Development and Optimization of a Luminescence-Based Assay to Evaluate Serum Bactericidal Activity Against Neisseria gonorrhoeae

Neisseria gonorrhoeae (Ng) is a human-adapted pathogen and the etiological agent of gonorrhoea, a sexually transmitted infection (STI) that affects millions of people worldwide each year. Over the past two decades, the global incidence of Ng infection has risen, driven by its ability to acquire resistance to available antibiotics. As a result, developing new therapeutic strategies and effective vaccines has become a critical global public health challenge. In this study, we developed a luminescence-based serum bactericidal assay (L-SBA) to evaluate complement-mediated bactericidal activity against several gonococcal strains. Bactericidal activity was measured by quantifying adenosine triphosphate (ATP) levels, which correlate with bacterial viability. The first phase focused on optimizing key parameters for the assay, such as bacterial growth conditions and bacterial concentration. In addition, the incubation time and appropriate complement source were selected to avoid complement toxicity and non-specific killing. After optimizing these conditions, the bactericidal activity of pre- and post-immunization sera from mouse and rabbit models was assessed, alongside monoclonal antibodies targeting specific Ng antigens. The data obtained confirmed the feasibility of the L-SBA, showing significant differences between pre- and post-immunization sera and variation among different immunization groups. Additionally, monoclonal antibodies exhibited bactericidal activity, consistent with previous findings. These promising results mark an important step in the development of the L-SBA for Neisseria gonorrhoeae. However, further optimization and a thorough process of assay qualification and validation will be needed before the test can be reliably incorporated into vaccine development pipelines.