Induction of Th2/Th17 responses: immunoprotective mechanisms of a hepatitis B core antigen-based multi-epitope vaccine against Klebsiella pneumoniae infection

Klebsiella pneumoniae (Kp) is a clinically prevalent opportunistic pathogen, with escalating antimicrobial resistance posing substantial challenges. Current vaccine strategies are constrained by the complexity of serotype diversity and the weak immunogenicity of candidate antigens, hindering effective protection. The construction of a multiepitope vaccine against K. pneumoniae, designated HBc-KpEpi, based on hepatitis B core antigen (HBc) virus-like particles, aims to overcome the weak immunogenicity of epitope peptides and to validate its ability to induce specific immune responses and protective efficacy.

After the multi-epitope vaccine HBc-KpEpi was expressed and purified, its purity and self-assembly status were characterized by SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. Its safety was evaluated using cytotoxicity assay, hemolysis assay, and detection of serum biochemical indicators. Female BALB/c mice (6 to 8 weeks old, weighing 18 to 20 g) were randomly divided into the following groups: PBS control group, KpEpi group, and HBc-KpEpi group(n=10). Each group received intranasal immunization with PBS, KpEpi, or HBc-KpEpi (30 μg/mouse) on days 0, 14, and 21, respectively. In 7 d after the final immunization, the mice were challenged with a lethal dose (6×10⁶ CFU) or a sublethal dose (5×10⁵ CFU) of the clinical strain YBQ via intratracheal instillation. Detection indicators included survival rate and changes in body weight, bacterial colonization in lung tissue, serum inflammatory cytokines (IL-6, TNF-α, and IL-1β), lung histopathological changes, titers of serum specific antibodies (IgG, IgG1, IgG2a), and proportions of splenic CD4⁺ T cell subsets (Th1, Th2, Th17). Meanwhile, the ability of the vaccine to induce the differentiation and maturation of mouse bone marrowderived dendritic cells (BMDCs) was evaluated through in vitro experiments.

The HBc-KpEpi protein was successfully prepared with high purity and self-assembled into regular particles. The survival rates in the HBc-KpEpi group were significantly higher than those in the PBS group (P < 0.05). The bacterial colonization in lung tissue was significantly lower in the HBc-KpEpi group than that of the PBS and KpEpi groups (P < 0.01). The HBc-KpEpi group exhibited significantly lower IL-6 level than the PBS group (P < 0.0001), with the levels of TNF-α and IL-1β showing downward trends. Lung histopathology showed that the alveolar structure remained relatively intact with markedly reduced inflammatory cell infiltration in the HBc-KpEpi group. After immunization, high titers of specific IgG antibodies were induced in the HBc-KpEpi group, with IgG1 as predominant subtype. Flow cytometry detection showed that the proportions of Th2 and Th17 cells in CD4⁺ T cells were significantly higher in the HBc-KpEpi group than in the KpEpi group (P < 0.05). In vitro experiments indicated that HBc-KpEpi significantly promoted the expression of MHC-Ⅱ, CD40, CD80, and CD86 on the surface of BMDCs (P < 0.0001)

The HBc-KpEpi vaccine significantly enhances the immunogenicity of epitope peptides, inducing cellular immune responses dominated by Th2 and Th17 as well as high titers of specific antibodies, and effectively reduce bacterial load, alleviate inflammatory damage, and improve the survival rate of mice.