Role of antilipopolysaccharide antibodies in serum bactericidal activity against Salmonella enterica serovar Typhimurium in healthy adults and children in the United States - PubMed (original) (raw)

Role of antilipopolysaccharide antibodies in serum bactericidal activity against Salmonella enterica serovar Typhimurium in healthy adults and children in the United States

Estela Trebicka et al. Clin Vaccine Immunol. 2013 Oct.

Abstract

Recent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection with Salmonella enterica serovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity against S. Typhimurium in a standard in vitro assay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) from S. Typhimurium but not from Escherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing of S. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing of S. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against an S. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

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Figures

Fig 1

Fig 1

Serum bactericidal activity against S. Typhimurium. (A) S. Typhimurium strain SL1344 was incubated with PBS, adult sera, or child sera for 60 min at 37°C. The numbers of surviving bacteria were determined by plating serial dilutions of the reaction mixtures. *, P = 0.0004; **, P = 0.0007; ***, P < 0.0001. (B) The S. Typhimurium human isolate BAA-189 was incubated with PBS or adult sera for 60 min at 37°C. The numbers of surviving bacteria were determined by plating serial dilutions of the reaction mixtures. *, P = 0.0015. The dashed lines indicate the starting inocula.

Fig 2

Fig 2

Serum hemolytic complement activity. Hemolytic complement activity against antibody-coated sheep erythrocytes was evaluated as described in Materials and Methods using bactericidal (B) adult samples, child samples, or the nonbactericidal (NB) adult sample. The results are shown as percentages of the activity of a commercial human complement used as a reference for this experiment.

Fig 3

Fig 3

Results of a bactericidal assay using IgG purified from adult sera. S. Typhimurium strain SL1344 was incubated with PBS or with purified IgG and a source of complement for 60 min at 37°C. The numbers of surviving bacteria were determined by plating serial dilutions of the reaction mixtures. *, P = 0.029. The dashed line indicates the starting inoculum.

Fig 4

Fig 4

Effect of LPS on serum bactericidal activity. (A) Adult and child serum samples were analyzed by ELISA to determine levels of IgM and IgG antibodies reactive with S. Typhimurium LPS. *, P = 0.011. (B) S. Typhimurium strain SL1344 was incubated with PBS or adult serum that had been preincubated or not preincubated with 100 μg/ml of S. Typhimurium (St) or E. coli (Ec) LPS. After 60 min at 37°C, the numbers of surviving bacteria were determined by plating serial dilutions of the reaction mixtures. *, P < 0.0001; **, P < 0.0001. The dashed line indicates the starting inoculum.

Fig 5

Fig 5

Comparison of serum bactericidal activities against S. Typhimurium strains JS107 (wild-type) and JS93 (oafA mutant). JS107 and JS93 were incubated at 37°C for 60 min with PBS or adult sera. The numbers of surviving bacteria were determined by plating serial dilutions of the reaction mixtures. The dashed line indicates the starting inoculum.

Fig 6

Fig 6

Inhibitory effect of the nonbactericidal sample. (A) The standard assay to test bactericidal activity against SL1344 was carried out using PBS, individual bactericidal samples (B), the nonbactericidal sample (NB; results from multiple experiments), or a mix of individual bactericidal samples with the nonbactericidal sample (B + NB). In some of the experiments, individual bactericidal samples were mixed with the nonbactericidal sample, which had been preabsorbed with either S. Typhimurium SL1344 (B + NBSt) or S. Dublin 2229 (B + NBSd). *, P = 0.0022. The dashed line indicates the starting inoculum. (B) Bactericidal (B) and nonbactericidal (NB) samples were analyzed by ELISA to determine levels of IgM and IgG antibodies reactive with S. Typhimurium LPS.

Fig 7

Fig 7

Role of S. Typhimurium LPS-specific IgM antibodies in the inhibitory effect of the nonbactericidal sample. (A) Results of the ELISA for S. Typhimurium LPS-specific IgM and IgG in the nonbactericidal sample (NB), in NB preabsorbed with S. Typhimurium SL1344 (NBSt), or in NB preabsorbed with S. Dublin 2229 (NBSd). The histograms indicate the mean absorbances of triplicates. (B) Flow cytometric analysis of IgM binding to the surface of SL1344 bacteria following incubation with PBS, NB, NB preabsorbed with S. Typhimurium SL1344 (NBSt), or NB preabsorbed with S. Dublin 2229 (NBSd). Percentages of bacteria staining positively for IgM are indicated.

Fig 8

Fig 8

Effect of the nonbactericidal sample on complement deposition on S. Typhimurium. S. Typhimurium SL1344 bacteria were incubated with PBS, a bactericidal sample (B), the bactericidal sample heat inactivated to eliminate complement (B-C), the nonbactericidal sample (NB), or a mix of the bactericidal and nonbactericidal samples (B+NB). Flow cytometry was carried out to detect the presence of the activated terminal complement components C5b to 9 on the bacterial surface. Percentages of bacteria staining positively for C5b to 9 are indicated.

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