New research addresses the need for Shigellosis vaccine development innovations
Shigellosis remains a pressing global health challenge, particularly in low- and middle-income countries where it disproportionately affects children under five. This bacterial infection, caused by Shigella flexneri and other species, is a leading cause of diarrheal mortality and is further complicated by the rise of antibiotic-resistant strains.
As vaccine development advances, assessing the functionality of vaccine-induced antibodies through robust in vitro methods like the serum bactericidal activity (SBA) assay becomes increasingly vital.
In a recent study, researchers from the GSK Vaccines Institute for Global Health optimized and characterized a high-throughput luminescence-based SBA (L-SBA) assay to evaluate functional antibody responses against seven prevalent S. flexneri serotypes. This research not only demonstrated the utility of the L-SBA but also validated its performance using Pel-Freez baby rabbit complement.
Developing an expanded, high-throughput S. flexneri L-SBA assay
The researchers used L-SBA to efficiently quantify functional antibody responses in human serum samples against a diverse set of S. flexneri serotypes, including 1b, 3a, 4a, 5b, 6, X, and Y. Key steps included:
- Complement selection: Baby rabbit complement was sourced from multiple suppliers, including Pel-Freez, to assess its impact on assay performance. Optimal concentrations varied by strain, ranging from 7% for S. flexneri 4a and 5b to 30% for S. flexneri 1b.
- Serum preparation: Sera were pooled from participants in clinical trials conducted in endemic regions and heat-inactivated to remove endogenous complement activity. Dilutions of the sera were prepared in phosphate-buffered saline (PBS) or Luria Bertani medium (LB), depending on the target serotype.
- Assay setup:
- Serial dilutions of the serum samples were incubated with bacterial cultures and complement under standardized conditions.
- A luminescence-based readout was used to measure bacterial viability after 3 hours, with IC50 values calculated to measure the serum dilution that achieved 50% bacterial killing.
- Validation and optimization:
- Repeatability and intermediate precision were assessed through independent replicates by two operators over multiple days.
- Linearity was evaluated by comparing theoretical and experimental IC50 values across serial dilutions.
- Specificity was confirmed using homologous and heterologous antigens, demonstrating a strong depletion of SBA titers (up to 60%) with homologous competitors while showing minimal cross-reactivity with heterologous controls.
Findings demonstrate reproducibility and specificity across serotypes
Reproducibility across complement sources
The study confirmed consistent performance of the L-SBA assay when using baby rabbit complement from different suppliers, including Pel-Freez. For example, comparisons between Pel-Freez and another complement source showed no significant differences in IC50 values for S. flexneri 1b or fold-change assessments for S. flexneri 3a. These findings highlight the reliability of Pel-Freez baby rabbit complement for Shigella immunological assays.
Robust precision and repeatability
The assay demonstrated high precision, with coefficients of variation for intermediate precision (CV% IP) below 7% and repeatability (CV% R) below 3.9% across all tested serotypes. Variance analysis confirmed that neither operator nor day significantly impacted results, establishing the assay’s reproducibility under different conditions.
Specificity for functional antibody responses
Strong specificity was observed, with homologous competitors reducing SBA titers by more than 60% across all serotypes, while heterologous antigens (e.g., S. sonnei or Salmonella typhimurium) caused minimal reductions (≤30%). These results confirm the assay’s ability to specifically measure functional antibody responses to S. flexneri serotypes.
Expanded strain coverage
The optimization of L-SBA conditions enabled the inclusion of five additional S. flexneri serotypes (4a, 5b, 6, X, and Y) alongside the previously characterized 1b and 3a. This expanded coverage provides a comprehensive tool for evaluating vaccine-induced immunity across diverse S. flexneri strains, addressing a critical need in Shigella vaccine development and public health surveillance.
Overall, these findings illustrate the effectiveness of the L-SBA assay in delivering consistent and precise measurements of functional antibody responses across diverse serotypes.
By incorporating high-quality complement sources into an efficient, cost-effective assay, the study showcases how the reliability and adaptability of L-SBA align with the rigorous demands of preclinical and clinical Shigella research.
Shaping the future of Shigella research and vaccine development
The expanded high-throughput L-SBA assay described in this research demonstrated its value as a reliable and adaptable method for assessing functional antibody response to a broad range of S. flexneri serotypes.
At Pel-Freez, we are proud to support groundbreaking research with high-quality biological reagents, such as the baby rabbit complement used in this study. By empowering vaccine researchers with reliable and scalable solutions, we remain committed to advancing the global fight against preventable diseases and improving health outcomes worldwide.
Learn more about this research: High-Throughput Luminescence-Based Serum Bactericidal Assay Optimization and Characterization to Assess Human Sera Functionality Against Multiple Shigella flexneri Serotypes.
Disclaimer
The views and opinions presented in this blog article should not be considered to represent an endorsement or guidance on behalf of GSK or the study authors.
