Abstract
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.
Highlights
Diarrheal diseases, such as shigelloses and salmonellosis, are the second leading cause of death worldwide, resulting in millions of deaths per year, mostly in developing countries [1]
In order to determine the possibility of assessing the serum bactericidal activity of human sera against S. sonnei by L-Serum Bactericidal Assay (SBA), NVGH2863, an anti-S. sonnei IgG human standard serum already in use to assess the quantity of human antibodies raised upon vaccination with S. sonnei Generalized Modules for Membrane Antigens (GMMA) in clinical trials [17,18] has been used to setup the assay conditions with human sera and characterize the assay prior moving on with testing the functionality of the clinical samples
Initial experiments were conducted to test the behavior in luminescence-based SBA (L-SBA) of NVGH2863 under experimental conditions already established with pre-clinical sera (20% exogenous baby rabbit complement and S. sonnei with stabilized LPS expression in vitro as target bacteria)
Summary
Diarrheal diseases, such as shigelloses and salmonellosis, are the second leading cause of death worldwide, resulting in millions of deaths per year, mostly in developing countries [1]. On top of the deaths in endemic countries, enteric diseases are causing diarrhea among travelers and military personnel in developed countries, further increasing the burden and the economic and social impact. Among the different approaches used to produce Shigella vaccines, many of the candidate vaccines target the serotype-specific O-Antigen (OAg) part of the lipopolysaccharide (LPS), as OAg has been identified as a key antigen recognized by the immune system after natural infection [3]. Multiple immune mechanisms may provide protection against Shigella and are not yet fully elucidated, it is well established that antibodies directed to OAg can fix complement and kill target bacteria in a serotype-specific manner [3,4]. As LPS antibody production can confer protection from homologous serotypes, a multivalent Shigella vaccine is necessary to induce antibodies to LPS OAg from multiple serotypes in order to confer broad protection
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