A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

Abstract

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ(+)CD3(+) T cells and IL-17(+)CD4(+) T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.

Fig 1. Both SP27 and MAP27 bind to anti-PGN mAb specifically.

(A) The sequences of SP27 (Biotin-SASPHHHSRLRSESGG) and SP27’ (Biotin-SPHHHSRLRSESSAGG). Underlined letters represent nonspecific flanking amino acids. (B) Both SP27 and SP27’ bind to anti-PGN mAb in a dose-dependent manner. (C) Both SP27 and SP27’ (100 μg/ml) specifically bind to anti-PGN mAb, but not to anti-LTA mAb. For ELISA assays in panel B and C, anti-PGN mAb or anti-LTA mAb was used to coat the wells at a concentration of 5 μg/ml, SP27, SP27’ or non-specific peptide L2 (in panel B, sequence: Biotin-HSGHWDFRQWWQPSGG) was then added at indicated concentrations and incubated at 37°C for 40 min, followed by detection with HRP-labeled streptavidin. (D) The structure diagram of MAP27. MAP27 was synthesized in a tetra-branched form that contains four copies of a sequence (SASPHHHSRLRSESGG) that mimics PGN epitope. (E) MAP27 binds to anti-PGN mAb specifically. (F) MAP27 binds to anti-S. aureus polyclonal antibodies specifically. For ELISA assays in panel E and F, MAP27 or MAPctrl was used to coat the wells of a microplate. Anti-PGN mAb or anti-S. aureus polyclonal antibodies was added, followed by detection with HRP-labeled antibodies. The absorbance was measured at OD_450nm. The results are shown as means ±SEM. * P<0.05, ** P<0.01, *** P<0.001.

Fig 2. BALB/c mice immunized with MAP27 produced anti-MAP27, anti-PGN and anti-S.aureus antibodies.

(A) Titers of anti-MAP27 serum during the period of immunization. 96-well plates were coated with MAP27. Serum samples from mice immunized with MAP27, MAPctrl or blank were pooled and added in a 10-fold serial dilution, followed by incubation with HRP-conjunctive goat anti-mouse IgG. (B) Sera from MAP27-immunized mice bind to PGN after the fifth MAP immunization. (C) Sera from MAP27-immunized mice bind to S. aureus after boosting with heat-killed S.aureus. For ELISA assays in panel B and C, 96-well plates were coated with PGN or sonicated S. aureus fragments. Serum samples were added in a 1/200 dilution as primary antibodies, followed by incubation with HRP-conjunctive goat anti-mouse IgG. The absorbance was measured at OD_450nm. The results are presented as means ±SEM. * P<0.05, ** P<0.01, n = 5–9 mice/group.

Fig 3. Immunization with MAP27 protects mice against S. aureus infection.

(A) Survival analysis of mice with or without immunization after S. aureus infection. Five days after boosting with heat-inactivated S. aureus, all of BALB/c mice (including MAP27-immunized, MAPctrl-immunized and blank control, n = 8–9 mice/group) were challenged with S. aureus (ATCC 25923, 5×10⁸ CFU/mouse) by intravenous injection. Survival rate were monitored for seven days after infection. (B) Bacterial numbers in kidney from three groups of mice (n = 4 mice per group) were measured after three days infection of S. aureus (2×10⁷ CFU/mouse). (C) Bacterial numbers in lung from different groups of mice (n = 5 mice per group) were measured after three days infection. * P<0.05, ** P<0.01, *** P<0.001.

Fig 4. Immunization with MAP27 promoted IFN-γ, IL-17A/F and CCL3 production in organs post infection with S. aureus.

Five days after boosting with heat-inactivated S. aureus, all of the mice (n = 5 mice per group) were infected with S. aureus (2×10⁷ CFU/mouse) for three days. Spleen and lung were aseptically taken and homogenized with sterile PBS. The supernatants were pooled and analyzed by ELISA. The concentrations of IFN-γ (panel A and D), IL-17A/F (panel B and E), and chemokine ligand 3 (CCL3, panel C and F) were measured in spleen and lung, respectively. * P<0.05, ** P<0.01, *** P<0.001.

Fig 5. Vaccination with MAP27 primed IFN-γ⁺CD3⁺ T cells and IL-17⁺CD4⁺ T cells after S. aureus infection.

Five days after the last boost with heat-inactivated S. aureus, all of the mice were infected with S. aureus (2×10⁷ CFU/mouse). Spleen cells (n = 5 mice per group) were harvested three days post infection and incubated with cocktail (including PMA and ionomycin) and BFA for 6 h. Cells were then fixed, permeabilized and stained with FITC-anti CD3, APC-anti CD4, PE-anti IFN-γ and PE-anti IL-17A mAb, respectively. Intracellular cytokine analysis was performed by flow cytometry. All lymphocytes were first gated based on FSC and SSC. CD3⁺ lymphocytes were then further gated from the lymphocyte cluster according to the fluorescence signal intensities. The results were presented as mean±SEM. (A) The percentage of IFN-γ⁺CD3⁺CD4⁺ cells. (B) The percentage of IFN-γ⁺CD3⁺CD4^- cells. (C) Representative flow cytometry plots showing the percentage of IFN-γ⁺CD3⁺ cells in spleen. (D) The percentage of IL-17A⁺CD3⁺CD4⁺ cells. (E) The percentage of IL-17A⁺CD3⁺CD4^- cells. (F) Representative flow cytometry plots showing the percentage of IL-17A⁺CD3⁺ cells in spleen. * P<0.05. ns: not significantly different between two groups (p>0.05).

Fig 6. In vitro MAP27 treatment promoted the production of IFN-γ in splenocytes from MAP27-immunized mice.

Five days after the last boost immunization, splenocytes isolated from the mice (n = 5 mice per group) were cultured at 2×10⁵/well in U-bottom 96-well plates and stimulated with MAP27 (100 μg/ml) for 24 h. The concentrations of IL-2 (A), IFN-γ (B), and IL-4 (C) in supernatant were measured by ELISA. (D) Frequency of IFN-γ-producing cells induced by MAP27. Splenocytes isolated from the mice (n = 5 mice per group) were cultured at 4×10⁵/well in pre-coated plates, and stimulated with MAP27 (10 μg/ml) plus anti-mouse CD28 antibody (1 μg/ml) for 24 h. The number of IFN-γ-producing cells was then measured by ELISPOT and shown in box and whisker plots. (E) Representation of one of the ELISPOT assays. ** P<0.01; *** P<0.001; ns: P>0.05.

Fig 7. Heat-inactivated S. aureus promoted Th1 and Th17 cell responses in the spleen of MAP27-immunized mice.

Five days after the last booster with heat-inactivated bacteria, the splenocytes isolated from the mice (n = 5 mice per group) were cultured (2×10⁵/well), and stimulated with 2×10⁴ CFU/well heated-inactivated S. aureus for 72 h. The concentrations of IL-2 (A), IFN-γ (B), IL-17A/F (C) and IL-4 (D) were measured by ELISA. The isolated splenocytes were also cultured at 4×10⁵/well in pre-coated plates and stimulated with heat-inactivated S. aureus for 24 h. Frequency of IFN-γ-producing (E) and IL-17A-producing cells (F) was determined by ELISPOT. Cell numbers were shown in box and Whisker plots. (G) Representation of one of the ELISPOT assays for IFN-γ-producing cells. (H) Representation of one of the ELISPOT assays for IL-17A-producing cells. * P<0.05; ** P<0.01; ns: P>0.05.