Intranasal Immunization of Mice with Multiepitope Chimeric Vaccine Candidate Based on Conserved Autotransporters SigA, Pic and Sap, Confers Protection against Shigella flexneri

Abstract

Shigellosis is a diarrheal disease and the World Health Organization prompts the development of a vaccine against Shigella flexneri. The autotransporters SigA, Pic and Sap are conserved among Shigella spp. We previously designed an in silico vaccine with immunodominat epitopes from those autotransporters, and the GroEL protein of S. typhi as an adjuvant. Here, we evaluated the immunogenicity and protective efficacy of the chimeric multiepitope protein, named rMESF, in mice against lethal infection with S. flexneri. rMESF was administered to mice alone through the intranasal (i.n.) route or accompanied with Complete Freund's adjuvant (CFA) intradermically (i.d.), subcutaneously (s.c.), and intramuscular (i.m.), as well as with Imject alum (i.m.). All immunized mice increased IgG, IgG1, IgG2a, IgA and fecal IgA titers compared to PBS+CFA and PBS+alum control groups. Furthermore, i.n. immunization of mice with rMESF alone presented the highest titers of serum and fecal IgA. Cytokine levels (IFN-γ, TNF-α, IL-4, and IL-17) and lymphocyte proliferation increased in all experimental groups, with the highest lymphoproliferative response in i.n. mice immunized with rMESF alone, which presented 100% protection against S. flexneri. In summary, this vaccine vests protective immunity and highlights the importance of mucosal immunity activation for the elimination of S. flexneri.

Keywords: GroEL; Shigella flexneri; autotransporters; bacillary dysentery; mucosal immunity; multiepitope chimeric vaccine.

Figure 1

Immunization strategy and sample collection. Days of vaccine administration, adjuvants and routes are shown. The challenge assay was developed 30 days after the last immunization. Blood and feces collection are represented in red and golden brown circles, respectively. For more details of control groups read the text. rMESF, recombinant multiepitope S. flexneri protein. CFA, complete Freund’s adjuvant. IFA, incomplete Freund´s adjuvant. Alum, Imject^TM alum adjuvant. i.n., intranasal. i.m., intramuscular. s.c., subcutaneous.

Figure 2

Cloning of chimeric gene (MESF), expression and purification of the recombinant multiepitope protein (rMESF). (A) Cloning of the MESF gene in pQE-80L expression vector with T4 ligase. Lane 1: 1 kb DNA ladder. Line 2: MESF gene. Line 3: pQE-80L. Line 4: product digestions of pQE-80L-rMESF with SmaI and KpnI after ligation. Line 5: full circular size of pQE-80L-rMESP (B). SDS–PAGE of the expressed rMESF in E. coli BL21 cells after the induction with 1 mM IPTG for 4 h at 37 °C. Line 1: molecular mass marker. Line 2: protein before induction with IPTG. Line 3: proteins after induction with IPTG showing the rMESF protein with 84.5 kDa. (C) Western blot confirming the expression of rMESF after the induction with IPTG. (D) Western blot confirming the presence of rMESF protein after the purification-elution by Ni-NTA affinity chromatography. IPTG, isopropylthiogalactoside; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 3

Antibody serum titers in controls and immunized mice. (A) Serum IgG. (B) IgG1. (C) IgG2a. (D) serum IgA. Controls, PBS+alum and PBS+CFA. rMESF+alum groups were immunized intramuscular (i.m.). rMESF +CFA were immunized through intradermic route (i.d.) and the first and second boosters were applied in this group through subcutaneous (s.c.) and i.m. routes, respectively with incomplete Freund’s adjuvant (IFA). rMESF groups were immunized with rMESF alone trough intranasal route (i.n.). The two booster doses were given to all experimental mice groups on the 14th and 28th days. Antibody titers were expressed as mean ± standard deviation (SD) of log₁₀ of the last reciprocal serum dilution above cut-off. Symbols and letters indicate statistically significant values of p < 0.0001 compared with the respective mice group, # vs. PBS+CFA, a vs. PBS+alum. b vs. rMESF +alum, c vs. rMESF +CFA. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant.

Figure 4

Antibody titers of fecal IgA in controls and immunized mice. Controls, PBS+alum and PBS+CFA. rMESF+alum groups were immunized intramuscular (i.m.). rMESF+CFA were immunized through intradermic route (i.d.) and the first and the second boosters were applied in this group through subcutaneous (s.c.) and i.m. routes, respectively with incomplete Freund’s adjuvant (IFA). rMESF groups were immunized with rMESF alone trough intranasal route (i.n.). The two booster doses were given to all experimental mice groups on the 14th and 28th days. Antibody titers were expressed as mean ± standard deviation (SD) of log₁₀ of the last inverse serum dilution above cut-off. rMESF+alum group remains below the cut-off value calculated, Symbols and letters indicate statistically significant values of p < 0.0001 as compared with the respective mice group, # vs. PBS+CFA, a vs. PBS+alum, b vs. rMESF +alum, c vs. rMESF +CFA. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant.

Figure 5

Cytokine levels quantified by ELISA sandwich. Production of (A) IL-4, (B) TNF-α, (C) IL-17 and (D) IFN-γ, present in the supernatant of splenocytes stimulated in vitro with 1 or 2 μg/mL of rMESF multiepitope fusion proteins for 72 h. Splenocytes were collected after 30 days of the last immunization (n = 5 mice/group). The absorbance was measured at 450 nm. Symbols and letters indicate statistically significant values with p values raging from <0.05 to 0.0001 (for more details read the text) as compared with the respective mice group. # vs. PBS+CFA, a vs. PBS+alum, b vs. rMESF+alum, c vs. rMESF+CFA. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant. Results are plotted as mean ± standard deviation.

Figure 6

Lymphocyte proliferation. Splenocytes were collected from mice groups (n = 5 mice/group) after 30 days of the last immunization and stimulated in vitro with 1 μg/mL or 2 μg/mL with rMESF multiepitope fusion protein. Symbols and letters indicate statistically significant values of p < 0.0001 compared with the respective mice group. # vs. PBS+CFA, a vs. PBS+alum, b vs. rMESF + alum, c vs. rMESF + CFA. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant. Results are plotted as mean ± standard deviation.

Figure 7

Protective efficacy of rMESF multiepitope fusion protein immunization on survival. Groups of female BALB/c mice (n = 5 mice/group) were immunized through different routes and adjuvant with 25 µg/mouse of rMESF. Thirty days after the last booster, mice were challenged with a S. flexneri lethal dose of 1 × 10⁷ CFU/mouse i.n. Mortality was observed for 30 days. Symbols and letters indicate statistically significant values of p < 0.0001 compared with the respective mice group. # vs. PBS+CFA, a vs. PBS+alum b vs. rMESF + alum, c vs. rMESF + CFA. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant. i.n., intranasally. The protective effect was represented as Kaplan-Meier survival curves.

Figure 8

Bacterial burden in lungs after challenge assay of control and immunized mice. Lungs were collected from survival mice and controls groups after 30 days and three days, respectively, of challenge with lethal dose of S. flexneri (1 × 10⁷ CFU/mouse), and bacterial loads were determined by 10-fold serial dilutions on LB agar plates incubated at 37 °C for 18 h. Symbols and Letters indicate statistically significant values of p < 0.0001 compared with the respective mice group. # vs. PBS+CFA, a vs. PBS+alum. PBS, phosphate-buffered saline. CFA, complete Freund’s adjuvant. CFU, colony forming units; i.n., intranasally.