Serum Bactericidal Assays To Evaluate Typhoidal and Nontyphoidal Salmonella Vaccines

Abstract

Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.

Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines

“…In the absence of HL-60 cells, there was no detectable bacterial killing. Thus, at a concentration of 12.5%, BRC was unable to mediate killing, as shown previously (16). When HL-60 cells were incubated with bacteria and serum without BRC, limited OPA titers were observed for immune mice (1:400) but not for nonimmune mice.…”

“…We previously developed a complementdependent serum bactericidal antibody (SBA) activity assay that quantifies serum antibody responses to typhoidal (S. Typhi and S. Paratyphi A) and nontyphoidal (S. Typhimurium and S. Enteritidis) Salmonella (16). Here, we describe an assay that measures the opsonophagocytic capacity of NTS antibodies based on the wellcharacterized pneumococcus opsonophagocytic activity (OPA), which employs HL-60 phagocytic cells and a standardized source of complement, i.e., baby rabbit complement (BRC) (17,18).…”

Overexpression of O‐polysaccharide chain length regulators in Gram‐negative bacteria using the Wzx‐/Wzy‐dependent pathway enhances production of defined modal length O‐polysaccharide polymers for use as haptens in glycoconjugate vaccines

“…Typhimurium (Lindberg ), expresses almost entirely short‐chain OPS (Boyd et al . ). We found that expression of wzzB from S .…”

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi

“…SBA. The serum bactericidal assay (SBA) was performed as previously described (57). In brief, log-phase cultures (optical density at 600 nm [OD 600 ], 0.5 at 37°C) of S. Typhi strain Ty2 were prepared in Luria-Bertani (LB) broth with 10 mM NaCl.…”