Surface Display of Cholera Toxin B Subunit Recombinant Escherichia coli Ghosts Further Enhances Resistance to Chlamydia abortus Infection in Mice

Abstract

Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge.

Keywords: CTB; Chlamydia abortus; bacterial ghost; mucosal immunity; vaccine.

Figure 1

Construction of surface display plasmid and identification of fusion protein. (A) Schematic diagram of recombinant plasmids pA-lpp’-MIP and pA-lpp’-MIP-CTB. Fusion protein expression was detected by Western blotting using monoclonal antibodies against MIP (B) and monoclonal antibodies against CTB (C). M: protein marker.

Figure 2

Analysis of display of MIP fusion proteins on bacterial surfaces. (A) MIP antibody was used to incubate bacteria, followed by Alexa Fluor 594-labeled anti-mouse IgG antibody (red) for indirect immunofluorescence analysis. Bacterial genomes were labeled with DAPI (blue). (B) Proteinase K-treated (+PK) or untreated C43-lpp’-MIP and C43-lpp’-MIP-CTB were incubated with MIP antibodies, followed by flow cytometry detection with Alexa Fluor 594-labeled anti-mouse IgG antibodies. Final data are shown as MFI. Results are expressed as mean ± standard deviation (SD). ** p < 0.01; ns, no significance.

Figure 3

Effects of rECGs on BMDCs’ maturation and activation. (A,B) After BMDCs were stimulated by rECGs in vitro, the CD80 and MHC II expression frequencies of CD11c⁺ cells were detected by flow cytometry. The final data are shown as the MFI. Content of cytokines IL-6 (C), IL-12p70 (D), and TNF-α (E) in BMDC culture supernatants. Results are expressed as means ± SD. * p < 0.5, ** p < 0.01, **** p < 0.0001; ns, no significance.

Figure 4

Humoral immune response induced by rECGs in mice. (A) Dynamics of MIP-specific IgG in serum samples collected 0, 14, 28, 42, and 56 days after the first immunization. Titers of MIP-specific IgG2a (B) and IgG1 (C) in serum samples 42 days after the first immunization. MIP-specific IgA titers in serum samples (D) and vaginal lavage fluid (E) 42 days after the first immunization. Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significance.

Figure 5

Cellular immune response induced by rECGs in mice. (A) Specific splenic lymphocyte proliferation response in immunized mice, with concanavalin A (ConA)-treated group serving as a positive control. Content of IFN-γ (B), IL-12p70 (C), TNF-α (D), IL-4 (E), and IL-10 (F) in lymphocyte culture supernatants. Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significance.

Figure 6

Protective effect of rECGs on mice infected with C. abortus. (A) Vaginal swabs were collected every 3 d post-challenge to enumerate the C. abortus-recoverable IFUs. The mean recoverable IFUs are expressed as log₁₀ IFU/mL ± SD. (B) Histopathological analysis of uterine tissue sections. Lysis and necrosis of the lamina propria are indicated with black arrows, uterine gland cavity occlusion is indicated with red arrows, and lamina propria edema is indicated with yellow arrows. (C) Quantitative isolation of C. abortus in uterine homogenates of mice 7 days after challenge. * p < 0.05, *** p < 0.001.