The short- and long-term passive protective efficacy of a mixture of heat-killed cells of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, S. boydii 4, and S. sonnei) were studied in neonatal mice. Neonatal mice from immunized dams exhibited significant short- and long-term passive protection against individual challenge by each of the six Shigella strains. High IgG and IgA titers against the lipopolysaccharide from each of the six Shigella strains were observed in sera from immunized dams.
Infection by Shigellae is a major cause of morbidity and mortality during childhood, especially in developing countries [1-3]. Following several decades of research, the goal of developing a vaccine against shigellosis has yet to be attained [1, 2, 4-6]. To develop a vaccine that confers broad-spectrum coverage requires the inclusion of all epidemic and endemic Shigella serotypes [1, 2, 6]. Recently, we reported that an oral vaccine candidate of heat-killed Shigella strains (S. dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, S. boydii 4, and S. sonnei) achieved significant levels of protection against shigellosis in a guinea pig colitis model . Similarly, Kaminski et al.  reported the successful development of a trivalent, formalin-inactivated Shigella whole-cell vaccine.
The current study confirmed the efficacy of the heat-killed hexavalent Shigella vaccine , by investigating protective immunity in an infant mouse model wherein the offspring of immunized dams were challenged by wild strains of Shigellae. The level of protection was measured as the degree of colonization of the challenged bacteria in the bowel [9, 10]. Serum IgG and IgA titers of the immunized dams were also monitored during and after immunization periods.
Fifty female mice (Swiss albino, 7–8 weeks old) were divided into two groups (immunized and control), containing 25 mice each. The Animal Ethical Committee of the National Institute of Cholera and Enteric Diseases, Kolkata, India, approved detailed protocols of the animal experiments. All results presented were from two independent experiments. Each female mouse in the immunized group was immunized with a mixture of heat-killed cells of S. dysenteriae 1 (NT4907Δstx), S. flexneri 2a (B294), S. flexneri 3a (C519), S. flexneri 6 (C347), S. sonnei phase I (IDH00968), and S. boydii 4 (BCH612). Non-immunized male mice were housed in parallel with experimental female mice for the duration of each experiment. Bacterial culture conditions and the preparation of a mixture of heat-killed cells containing six Shigella strains were as described previously .
Oral immunization of female mice with a mixture of heat-killed cells of Shigella strains (107 cfu in 200 µL PBS containing an equal number of six Shigella strains) was carried out on days 0, 7, 14, and 21. In the control group, mice were given 200 µL sterile PBS. From days 41 to 55 after the initial immunization, one female mouse from the immunized and control groups and two age-matched male mice for each female mouse were housed for mating. Then, female mice were separated from males after pregnancy and monitored for birth. After birth, 4-day-old neonatal mice from the control and immunized groups were challenged with 109 cfu Sereny positive wild-type strain of either S. dysenteriae 1 (NT4907), S. flexneri 2a (B294), S. flexneri 3a (C519), S. flexneri 6 (C347), S. sonnei phase I (IDH00968), or S. boydii 4 (BCH612) in 50 µL PBS.
The first challenge experiments using 4-day-old neonatal mice were conducted from days 67 to 75 and, after completion of the first challenge experiments, immunized female mice were subjected to a second mating from days 95 to 110. The second challenge experiments using 4-day-old neonatal mice were conducted from days 117 to 130.
At 18 hr after challenge, neonatal mice were killed, the bowel removed by dissection and mechanically homogenized in 1 mL PBS. Appropriate dilutions of the homogenate were plated on Hektoen enteric agar (Difco, Sparks, MD, USA) and MacConkey agar plates (Difco). Representative colonies were subjected to confirmation with appropriate Shigella serotyping antisera (Denka Seiken, Tokyo, Japan).
Results of the two challenge experiments are summarized in Table 1. Sixteen 4-day-old neonatal mice per group (immunized and control groups) were used in two experiments and were challenged by either S. dysenteriae1 (NT4907), S. flexneri 2a (B294), S. flexneri 3a (C519), S. flexneri 6 (C347), S. sonnei phase I (IDH00968), or S. boydii 4 (BCH612). The number of colonized challenge bacteria in the bowel was counted.
|Immunogen||Experimental group||Strain for oral challenge||No. of neonates examined after first challengea||No. of Shigella recovered after first challenge (cfu/g of bowel; mean ± SD)b||No. of neonates examined after second challengea||No. of Shigella recovered after second challenge (cfu/g of bowel; mean ± SD)b|
|Hexavalent||Immunized||S. dysenteriae 1||16||23.3 ± 21.6||16||21.0 ± 22.1|
|Controlc||16||(1.9 ± 3.0) × 1010||16||(1.8 ± 3.2) × 1010|
|Hexavalent||Immunized||S. flexneri 2a||13d||10.6 ± 1.9||13d||11.7 ± 4.3|
|Control||16||(1.9 ± 2.8) × 1010||16||(1.5 ± 2.4) × 109|
|Hexavalent||Immunized||S. flexneri 3a||16||21.5 ± 21.2||16||14.8 ± 12.7|
|Control||16||(4.3 ± 3.0) × 1010||16||(2.4 ± 3.2) × 1010|
|Hexavalent||Immunized||S. flexneri 6||16||11.6 ± 5.7||16||16.2 ± 12.6|
|Control||16||(2.4 ± 2.9) × 1010||16||(2.1 ± 2.4) × 1010|
|Hexavalent||Immunized||S. sonnei||14d||10.4 ± 1.6||14d||14.1 ± 11.5|
|Control||16||(3.3 ± 2.3) × 1010||16||(1.6 ± 2.6) × 109|
|Hexavalent||Immunized||S. boydii 4||16||11.2 ± 3.4||16||10.8 ± 2.5|
|Control||16||(2.5 ± 2.6) × 1010||16||(1.4 ± 2.7) × 1010|
After the first challenge, colonization levels ranged from 1.9 ± 2.8 to 4.3 ± 3.0 × 1010 cfu/g of bowel in the control group, compared with 10.4 ± 1.6 to 23.3 ± 21.6 cfu/g of bowel in the immunized group with the following exceptions: in three mice challenged with S. flexneri 2a and two mice challenged with S. sonnei, the number of colonized bacteria were 2.6 ± 3.7 × 108 and 1.0 ± 1.4 × 108 cfu/g of bowel, respectively.
Similar results were obtained after the second challenge. In the control group, colonization levels were between 1.5 ± 2.4 × 109 to 2.4 ± 3.2 × 1010 cfu/g of bowel and 10.8 ± 2.5 to 21.0 ± 22.1 cfu/g of bowel in the immunized group with the following exceptions: in three mice challenged with S. flexneri2a and two mice challenged with S. sonnei, the number of colonized bacteria were 7.9 ± 9.6 × 108 and 8.8 ± 8.6 × 108 cfu/g of bowel, respectively.
Statistical analysis of data is presented as mean ± SD. Differences between the control and experimental groups were evaluated using the Student's t-test. Statistical analyses were done using JMP Pro version 11 (SAS Institute, Cary, NC, USA). Values of P ≤ 0.05 and P < 0.001 were considered to be statistically significant, and highly significant, respectively.
Serum IgG and IgA responses against LPS of immunized Shigellae in immunized and control mice were measured by ELISA, as described previously . Blood was collected from the lateral tail vain of each mouse on days 0, 7, 14, 21, 28, 35, 77, and 150. Serum IgG and IgA levels are shown in Figures 1a and b. During four successive immunizations with a mixture of six heat-killed Shigella strains, both serum IgG and IgA titers against LPS from each of six Shigella strains increased, reaching a maximum 1 week after the last immunization. Levels remained high for several weeks (especially, for serum IgG), then declined slightly and persisted at a relatively high level until day 150.
The primary aim of the present study was to investigate the potential use of multivalent heat-killed Shigella strains as a new oral vaccine candidate for shigellosis. Previously, we demonstrated that a mixture of six heat-killed Shigella strains induced relevant serum and mucosal immune responses and showed protection against challenge with wild Shigellae strains in a guinea pig model . Herein, we extended the previous study to investigate short- and long-term immunogenicity and protective efficacy of hexavalent heat-killed Shigella strains in a mouse model previously developed for Vibrio cholerae infection  and Shigellae infection . As shown in Figure 1, the same formulation  of hexavalent heat-killed Shigella strains induced both serum and mucosal immune responses and showed protection for a long period, until day 150. With regard to the protective efficacy, three of 16 mice (18.8%) challenged with S. flexneri 2a and two of 16 mice (12.5%) challenged with S. sonnei were not protected in either the first or the second challenge (Table 1). Similar results were obtained in the challenge experiment of the previous report  where two immunized groups of guinea pigs immunized with the mixture of six heat-killed Shigella strains exhibited 87.5% protection after rectal challenge with S. flexneri 2a and S. sonnei, respectively, whereas the other four groups of guinea pigs immunized with S. dysenteriae 1, S. flexneri 3a, S. flexneri 6, and S. boydii exhibited full protection. These results strongly suggest that the number of S. flexneri 2a and S. sonnei bacteria were low in the formulation of the hexavalent Shigella strains to elicit sufficient protection.
The success of oral vaccination using a combination of heat and formalin-killed multiple strains of V. cholerae has been reported [11, 12]. This strategy to develop vaccine(s) in enteric diseases is ideal in terms of inexpensiveness, simplicity of administration, and ease of storage and transport. To develop a multiplex heat-killed Shigella vaccine candidate, several issues such as an appropriate number of each serogroups/serotypes of Shigella strains, cross-reactivity among serogroups/serotypes and number of serogroups/serotypes included in the multiplex vaccine remain to be studied. A study along this line is in progress in our laboratory.
This study was supported by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), Ministry of Education, Culture, Sports, Science and Technology, Japan.
No authors have any conflict of interests to disclose.