Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines

Abstract

Nontyphoidal Salmonella (NTS) invasive infections are an important cause of morbidity and mortality in sub-Saharan Africa. Several vaccines are in development to prevent these infections. We describe an NTS opsonophagocytic killing assay that uses HL-60 cells and baby rabbit complement to quantify functional antibodies elicited by candidate NTS vaccines. Invasive nontyphoidal Salmonella (NTS) infections are increasingly being recognized as an important cause of morbidity and mortality in infants and HIV-infected adults and children in subSaharan Africa (1). Multiple vaccines are currently in development, including live-attenuated vaccines, conjugate vaccines, and generalized modules for membrane antigens against invasive S. Typhimurium and S. Enteritidis (2-7). As these vaccines enter clinical development, it is important to establish their immunogenicity through relevant, well-characterized immunological assays. There is a growing realization within the vaccinology community that in addition to determining antibody levels through binding assays, it is important to determine the functional, antimicrobial capacity of these antibodies.Although many studies have evaluated anti-Salmonella serum bactericidal and opsonophagocytic antibody activity (2,(8)(9)(10)(11)(12)(13)(14)(15), there is a need for reproducible assays that can be used routinely to characterize functional antibody responses to Salmonella in a standardized manner. 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).The Salmonella OPA assay was evaluated using serum samples from mice that had been vaccinated with live-attenuated NTS vaccines (7). BALB/c mice were orally immunized with 10 9 CFU of S. Typhimurium CVD 1931 or S. Enteritidis CVD 1944 suspended in phosphate-buffered saline (PBS) or given PBS alone on days 0, 28, and 56, as previously described (7) [19]) from overnight cultures were diluted 1 in 1,000 in Hy-Soy medium (0.5% sodium chloride, 1% Hy-Soy [Kerry, Clackmannshire, United Kingdom], and 0.5% Hy-Yest [Kerry]), grown at 37°C to an optical density at 600 nm (OD 600 ) of 0.3, and then diluted to 3 ϫ 10 4 CFU/ml in OPB. Immune and nonimmune sera were heat-inactivated at 56°C for 20 min, and 2-fold serial dilutions in OPB (25 l/well final volume) were performed in a Ubottomed 96-well microplate (Sigma-Aldrich, St. Louis, MO). The bottom row was left without sera to act as a negative control. To each well, 3 ϫ 10 2 CFU of bacterial suspension in 10 l OPB was added. Opsonization of bacteria was allowed to occur for 15 min at 37°C with 5% CO 2 . After incubation, 25 ...

Improving Our Understanding of Salmonella enterica Serovar Paratyphi B through the Engineering and Testing of a Live Attenuated Vaccine Strain

“…The opsonophagocytic capability of sera was assayed by using the method of Ramachandran et al ( 49 ). Briefly, HL-60 cells were propagated in RPMI 1640 (Corning CellGro, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; HyClone, Waltham, MA) and 1% GlutaMax, 1% sodium pyruvate, and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA).…”

Modeling the Potential for Vaccination to Diminish the Burden of Invasive Non-typhoidal Salmonella Disease in Young Children in Mali, West Africa

“…Bacteremic organisms that reach the meninges, synovia and pleura can cause meningitis, septic arthritis and empyema, respectively. The NTS conjugate vaccines under development elicit serum antibodies that exhibit both bactericidal and opsonophagocytic functional properties [ 31 – 32 , 36 ], like the antibodies stimulated by Hib conjugate vaccines [ 25 , 37 – 40 ]. Thus, there exist striking pathogenetic, clinical and epidemiologic similarities between iNTS and Hib pathogens and similar functional activities are exhibited by the antibodies stimulated by the parenteral NTS conjugate vaccines (in animals) and by Hib conjugate vaccine in human infants.…”

Syntheses of Salmonella Paratyphi A Associated Oligosaccharide Antigens and Development towards Anti‐Paratyphoid Fever Vaccines

“…[56] Opsonophagocytic antibody activity is an important functional mechanism for antibodymediated clearance of S. Paratyphi A. [57] Antisera from Qbglycan 1 vaccinated rabbits markedly enhanced uptake of S. Paratyphi Abacteria by murine J774 macrophage, as compared to pre-immunization sera from theser abbits as well as sera generated after immunization with Qb particles alone ( Figure 9). To demonstrate the protective efficacy of anti-Qb-glycan 1 rabbit antibodies, we used a S. Paratyphi Am urine challenge model.…”