Invasive African nontyphoidal Salmonella requires high levels of complement for cell-free antibody-dependent killing

Abstract

Nontyphoidal isolates of Salmonella (NTS), particularly Salmonella Typhimurium, are a major cause of invasive bacteremia in Africa. Despite this, no vaccine against NTS is currently available for use in humans. If a NTS vaccine is to be developed in a timely manner, there is a need to develop assays to assess its in vivo efficacy. Assessment of potential efficacy of candidate vaccines in preclinical models is important for proof-of-concept and reduces attrition of vaccines in clinical trials. Serum bactericidal assays (SBA) are often used to assess the functional activity of vaccine-induced antibody responses targeted against Gram-negative bacteria with results given as the maximum dilution of serum that can effect bacterial killing. Previously we have found evidence for a protective role for antibody-induced complement-mediated killing of NTS in African children using an undiluted whole serum SBA. However, endogenous complement in diluted human sera is limiting and insufficient to effect bactericidal activity against S. Typhimurium beyond two two-fold dilutions. In the current study, we examined the requirements for SBA against NTS using baby rabbit serum (BRS) as an exogenous source of complement. We found that the amount of complement required for antibody-mediated bactericidal activity is much higher for the invasive African S. Typhimurium isolate D23580, compared with the laboratory S. Typhimurium LT2 and Salmonella Paratyphi A CVD1901. While 20% BRS was sufficient to kill LT2 and CVD1901, 75% BRS was needed to kill D23580. Our findings demonstrate that one concentration of exogenous complement is not suitable for SBA against all Salmonella isolates. To develop SBA to assess the in vivo efficacy of Salmonella vaccines, it is necessary to optimize the assay for the Salmonella isolates against which the vaccine is targeted.

The Role of the st313-td Gene in Virulence of Salmonella Typhimurium ST313

“…This indicates that st313-td influences the uptake and/or early survival in macrophages but only in activated cells. Notably, in a previous study it was demonstrated that S. Typhimurium D23580, assigned to ST313 and carrying st313-td requires high levels of complement for antibody-mediated bactericidal activity compared with S. Typhimurium LT2 [19]. Thus, st313-td might play a role in virulence by either decreasing the binding of the bacteria to antibodies causing less uptake in macrophages or by diminishing the susceptibility to complement mediated lysis.…”

“…Gallinarum [10]. For these reasons, S. Typhimurium ST313 D23580 has been extensively used in studies on host pathogen interaction to gain more knowledge about NTS infection and to elucidate the reason for the apparent increased pathogenicity of this clone [15]–[19]. Some of these studies suggest that S. Typhimurium ST313 might have adapted to occupy an ecological and immunological niche provided by HIV, malaria and malnutrition in Africa [11].…”

Serum Bactericidal Assays To Evaluate Typhoidal and Nontyphoidal Salmonella Vaccines

“…Other groups have successfully used 3-to 4-week-old baby rabbit complement (BRC) in SBA assays to evaluate S. Typhi vaccines (23,24). Similarly, BRC was employed in an S. Typhimurium SBA assay, although very high concentrations (75%) of complement were required to mediate killing by the antibody (25). While these assays have allowed the demonstration of SBA responses against S.…”

“…Typhi, S. Paratyphi A, and S. Typhimurium, no assay has been reported to determine SBA responses against S. Enteritidis (14,(23)(24)(25). Furthermore, none of these assays has been standardized to measure vaccine-induced endpoint SBA responses against Salmonella serovars.…”

Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

“…Microheterogeneities in the polysaccharide compositions of LPSs have been described even within the same serotype (36), and it is possible that such variations affect antibody binding and function, especially since the exact epitope to which antibody binds may alter bactericidal activity (5,8). In this context, it may be relevant that recent experiments have shown that antibody-mediated killing of the S. Typhimurium isolate used to assess bactericidal activity in the Malawi study required unusually large amounts of complement (37). This characteristic may also contribute to the differences from our results.…”