Vaccination is considered the most effective means to fight against the multidrug-resistant strains of Klebsiella pneumoniae. In recent years, a potential protein glycan coupling technology has been extensively used in the production of bioconjugated vaccines. Here, a series of glycoengineering strains derived from K. pneumoniae ATCC 25955 were designed for protein glycan coupling technology. The capsule polysaccharide biosynthesis gene cluster and the O-antigen ligase gene waaL were deleted via the CRISPR/Cas9 system to further weaken the virulence of host stains and block the unwanted endogenous glycan synthesis. Particularly, the SpyCatcher protein in the efficient protein covalent ligation system (SpyTag/SpyCatcher) was selected as the carrier protein to load the bacterial antigenic polysaccharides (O1 serotype), which could covalently bind to SpyTag-functionalized nanoparticles AP205 to form nanovaccines. Furthermore, two genes (wbbY and wbbZ) located in the O-antigen biosynthesis gene cluster were knocked out to change the O1 serotype of the engineered strain into the O2 serotype. Both KPO1-SC and KPO2-SC glycoproteins were successfully obtained as expected using our glycoengineering strains. Our work provides new insights into the design of nontraditional bacterial chassis for bioconjugate nanovaccines against infectious diseases.

CpG is a widely used adjuvant that enhances the cellular immune response by entering antigen-presenting cells and binding with receptors. The traditional physical mixing of the antigen and CpG adjuvant results in a low adjuvant utilization rate. Considering the efficient delivery capacity of nanovaccines, we developed an attractive strategy to covalently load CpG onto the nanovaccine, which realized the co-delivery of both CpG and the antigen. Briefly, the azide-modified CpG was conjugated to a bioconjugate nanovaccine (NP-OPS) against Shigella flexneri through a simple two-step reaction. After characterization of the novel vaccine (NP-OPS-CpG), a series of in vitro and in vivo experiments were performed, including in vivo imaging, lymph node sectioning, and dendritic cell stimulation, and the results showed that more CpG reached the lymph nodes after covalent coupling. Subsequent flow cytometry analysis of lymph nodes from immunized mice showed that the cellular immune response was greatly promoted by the nanovaccine coupled with CpG. Moreover, by analyzing the antibody subtypes of immunized mice, NP-OPS-CpG was found to further promote a Th1-biased immune response. Thus, we developed an attractive method to load CpG on a nanovaccine that is simple, convenient, and is especially suitable for immune enhancement of vaccines against intracellular bacteria.

African swine fever (ASF) is an acute, febrile, and lethal pig disease induced by the African swine fever virus (ASFV). In the absence of an effective vaccine, early diagnosis is essential for the prevention and control of ASF disease. The p54 protein is important for ASFV diagnosis and vaccine design. In this study, ASFV p54 protein was constructed, expressed, purified, and used to generate three mAbs, namely 9A3, 5H2, and 2G6. Epitope mapping was performed using alanine mutants; the minimal linear epitope recognized by 9A3 and 5H2 was ⁵⁶KKKAAAI⁶², and the minimal linear epitope recognized by 2G6 was ¹⁰⁸TNRPATN¹¹⁴. Of these, ⁵⁶KKKAAAI⁶² was identified as a new linear epitope for the first time. The epitopes were highly conserved in at least genotypes I and II. Alanine-scanning mutagenesis further revealed that residues 56K, 57K, 60A, 61A, 62L, 108T, 110R, 111P, 113T, and 114N were the core sites involved in antibody recognition. Overall, the mAbs and epitopes of the p54 protein identified in this study provide theoretical support for the development of ASFV vaccines based on the B cell epitope, the development of ASFV therapeutic antibody drugs, and the development of ASFV diagnostic tools.