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. 2008 Apr;118(4):1553-62.
doi: 10.1172/JCI33998.

The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children

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The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children

Calman A MacLennan et al. J Clin Invest. 2008 Apr.

Abstract

Nontyphoidal strains of Salmonella (NTS) are a common cause of bacteremia among African children. Cell-mediated immune responses control intracellular infection, but they do not protect against extracellular growth of NTS in the blood. We investigated whether antibody protects against NTS bacteremia in Malawian children, because we found this condition mainly occurs before 2 years of age, with relative sparing of infants younger than 4 months old. Sera from all healthy Malawian children tested aged more than 16 months contained anti-Salmonella antibody and successfully killed NTS. Killing was mediated by complement membrane attack complex and not augmented in the presence of blood leukocytes. Sera from most healthy children less than 16 months old lacked NTS-specific antibody, and sera lacking antibody did not kill NTS despite normal complement function. Addition of Salmonella-specific antibody, but not mannose-binding lectin, enabled NTS killing. All NTS strains tested had long-chain lipopolysaccharide and the rck gene, features that resist direct complement-mediated killing. Disruption of lipopolysaccharide biosynthesis enabled killing of NTS by serum lacking Salmonella-specific antibody. We conclude that Salmonella-specific antibody that overcomes the complement resistance of NTS develops by 2 years of life in Malawian children. This finding and the age-incidence of NTS bacteremia suggest that antibody protects against NTS bacteremia and support the development of vaccines against NTS that induce protective antibody.

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Figures

Figure 1
Figure 1. Cases of NTS bacteremia in Malawian children.
Shown are 352 cases of consecutive blood culture–confirmed NTS bacteremia by age in children presenting to QECH between August 1, 2003, and July 31, 2004 (ages not available for 10 children).
Figure 2
Figure 2. Killing of NTS by blood and serum from Malawian adults and by C9-deficient serum at 45, 90, and 180 minutes.
(A) Killing of 8 invasive Malawian S. Typhimurium isolates by whole blood from a healthy adult Malawian donor. (B) Killing of S. Typhimurium isolate D23580 by whole blood (squares), serum (circles), and blood cells washed in RPMI to remove antibody and complement (triangles). (C) Effect of heat inactivation of serum on ability to kill D23580: fresh serum (circles), heat-inactivated serum (squares), and heat-inactivated serum reconstitution with lyophilized complement (triangles). (D) Inability of C9-deficient serum (triangles) to kill D23580. Killing of Salmonella was enabled by addition of exogenous C9 (squares). (E) Killing of D23580 by fresh and heat-inactivated serum: 100% fresh (triangles); 60% fresh, 40% heat-inactivated (diamonds); 20% fresh, 80% heat-inactivated (squares); 10% fresh, 90% heat-inactivated (circles); and 100% heat-inactivated (x). Negative values correspond with a decrease in viable salmonellae compared with the initial concentration of 106 salmonellae/ml. In A, B, C, and E, data indicate blood and/or serum from 1 healthy adult and are representative of 4 healthy adult donors tested. Where error bars are present, data are mean ± 1 SD of 3 experiments.
Figure 9
Figure 9. Killing of smooth wild-type NTS and a rough NTS mutant by Malawian adult and child serum, heat-inactivated serum, and C9-deficient serum.
In vitro killing by serum of smooth wild-type S. Typhimurium D23580 and a deep rough mutant of D23580 with disrupted galE gene at 45, 90, and 180 minutes (initial concentration, 106 salmonellae/ml). (A) Killing of mutant rough D23580 by adult serum (circles) and child serum lacking Salmonella-specific antibody (diamonds) and of wild-type smooth D23580 by adult serum (squares) and child serum lacking Salmonella-specific antibody (triangles). (B) Killing of rough D23580 by child serum lacking Salmonella-specific antibody (squares) was maintained in the presence of 10 mM EGTA and MgCl2 (triangles). Killing of rough D23580 did not occur in heat-inactivated serum (circles) or C9-deficient serum (diamonds), but addition of exogenous C9 to C9-deficient serum induced killing (x). Data are mean ± 1 SD of 3 experiments.
Figure 10
Figure 10. Age-based comparison of healthy Malawian children with cases of NTS bacteremia in Malawian children: serum bactericidal activity and serum anti-Salmonella IgG and IgM titers.
Serological data are from 65 Malawian children as shown in Figure 4 grouped by age (0–6, 6–12, 12–18, and 18–24 months; 2–3, 3–4, 4–5, and 5–10 years). Shown are mean and 95% CI serum bactericidal activity against S. Typhimurium isolate D23580 at 180 minutes (solid line) and geometric mean and 95% CI anti-D23580 IgG (short dashes) and IgM (long dashes) titers. Below, age distribution of NTS bacteremia cases is reproduced from Figure 1 for reference.
Figure 8
Figure 8. The rck gene of invasive NTS isolates from Malawi.
(A) Partial nucleotide sequence of 120-kb plasmid of D23580 demonstrating the 555-bp sequence (gray shading) of rck (15) and oligonucleotide primer sequences used to PCR amplify across the rck gene (arrows). (B) Predicted 668 bp product, as demonstrated by 1% agarose gel, of amplification shown in A in all 8 Malawian S. Typhimurium clinical isolates (lanes 1–8) and 1 Malawian clinical S. Enteritidis isolate (lane 9). M, marker; N, negative control.
Figure 4
Figure 4. Killing of NTS and anti-Salmonella IgG and IgM titers for serum from Malawian children.
(A) In vitro killing of S. Typhimurium isolate D23580 at 180 minutes (initial concentration 106 salmonellae/ml), (B) anti-Salmonella D23580 IgG levels, and (C) anti-Salmonella D23580 IgM levels for sera from 65 healthy Malawian children compared with age. Each point corresponds to serum from 1 child. Numbers at top right denote the number of points in the indicated quadrants, as divided by patient age (more or less than 16 months) and normal or impaired killing (more or less, respectively, than 1.2 log10 kill or 1.5 U Ig titer as appropriate).
Figure 3
Figure 3. Killing of NTS by whole blood and serum from Malawian children.
In vitro killing of S. Typhimurium isolate D23580 (initial concentration 106 salmonellae/ml) at 180 minutes by (A) whole blood compared with serum and (B) whole blood with test aliquot lysed or unlysed prior to determination of viable salmonellae. Paired blood and serum are from 65 healthy Malawian children. Each point corresponds to blood and serum from 1 child. Lines of equivalence are shown.
Figure 5
Figure 5. Killing of NTS by serum from Malawian children compared with anti-Salmonella IgG and IgM titers and complement C3 and membrane attack complex deposition in vitro killing of S. Typhimurium isolate D23580 by serum at 180 minutes.
(A and B) NTS killing compared with specific anti-Salmonella D23580 IgG (A) and IgM (B) levels. (C and D) NTS killing compared with complement C3 (C) and C5b-9 membrane attack complex (MAC; D) deposition on D23580. The initial concentration was 106 salmonellae/ml. Each point corresponds to serum from 1 child, either less than (filled circle) or more than (open circle) 16 months of age. Numbers at top right denote the number of points in the indicated quadrants, as divided by threshold for normal killing (more or less than 1.5 U Ig, 100 U C3, and 0.6 U C5b-9 membrane attack complex) and normal or impaired killing (more or less, respectively, than 1.2 log10 kill).
Figure 6
Figure 6. Effect of the EGTA/MgCl2 combination, anti-Salmonella IgG, and mannose-binding lectin on killing of NTS by control adult serum and child serum lacking specific antibody to Salmonella.
In vitro killing of S. Typhimurium isolate D23580 by serum was determined at 45, 90, and 180 minutes (initial concentration, 106 salmonellae/ml). (A) Killing of Salmonella by control adult serum (squares) and inhibition of killing by addition of 10 mM EGTA and MgCl2 (triangles). (B) Absence of killing of Salmonella by child serum lacking Salmonella-specific antibody (squares). Addition of 0.2 g/l exogenous IgG containing anti-Salmonella antibody enabled killing of Salmonella in child serum lacking Salmonella-specific antibody (triangles), but not in heat-inactivated serum (circles). (C) Inability of child serum lacking Salmonella-specific antibody to kill Salmonella (squares) was unaffected by addition of 5 μg/ml exogenous mannose-binding lectin (triangles). Data are mean ± 1 SD of 3 experiments.
Figure 7
Figure 7. P22 bacteriophage sensitivity and lipopolysaccharide profiles of invasive NTS isolates from Malawi.
(A) P22 bacteriophage sensitivity of 8 Malawian clinical isolates of S. Typhimurium from bacteremic children (lanes 1–8) compared with smooth S. Typhimurium strain SL1344 (lane 9), E. coli strain C600 (lane 10), and rough S. Typhimurium strain BA85 (lane 11). P22 was streaked across LB agar between the arrows and allowed to dry before the bacterial strains were cross-streaked. The plate was left overnight at 37°C before photographing. (B) Comparison of lipopolysaccharide profiles of 8 Malawian clinical S. Typhimurium isolates from bacteremic children (lanes 1–8) with laboratory smooth (SL1344; lane 9) and rough (BA85; lane 10) strains of S. Typhimurium. Lipopolysaccharide was prepared from S. Typhimurium and separated by SDS-PAGE prior to silver staining.

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