Application of an O-Linked Glycosylation System in Yersinia enterocolitica Serotype O:9 to Generate a New Candidate Vaccine against Brucella abortus

Abstract

Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet available, and live attenuated vaccines commonly used in animals can cause human infection. N- and O-linked glycosylation systems have been successfully developed and exploited for the production of successful bioconjugate vaccines. Here, we applied an O-linked glycosylation system to a low-pathogenicity bacterium, Yersinia enterocolitica serotype O:9 (Y. enterocolitica O:9), which has repeating units of O-antigen polysaccharide (OPS) identical to that of Brucella abortus (B. abortus), to develop a bioconjugate vaccine against Brucella. The glycoprotein we produced was recognized by both anti-B. abortus and anti-Y. enterocolitica O:9 monoclonal antibodies. Three doses of bioconjugate vaccine-elicited B. abortus OPS-specific serum IgG in mice, significantly reducing bacterial loads in the spleen following infection with the B. abortus hypovirulent smooth strain A19. This candidate vaccine mitigated B. abortus infection and prevented severe tissue damage, thereby protecting against lethal challenge with A19. Overall, the results indicated that the bioconjugate vaccine elicited a strong immune response and provided significant protection against brucellosis. The described vaccine preparation strategy is safe and avoids large-scale culture of the highly pathogenic B. abortus.

Keywords: Brucella abortus; O-linked glycosylation; Yersinia enterocolitica serotype O:9; bioconjugate vaccine; brucellosis.

Figure 1

Analysis of glycosylated recombinant cholera toxin B subunit (rCTB) and CTB-OPS_Ba expressed in YeO9_52212. (A) YeO9_52212 was transformed with pET-CTB4573H or pET-pglL-CTB4573H and then induced with IPTG. A wild type strain was treated in the same way (negative control). Coomassie Blue staining (left) and western blotting (right) were performed. (B) The CTB-OPS_Ba (C-OPS_Ba) glycoprotein was purified from strain YeO9_52212 co-expressing PglL and rCTB. Samples were separated by 12% SDS-PAGE and analyzed by Coomassie Blue staining or western blotting using anti-His, anti-B. abortus (anti-Ba) or anti-Y. enterocolitica O:9 (anti-Ye) antibodies. (C) Coomassie Blue staining after native gel electrophoresis of C-OPS_Ba.

Figure 2

IgG responses against B. abortus A19 lipopolysaccharide (LPS). (A) IgG titers against A19 LPS were measured in sera from PBS-, OPS_Ba-, OPS_Ba+Al-, C-OPS_Ba-, and C-OPS_Ba+Al-vaccinated mice. (B) IgG subclass titers (IgG1, IgG2a, IgG2b, and IgG3) against A19 LPS were measured in sera from PBS-, OPS_Ba-, and C-OPS_Ba-vaccinated mice. Each value represents the mean ± standard deviation of log₂-transformed titers in the sera of individual mice (shown as data points on the graphs) from each group (n = 10). The unpaired t-test was used to evaluate differences between IgG titers (**, p <0.01; ****, p <0.0001; ns, no statistically significant difference).

Figure 3

Immune responses of mice following non-lethal B. abortus A19 infection. Immunized mice were infected intraperitoneally with 1.03 × 10⁷ CFU of A19 on the 14th day following the third immunization. As a control, another group of naive mice was injected intraperitoneally with normal saline. (A) After infection, the sera of mice in each group were collected on the 1st, 3rd, 5th, and 7th day and the TNF-α levels were measured. The unpaired t-test was used to evaluate differences between TNF-α levels at different time points. Each value represents the mean ± standard deviation (n = 3). (B) On the 7th day post-infection, mouse spleens were collected and weighed and the bacterial loads were measured. Each value represents the mean ± standard deviation of spleen weight or log₁₀-transformed bacterial loads (CFU/spleen) of individual mice (shown as data points on the graphs) from each group (n = 5 per group). The unpaired t-test was used to evaluate differences between spleen weights or bacterial loads (***, p <0.001; ****, p <0.0001; ns, no statistically significant difference). (C) The livers and spleens of infected mice and normal mice (Control) were fixed with 4% paraformaldehyde, paraffin sectioned, and then stained with hematoxylin and eosin. The yellow boxes in the top panels represent the field of view in the corresponding figures below, which were magnified four times. Green arrows indicate multinucleated giant cells and blue arrows indicate hyperplastic nodules.

Figure 4

Survival of BALB/c mice after challenge with a lethal dose of B. abortus A19. Mice were challenged 2 weeks after final immunization by intraperitoneal injection with different doses of A19 and survival was monitored. (A) Schematic diagram of the challenge experiment. (B) Mice were immunized with PBS, OPS_Ba, OPS_Ba+Al, C-OPS_Ba or C-OPS_Ba+Al and challenged with ~1.54 × 10⁸ CFU/mouse (3 × LD₅₀) of A19 (n = 10). (C) Mice were immunized with C-OPS_Ba, C-OPS_Ba+Al or PBS then challenged with approximately 2.51 × 10⁸ CFU/mouse (5 × LD₅₀) of A19 (n = 10).