Supplementary file <b>Immunogenic Potential of 65.5 kDa Pili Protein of </b><b><i>Klebsiella pneumoniae</i></b><b>: Evaluation of IgG, IgG1, and IgG2a Levels in BALB/c Mice</b>

<i>Klebsiella pneumoniae</i> is a significant etiological agent of both community-acquired and nosocomial infections, characterized by its pronounced vulnerability to drug resistance. Preventive measures, such as vaccine research, are essential since no effective and viable vaccine currently exists. Pili, a critical virulence component in <i>Klebsiella pneumoniae</i>, promote bacterial adherence and have very low variability relative to other virulence factors, rendering them a viable target for vaccination. The 65.5 kDa pili protein has been recognized as a possible antigen owing to its significant antigenicity. This study sought to assess the immunogenic response by quantifying IgG, IgG1, and IgG2a levels in BALB/c mice following the delivery of the 65.5 kDa pili protein. A total of 24 mice were categorized into three groups: PBS, adjuvant, and pili. Serum concentrations of IgG, IgG1, and IgG2a were assessed via ELISA. Statistical analysis was conducted utilizing the ANOVA test with a 95% confidence interval. The data indicate that vaccination with the 65.5 kDa pili protein elicits a robust humoral immune response characterized by increased IgG and IgG1 production, suggesting a Th2-biased response, while showing no significant impact on IgG2a levels, which are typically associated with Th1-mediated immunity. In summary, the 65.5 kDa pili protein shows promise as a vaccine candidate for <i>Klebsiella pneumoniae</i> by inducing a strong IgG and IgG1-mediated immunological response in BALB/c mice. Future studies should use flow cytometry to assess immune cell activation, cytokine profiling for Th1/Th2 responses, and antigen-specific assays to explore T-cell activation. Research on dose optimization, adjuvant screening, immunity duration, and challenge studies with virulent <i>Klebsiella pneumoniae</i> is crucial. Proteomic analysis and epitope mapping can aid in refining vaccine design.