Streptococcus pyogenes (Strep A) is a Gram-positive bacterium responsible for >500 thousand deaths every year, and primarily affects those living in resource-limited settings (Antimicrobial Resistance, C, 2022). Strep A manifests along a spectrum from asymptomatic carriage, mild superficial infections (sore throat and skin sores) to severe and potentially fatal invasive disease. Furthermore, repeated exposure in susceptible individuals can lead to pathological autoimmune sequelae including rheumatic fever that may result in rheumatic heart disease RHD (Macleod et al., 2019). According to the World Health Organization (WHO), Strep A is one of the most neglected diseases relative to its burden worldwide (Palafox et al., 2017). Strep A is also a key driver of antibiotic prescriptions and resistance, responsible for >400 million antibiotic prescriptions every year (Dooling et al., 2014). Thus, developing a vaccine is a global health priority, and an effective vaccine would reduce the burden of superficial infections, invasive disease and pathological autoimmune sequelae.

There has been increasing effort over the last decade to develop a Strep A vaccine via different approaches, with promising results obtained in preclinical studies (Dale et al., 2011; van Sorge et al., 2014; Dale and Walker, 2020; Sekuloski et al., 2018). GSK Global Health Vaccines R&D (GVGH) is developing a Strep A vaccine that is fully aligned with the WHO preferred product characteristics and technology roadmap. The current candidate is an M protein-free multicomponent vaccine targeting surface and secreted virulence factors identified by reverse vaccinology (Bensi et al., 2012). It consists of three recombinant protein antigens - Streptolysin O (SLO), S. pyogenes cell envelope protease (SpyCEP) and S. pyogenes adhesion and division protein (SpyAD), as well as one glycoconjugate, the Group A carbohydrate conjugated to carrier protein CRM197 (Kabanova et al., 2010). Together these antigens have been shown to elicit protection in animal models and are highly conserved in clinical Strep A isolates (Bensi et al., 2012; Cunningham, 2000; Lancefield, 1933; Braun, 1983; Davies et al., 2019; Lacey et al., 2023), meaning that the coverage of this vaccine should be virtually 100%.

To support vaccine development and better understand the immune response after natural Strep A exposure, multiple serological-based studies have been performed. These have confirmed that antibodies against key vaccine antigen targets are usually present in convalescent individuals (Keeley et al., 2023; Salie et al., 2023; Hysmith et al., 2017; Whitcombe et al., 2022a; Whitcombe et al., 2022b). However, determining the presence of binding antibodies alone is insufficient to infer potential functional activity. Antibody function is critical to establish for vaccine antigens, especially during early clinical assessments of the new vaccine. SLO is responsible for eukaryotic cell disruption and immune evasion (Chiarot et al., 2013; Feil et al., 2014; Uchiyama et al., 2015) and has >99% carriage in clinical Strep A collections with <2% sequence divergence (Davies et al., 2019). For these reasons it is included in a number of combination vaccine candidates in addition to the GVGH Strep A vaccine (Bensi et al., 2012; Rivera-Hernandez et al., 2016; Bi et al., 2019). Vaccine induced antibodies that can bind and neutralise SLO activity represent an important target to prevent the cellular damage caused by Strep A infections. However, standardized assays that can measure functional anti-SLO antibodies in large vaccine trials are lacking.

Hemolysis inhibition assays are a well-established method for quantifying the in vitro ability of a compound to inhibit red blood cell lysis in presence of active toxin, and have been used extensively for multiple pathogens to determine functional activity of vaccine induced sera (Bensi et al., 2012; Paton et al., 1983). Here we describe the optimization and characterization of an in vitro hemolysis inhibition method for testing functional anti-SLO antibodies in human sera (Bensi et al., 2012). Precision, specificity and linearity were determined and the correlation between anti-SLO antibody titers and functionality was explored, as well as the impact of critical reagents such as the source of red blood cells.