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

Abstract

17Despite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is 18 still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of 19 disease coupled with the rise of antimicrobial resistance renders the introduction of an effective 20 vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not 21 been established yet, the demonstration of bactericidal activity of antibodies induced upon 22 vaccination may provide one means of functionality of antibodies induced on protecting against 23 Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-24 induced antibodies is the Serum Bactericidal Assay (SBA). 25Here we present the development and intra-laboratory characterisation of a high-throughput 26 luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving 27 bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high 28 specificity of the assay against homologous strain without any heterologous aspecificity detected 29 against species-related and not species-related strains. We assessed linearity, repeatability and 30 reproducibility of L-SBA on human sera. 31This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The 32 method has the potential of being applicable with similar performances to determine bactericidal 33 activity of any non-clinical and clinical sera that rely on complement mediated killing. 34 35 IMPORTANCE 36Shigella is an important cause of diarrhoea worldwide and antimicrobial resistance is on rise, thus 37 efforts by several groups are ongoing to produce a safe and effective vaccine against shigellosis. 38Although a clear immune correlate of protection has not been established, demonstration of 39 bactericidal capacity of sera from patients immunised with Shigella vaccines may provide one means 40 characterisation L-SBA on human sera of protecting against shigellosis. We have developed and fully characterised a novel high-throughput 41 L-SBA method for evaluation of functionality of antibodies raised against S. sonnei in human sera. 42This work will allow the clinical testing of human sera raised against GMMA-based and potentially 43 all vaccines producing antibodies than can work via complement mediated manner. 44 45 46 47 48 Diarrheal diseases, such as shigelloses and salmonelloses, are the second leading cause of death 49 worldwide, resulting in millions of deaths per year, mostly in developing countries (1). Shigella is 50 major cause of sustained endemic bacterial diarrhoea, especially in low and middle-income countries 51 where accessibility to clean water is restricted. Although the improvement of hygienic conditions in 52 the last decade has dramatically reduced the burden of the disease, Shigella is still responsible for 53 more than 200,000 deaths, with a third of them being in young children (1). On top of deaths...