A High-throughput <em>Shigella</em>-specific Bactericidal Assay

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

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Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera against Shigella

10.1101/2020.04.03.024950 ◽

2020 ◽

Author(s):

O. Rossi ◽

E. Molesti ◽

A. Saul ◽

C. Giannelli ◽

F. Micoli ◽

...

Keyword(s):

Antimicrobial Resistance ◽

High Throughput ◽

Bactericidal Activity ◽

High Specificity ◽

Effective Vaccine ◽

Correlate Of Protection ◽

Bactericidal Assay ◽

Immune Correlate ◽

Human Sera ◽

Serum Bactericidal Assay

ABSTRACTDespite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not been established yet, the demonstration of bactericidal activity of antibodies induced upon vaccination may provide one means of functionality of antibodies induced on protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA).Here we present the development and intra-laboratory characterisation of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against homologous strain without any heterologous aspecificity detected against species-related and not species-related strains. We assessed linearity, repeatability and reproducibility of L-SBA on human sera.This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine bactericidal activity of any non-clinical and clinical sera that rely on complement mediated killing.IMPORTANCEShigella is an important cause of diarrhoea worldwide and antimicrobial resistance is on rise, thus efforts by several groups are ongoing to produce a safe and effective vaccine against shigellosis. Although a clear immune correlate of protection has not been established, demonstration of bactericidal capacity of sera from patients immunised with Shigella vaccines may provide one means of protecting against shigellosis. We have developed and fully characterised a novel high-throughput L-SBA method for evaluation of functionality of antibodies raised against S. sonnei in human sera. This work will allow the clinical testing of human sera raised against GMMA-based and potentially all vaccines producing antibodies than can work via complement mediated manner.

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Development of an Automated, High-Throughput Bactericidal Assay That Measures Cellular Respiration as a Survival Readout for Neisseria meningitidis

Clinical and Vaccine Immunology ◽

10.1128/cvi.05028-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1252-1260 ◽

Cited By ~ 21

Author(s):

Puiying A. Mak ◽

George F. Santos ◽

Kelly-Anne Masterman ◽

Jeff Janes ◽

Bill Wacknov ◽

...

Keyword(s):

Data Analysis ◽

Neisseria Meningitidis ◽

High Throughput ◽

Agar Plate ◽

Cellular Respiration ◽

Strongly Correlated ◽

Kinetic Assay ◽

Bacterial Colony ◽

Content Type ◽

Bactericidal Assay

ABSTRACTComplement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity againstNeisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.

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Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)

BioTech ◽

10.3390/biotech10030019 ◽

2021 ◽

Vol 10 (3) ◽

pp. 19

Author(s):

Maria Grazia Aruta ◽

Martina Carducci ◽

Francesca Micoli ◽

Francesca Necchi ◽

Omar Rossi

Keyword(s):

High Throughput ◽

Bactericidal Activity ◽

High Performance ◽

Traditional Methods ◽

Bactericidal Assay ◽

Colony Forming Units ◽

Serum Bactericidal Assay ◽

Live Bacteria

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens.

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