2 in each panel has strains without FLAG-tagged proteins. Lanes 3–4 in panel A and lane 3 in panel B refer to 2457T3XFLAG ShET2-1. Lanes 3–4 in panel C refer to Moseley3XFLAG ShET2-1. Lanes 5–6 in panel A and lane 4 in panel B refer to 2457T3XFLAG ShET2-2. Lanes 5–6 in panel C refer to Moseley3XFLAG ShET2-2. Western blots were developed with anti-FLAG MAbs.

Fig. 3

Shigella infections in gnotobiotic piglets. 3-Day-old gnotobiotic piglets were orally inoculated with either 5 × 10⁹ CFU of Moseley or Moseley2ΔShET2 strain. Fecal consistency was scored daily for 7 days after inoculation. Daily mean fecal scores were recorded based on the fecal consistency from post-inoculation day (PID) 1 to PID7. Scores above 2 were considered diarrheic. Statistical analysis was carried out using a mixed model (SAS software) as described in Section 2. The scores for Moseley were significantly higher than Moseley2ΔShET2 over the course of the study (P < 0.0001).

Fig. 4

In vitro characterization of WRSs2 and WRSs3. Panel A: HeLa cell invasion assay. Invasiveness is plotted as a percentage relative to S. flexneri 2a strain 2457T, which is set at 100%. Strain ΔIpaB is a Moseley strain lacking the ipaB gene and is included as a noninvasive control strain. The figure shown is a representative experiment in which three colonies of each strain were grown and each culture was inoculated in duplicate in a 24-well tissue culture plate containing HeLa cells. Each bar represents the average number of cfu recovered for each strain and the error bars represent the calculated standard deviations. Panel B: MS analysis of lipid A from Moseley and from WRSs3. The ions above 2400 m/z are probably LPS (not shown). The ions between 1300 and 1900 m/z are associated with lipid A. The 1769.8 peak is probably hexaacylated with two C12 chains instead of one. The composition of the peaks at 1568 m/z and 1587 m/z which represent pentacylated products are indicated in panel B. The 1360 m/z peak has four acyl chains. Panels C and D: Phenotype of WRSs3 in cultured macrophages. RAW 264.7 macrophages were infected with log-phase wild-type Moseley (white bars), WRSs2 (gray bars), or WRSs3 (black bars). Culture supernatants were analysed at 2 or 4 h post-infection for Shigella-induced cytotoxicity on macrophages, indicated by lactate dehydrogenase (LDH) activity (panel C), and inflammatory potential of Shigella-infected macrophages, indicated by TNFα release (panel D). Data presented are the means of two independent experiments, each done in triplicate. Each error bar represents one standard deviation from the mean. Differences between wild-type Moseley (n = 12) and WRSs2 or WRSs3 (n = 6) were assessed for statistical significance using a two-tailed Mann–Whitney U-test. *P < 0.05.

Fig. 5

Efficacy of WRSs2 and WRSs3 tested in guinea pig eyes. Guinea pigs ocularly immunized with WRSS1, WRSs2 or WRSs3 and saline control guinea pigs were challenged 26 days after the final immunization with 1 × 10⁸ CFU/eye of wild-type S. sonnei 53G. The reactogenicity in the eyes of each guinea pig were scored over 7 days (x-axis) by the Sereny test-based score (y-axis) as described in Section 2. The asterisks indicate significant difference in reactogenicity between the control animals and animals vaccinated with different S. sonnei vaccine candidates.

Fig. 6

Antibody titer responses in guinea pigs immunized with S. sonnei vaccine candidates. Serum IgG- and IgA-specific antibody titers against S. sonnei LPS and S. sonnei invasin-LPS complex (Invaplex) were measured in guinea pigs immunized with WRSS1, WRSs2 or WRSs3. The immune responses were determined for days 0 (D0), 14 (D14), and 28 (D28). The geometric mean titer (GMT) for each group of guinea pigs was calculated and is shown on the y-axis. Serum immune responses were also measured 2 weeks after challenge of immunized guinea pigs with 53G (CLG). 95% confidence intervals are indicated by error bars. No significant differences were found between the three vaccine candidates.

Fig. 7

Mucosal responses measured in eye washes of guinea pigs vaccinated with S. sonnei vaccine candidates. Eyewashes from immunized animals were used to determine the IgA specific antibody against S. sonnei LPS and Invaplex. The GMT (y-axis) were determined for day 0 (D0), 14 (D14), and 28 (D28) for each group of guinea pigs. The error bars indicate 95% confidence intervals. The asterisk over the bar representing WRSS1 D14 GMT in the anti-Invaplex IgA indicates significance with respect to WRSs2 and WRSs3. The asterisk over the bar representing WRSS1 D14 GMT in the anti-LPS IgA panel indicates significance with respect to only WRSs2.

Fig. 8

Stability of the Form I phenotype in vaccine candidates. Panel A: Overnight cultures of 10 Form I isolates of each strain were subcultured every 24 h and at each subculture, aliquots of each culture were plated out on three TSA plates. The plates were incubated at 37 °C for 16 h. After determining the colony count on each plate, Form II colonies were scored under the microscope and indicated as a percentage of the total colony count (y-axis). Each bar represents the average of the ten isolates of each strain and the error bars represent the standard deviations for each strain. Panel B: Five colonies of each strain were grown to an OD₆₀₀ = 1.0 and dilutions plated out on TSA plates that were incubated at 37 °C for 12, 14, 16, or 18 h. At each time point the plates were removed, total colony counts obtained and Form II percentage scored as described under panel A. Each bar represents the average of Form II colonies from the five TSA plates representing an individual strain at each time point and the error bars are the standard deviations. Panel C: The plates in panel B were subjected to colony blot assay with MAb to IpaB. The number of Form II colonies were determined from the total count (Form I and II colonies) minus the number of pink spots representing IpaB positive colonies (Form I only). Each bar represents the average percentage of Form II colonies from the five TSA plates representing an individual strain at each time point and the error bars are the standard deviations.