Intranasal Immunization With Nanoparticles Containing an Orientia tsutsugamushi Protein Vaccine Candidate and a Polysorbitol Transporter Adjuvant Enhances Both Humoral and Cellular Immune Responses

Figures

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  Figure 1. Synthesis of PST and cloning of recombinant proteins from Orientia tsutsugamushi. (A) Reaction scheme for PST preparation via a Michael reaction between SDA and branched LMW PEI (feed molar ratio of SDA:PEI = 4:1). (B) 1H NMR spectrum of PST. (C) Comparison of 1H NMR spectra among SDA, branched LMW PEI, and PST. Frame near 6−7 ppm indicates acrylate groups. Acrylate groups of SDA are shown enlarged on the right side of the panel. (D) Target recombinant proteins, TSA56 and ScaA, were synthesized using E. coli and a pET28 cloning vector system. Truncated forms of recombinant TSA56 (amino acids 88–479) and ScaA (amino acids 607–994) were produced. Expected sizes of synthesized proteins were confirmed by Coomassie blue staining and western blotting.

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  Figure 2. Physicochemical characterization of PST with TSA56 or ScaA. (A, B) PST with TSA56 or ScaA, mixed at weight ratios of 1:1, 1:5, and 1:10, were incubated at room temperature for 30 min to allow complex formation. Size distributions of (A) TSA56-PST and (B) ScaA-PST complexes at different weight ratios were measured by DLS. (C) Gating strategy for cytotoxicity assay. (D) Cytotoxicity of TSA56-PST and ScaA-PST was analyzed in BMDCs stained with annexin V/7-AAD using flow cytometry (n=5). Results are presented as mean ± SEM. Significant differences compared with CON group within each gating (Live, Early apoptosis, Necrosis) determined by t-test. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 3. Ag-specific Ab responses in mice immunized with TSA56-PST or ScaA-PST. Mice (n=5 mice/group pooled data from 2 independent experiments) were immunized three times at 2-wk intervals with TSA56, ScaA, TSA56-PST, or ScaA-PST. (A) Schematic of vaccination protocol. TSA56- or ScaA-specific (B) IgG2a, (C) IgG2b, and (D) IgA in serum were determined by ELISA. Results are presented as means ± SEMs. Significant differences were analyzed by two-way analysis of variance, followed by Tukey’s multiple comparison test. Significant difference is noted only within the time points. **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 4. Ag-specific T-cell responses in mice vaccinated with TSA56-PST or ScaA-PST. Mice (n=5 mice/group pooled data from 2 independent experiments) were immunized three times at 2-week interval with TSA56, ScaA, TSA56-PST, or ScaA-PST. Two weeks after the third vaccination, spleens were collected; single cells from splenocytes were labeled with CTV and treated with TSA56 or ScaA protein (10 µg/ml) for 72 h. Then, changes in CD4+ and CD8+ T cell populations were examined by flow cytometry. (A) Gating strategy for the proliferation of CD4+ and CD8+ T cells. (B, C) The total populations of CD8+ and CD4+ T cells were examined after (B) TSA56 or (C) ScaA protein restimulation. (D, E) After TSA56 protein restimulation, the changes in proliferation (CTVloCD44+) among (D) CD4+ or (E) CD8+ T cells were examined. (F, G) After ScaA protein restimulation, the change in proliferation (CTVloCD44+) among (F) CD4+ T cells or (G) CD8+ T cells were measured. (D-G) Proliferation results are presented with each of the representative dot plot. Results are presented as mean ± SEM. Significant differences were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. a,bDifferent letters…

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  Figure 5. Protective immunity against O. tsutsugamushi in mice immunized with TSA56-PST or ScaA-PST. Mice (n=5) were immunized with TSA56, ScaA, TSA56-PST, or ScaA-PST for three times at 2-wk interval. Two weeks after the final immunization, mice were challenged by intraperitoneal infection with O. tsutsugamushi Boryong strain (5×106 ICU, 100% lethal dose). (A) Schematic vaccination schedule. (B) Survival of mice after the lethal challenge with O. tsutsugamushi. Mortality among the group of mice was monitored for 3 weeks; the survival rate was calculated as the ratio of live to total challenged mice within each group. Significant differences in survival rate were determined by the log-rank (Mantel-Cox) test.