Induction of Protective Antiplague Immune Responses by Self-Adjuvanting Bionanoparticles Derived from Engineered Yersinia pestis

Abstract

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 × 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 × 10³ CFU (50 LD₅₀) of virulent Y. pestis This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Keywords: Y. pestis; lipid A; outer membrane vesicles; plague vaccine; protective immunity.

Fig 1

Comparison of morphological alterations in Y. pestis strains by TEM imaging. Samples of strains Y. pestis KIM6+ (A), χ10015 (ΔlpxP:: P_lpxLlpxL) (B), and χ10027 (ΔlpxP:: P_lpxLlpxL ΔlacZ:: P_lpplpxE) (C) were prepared by conventional staining with 1% aqueous uranyl acetate as described in the Materials and Methods. The results are representative of three repeated experiments.

Fig 2

Analysis of Y. pestis outer membrane vesicles (OMVs). (A) TEM of OMVs purified from Y. pestis KIM6+ culture supernatants. (B) Subcellular distribution of proteins present in Y. pestis KIM6+ OMVs as a percentage of the total proteins identified by mass spectrometry listed in Table S2. (C) Amounts of protein and relative lipid contents in OMVs purified from different Y. pestis strains (Y. pestis KIM6+, χ10015 [ΔlpxP::P_lpxLlpxL] and χ10027 [ΔlpxP::P_lpxLlpxL ΔlacZ::P_lpplpxE]). All the values were normalized according to the total bacterial number (×10¹¹ CFU). (D) Whole-cell lysates or OMVs isolated from Y. pestis KIM6+, χ10015, and χ10027 were examined for the presence of the outer membrane proteins Psn, OmpA, Pla, and Caf1 (F1) by immunoblotting. (E) Whole-protein profiles of OMVs from different Y. pestis strains as shown in an SDS-PAGE gel. Rabbit polyclonal Psn and Pla antibodies (lab stock), rabbit polyclonal OmpA antibody (LS‑C369146, LSBio), and mouse monoclonal F1 antibody (YPF19, Santa Cruz Biotechnology). The results are representative of three experiments. Statistical significance: ns, no significance; ****, P < 0.0001.

Fig 3

Subcellular location analyses of the oversynthesis of LcrV antigen in Y. pestis mutants. (A) Maps of the Asd+ plasmids pSMV12 (harboring the native lcrV gene of Y. pestis) and pSMV13 (harboring the N-terminal β-lactamase signal sequence [bla SS] and lcrV fusion to facilitate LcrV secretion by the T2SS). (B) Comparison of LcrV amounts in different cell fractions. The total cell lysates and subcellular fractions, including the cytoplasmic and periplasmic fractions, were prepared from YPS1, YPS2, and YPS3 strains individually harboring pYA3342 (an empty plasmid), pSMV12, or pSMV13 (Table 1). The cells were grown in HIB at 28°C for 14 h and then incubated at 37°C for 4 h, as described in the supplemental methods. Fractions with 25-μl volumes from cultures grown to an OD₆₀₀ of 0.8 were evaluated by immunoblotting with LcrV-specific polyclonal rabbit antibody. GroEL was used as a cytoplasmic marker for fractionation. (C) Comparison of the LcrV amounts in the OMV fractions isolated from YPS1, YPS2, and YPS3 strains individually harboring pSMV12 or pSMV13 (Table 1). OMVs were isolated from bacterial cultures as described in the Materials and Methods. Five-microliter volumes of OMVs normalized according to the bacterial numbers were evaluated by immunoblotting with LcrV-specific polyclonal rabbit antibody. (D) Amounts of protein and relative lipid contents in OMVs purified from YPS1, YPS2, and YPS3 strains individually harboring pSMV13. All the values were normalized according to the total bacterial numbers (×10¹¹ CFU). (E) Comparison of Psn, LcrV, and F1 synthesis in the OMV fractions isolated from YPS1, YPS2, and YPS3 strains individually harboring pSMV13. (F) Whole-protein profiles of OMVs from YPS1, YPS2, and YPS3 strains individually harboring pSMV13 were examined by SDS-PAGE gels. The results are representative of three experiments. Statistical significance: ns, no significance; ****, P < 0.0001.

Fig 4

Total IgG titers in LcrV-, rF1-V-, or OMV-immunized mice and the survival of mice challenged by virulent Y. pestis. (A) Immunization scheme used for the mouse study. (B) LcrV, YPL (Y. pestis whole-cell lysate), and F1-specific total IgG titers. (C) Immunized and PBS (sham) groups of Swiss-Webster mice (10 mice per group, equal numbers of males and females) were subcutaneously challenged with 8 × 10⁵ CFU of Y. pestis KIM6+(pCD1Ap) (8 × 10⁴ LD₅₀). (D) Immunized and PBS (sham) groups of Swiss-Webster mice (10 mice per group, equal numbers of males and females) were intranasally challenged with a low dose (L: 5 × 10³ CFU, 50 LD₅₀) or a high dose (H: 5 × 10⁴ CFU, 500 LD₅₀) of Y. pestis KIM6+(pCD1Ap). Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 5

Antibody isotypes in immunized mouse sera collected at days 14 and 28 after prime and booster immunization. (A) Anti-LcrV IgG1, IgG2a, and IgG2b. (B) Anti-YPL IgG1, IgG2a, and IgG2b. (C) Anti-F1 IgG1, IgG2a, and IgG2b. The statistical significance among the groups at day 14 and day 28 was analyzed by two-way multivariate ANOVA with a Tukey post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.

Fig 6

Analysis of antigen-specific T cells obtained from lungs and associated cytokine responses. On day 42 after the initial immunization, lymphocytes were aseptically isolated from mice and stimulated in vitro with 20 μg/ml purified recombinant LcrV protein for 72 h to detect specific CD4⁺ and CD8⁺ T cells encoding IFN-γ, IL-2, IL-4, IL-17, and TNF-α. Sham mouse lung cells were considered controls. (A) CD4⁺ T cell numbers in lungs and CD4⁺ IFN-γ⁺, CD4⁺ IL-2⁺, CD4⁺ IL-4⁺, CD4⁺ IL-17⁺, and CD4⁺ TNF-α⁺ cell numbers. (B) CD8⁺ T cell numbers in lungs and CD8⁺ IFN-γ⁺, CD8⁺ IL-2⁺, CD8⁺ IL-4⁺, CD8⁺ IL-17⁺, and CD8⁺ TNF-α⁺ cell numbers. Each symbol represents a data point obtained from an individual mouse, with horizontal mean value bars ± SD. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 7

In vivo responses after Y. pestis pulmonary challenge. Sham-, LcrV-, or OMV-immunized Swiss-Webster mice (3 mice per group) were infected i.n. with 3 × 10³ CFU of Y. pestis KIM6+(pCD1Ap). The groups of immunized mice infected with PBS served as negative controls. On day 2 postchallenge, different tissues (lungs, livers, and spleens) and bronchoalveolar lavage fluid (BALF) were collected from the euthanized mice. (A) Bacterial burden was evaluated in the lungs, livers, and spleens. (B) CD4⁺CD44⁺ cell numbers in the lungs of mice with or without infection were analyzed. (C) Alveolar macrophages in the BALF of mice with or without infection. (D) Neutrophils in the BALF of mice with or without infection. (E) Alveolar macrophages in the lungs of mice with or without infection. (F) Neutrophils in the lungs of mice with or without infection. Statistical significance: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.

Fig 8

Comparison of cytokine and chemokine levels in the BALF from mice with and without pulmonary Y. pestis challenge. Sham-, LcrV-, or OMV-immunized Swiss-Webster mice (3 mice per group) were infected i.n. with 3 × 10³ CFU of Y. pestis KIM6+(pCD1Ap). The groups of immunized mice infected with PBS served as negative controls. On day 2 postchallenge, BALF from each euthanized mouse was collected at 48 h postinfection, filtered through a 0.22-μm syringe filter and checked for sterility before transfer to the BSL2 lab for analysis. A Bio-Plex Pro mouse cytokine assay kit (Bio-Plex) was used to detect the cytokines and chemokines, such as IL-1α, IL-1β, IL-6, IL-17, IFN-γ, G-CSF, KC, and MIP-1α, in the BALF collected from mice according to the manufacturer’s instructions. The statistical significance among the groups was analyzed by two-way multivariate ANOVA with a Tukey post hoc test. ****, P < 0.0001. Abbreviations: IFN, interferon; G-CSF, granulocyte colony-stimulating factor; KC, keratinocyte chemoattractant; MIP-1-α, macrophage inflammatory protein 1-alpha.