Setup and Characterization of a High-Throughput Luminescence-Based Serum Bactericidal Assay (L-SBA) to Determine Functionality of Human Sera against Shigella flexneri

Abstract

Shigellosis represents a major public health problem worldwide. The morbidity of the disease, especially in children in developing countries, together with the increase of antimicrobial resistance make a vaccine against Shigella an urgent medical need. Several vaccines under development are targeting Shigella lipopolysaccharide (LPS), whose extreme diversity renders necessary the development of multivalent vaccines. Immunity against Shigella LPS can elicit antibodies capable of killing bacteria in a serotype-specific manner. Therefore, although a correlation of protection against shigellosis has not been established, demonstration of vaccine-elicited antibody bactericidal activity may provide one means of vaccine protection against Shigella. To facilitate Shigella vaccine development, we have set up a high-throughput serum bactericidal assay based on luminescence readout (L-SBA), which has been already used to determine the functionality of antibodies against S. sonnei in multiple clinical trials. Here we present the setup and intra-laboratory characterization of L-SBA against three epidemiologically relevant Shigella flexneri serotypes using human sera. We assessed the linearity, repeatability and reproducibility of the method, demonstrating high assay specificity to detect the activity of antibodies against each homologous strain without any heterologous aspecificity against species-related and non-species-related strains; this assay is ready to be used to determine bactericidal activity of clinical sera raised by multivalent vaccines and in sero-epidemiological studies.

Serum susceptibility of Escherichia coli and its association with patient clinical outcomes

“…Luminescence-based strategies to measure bacterial killing (including complement-mediated killing) have been published, though this approach is impractical for studying large numbers of bacterial strains given the requirement for introducing a luciferase gene into the bacterial strains of interest [ 44 – 46 ]. Prior work has also increased the throughput of SBAs through automated colony counting [ 47 , 48 ], or by detecting ATP release from dead/dying bacteria [ 49 – 53 ]. Here we demonstrate a technique that avoids the need for any plating and colony counting yet retains the ability to count individual viable bacteria.…”

Current challenges and improvements in assessing the immunogenicity of bacterial vaccines

“…The Luminescence-SBA (L-SBA) applies to the same principles of the C-SBA but evaluates the antibody bactericidal activity by quantifying the ATP level as a correlate of bacterial survival ( Figure 2B ) ( Necchi et al, 2017 ). In L-SBA the level of luminescence detected is directly proportional to the number of bacteria present in the assay wells, which is inversely proportional to the level of functional antibodies present in the serum ( Mancini et al, 2022 ). Therefore, serum bactericidal titer obtained by the luminescence readout method strongly correlates with the data obtained by the conventional agar plate-based assay ( Necchi et al, 2017 ).…”

Functional assays to evaluate antibody-mediated responses against Shigella: a review

“…High SBA titres have been associated with some degree of protection from moderate to severe disease in several studies, suggesting that SBA may be a functional correlate of protection recapitulating a positive role of antibodies during the pathogenesis of Shigella ( Shimanovich et al., 2017 ; Clarkson et al., 2020 ). Bactericidal activity is mainly attributed to anti-LPS/OAg antibodies, probably since LPS provides abundant and repetitive surface antigens that favour antibody hexamerization and thus complement deposition ( Rossi et al., 2020 ; Mancini et al., 2022 ).…”

“…These readouts are usually derived from reactions with metabolic reagents by the live bacteria. Necchi et al developed a luminescence-based SBA (L-SBA) protocol which detects adenosine triphosphate (ATP) production by live bacteria (Necchi et al, 2018) and the assay has been optimised and characterised (Rossi et al, 2020;Mancini et al, 2022) to analyse clinical samples from multiple studies (Frenck et al, 2021;Micoli et al, 2021;Kapulu et al, 2022). Resazurin (also known as alamarBlue) offers an alternative colorimetric and fluorometric endpoint (Romero-Steiner et al, 2004).…”