Antibodies in Action: Role of Human Opsonins in Killing Salmonella enterica Serovar Typhi

Abstract

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ⌬aroC ⌬ssaV). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2-to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.

Live Oral Salmonella enterica Serovar Typhi Vaccines Ty21a and CVD 909 Induce Opsonophagocytic Functional Antibodies in Humans That Cross-React withS. Paratyphi A andS. Paratyphi B

“…In contrast, the antibody functionality data presented in this study show that Ty21a and CVD 909 induce antibodies that enhance opsonophagocytosis and intracellular Our results show that postvaccination sera not only enhanced the opsonophagocytosis of S. Typhi into macrophages but also decreased their intracellular survival. These observations with Ty21a and CVD 909 vaccinees are similar to those reported with postvaccination sera from M01ZH09-immunized volunteers (33). Interestingly, we noted that the survival of S. Paratyphi A or S. Paratyphi B inside macrophages was not inhibited by opsonization with antibodies.…”

“…Internalization of S. Typhi by differentiated THP-1 macrophages (macrophages) is mediated mainly by phagocytosis (33). We therefore evaluated whether a similar process of phagocytosis is involved in the internalization of S. Paratyphi A and S. Paratyphi B and whether opsonization with complement-inactivated normal human sera increases the internalization of bacteria.…”

“…On the following day, the bacterial cultures were diluted 1:50 in LB and grown to log phase (optical density at 600 nm [OD 600 [, ϳ0.4 to 0.6 Opsonophagocytosis and bacterial killing assays. The assays were performed as originally described by Lindow et al with some modifications (33). In brief, THP-1 monocytes were seeded (5 ϫ 10 5 /well) onto 24-well plates and differentiated into macrophages by adding 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 48 h. Cells were then washed twice with PBS and incubated at 37°C in 5% CO 2 and in 0.5 ml RPMI complete medium without antibiotics for 2 to 4 h. Serum samples were preheated at 56°C for 30 min to destroy complement.…”

“…Levine et al described the presence of plasma-dependent mononuclear cell killing of S. Typhi in subjects immunized orally with attenuated S. Typhi vaccine strains (32). More recently, antibody-mediated en-hanced phagocytosis and intracellular killing of S. Typhi by macrophages was described following immunization with live oral S. Typhi strain M01ZH09 (33). However, the cross-reactivity of typhoid vaccine-induced antibodies in opsonin-mediated intake (opsonophagocytosis) and intracellular killing of S. Paratyphi A and S. Paratyphi B has not been reported.…”

Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines

“…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).…”