Live oral Salmonella enterica serovar Typhi vaccines Ty21a and CVD 909 induce opsonophagocytic functional antibodies in humans that cross-react with S. Paratyphi A and S. Paratyphi B

Abstract

Live oral Salmonella enterica serovar Typhi vaccine Ty21a induces specific antibodies that cross-react against Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Paratyphi B, although their functional role in clearance remains unknown. We utilized an in vitro assay with THP-1 macrophages to compare the phagocytosis and survival of Salmonella opsonized with heat-inactivated human sera obtained before and after vaccination with Ty21a or a live oral S. Typhi vaccine, CVD 909. Opsonization with postvaccination sera predominantly increased the phagocytosis of S. Typhi relative to the corresponding prevaccination sera, and increases were also observed with S. Paratyphi A and S. Paratyphi B, albeit of lower magnitudes. Relative to prevaccination sera, opsonization with the postvaccination sera reduced the survival inside macrophages of S. Typhi but not of S. Paratyphi A or S. Paratyphi B. Higher anti-S. Typhi O antigen (lipopolysaccharide [LPS]) IgG, but not IgA, antibody titers correlated significantly with postvaccination increases in opsonophagocytosis. No differences were observed between immunization with four doses of Ty21a or one dose of CVD 909. Ty21a and CVD 909 induced cross-reactive functional antibodies, predominantly against S. Typhi. IgG anti-LPS antibodies may be important in phagocytic clearance of these organisms. Therefore, measurement of functional antibodies might be important in assessing the immunogenicity of a new generation of typhoid and paratyphoid A vaccines. (The CVD 909 study has been registered at ClinicalTrials.gov under registration no. NCT00326443.).

Figures

FIG 1

Internalization of Salmonella strains into differentiated THP-1 macrophages is mediated by phagocytosis and is enhanced by opsonization with human sera. (A) Differentiated THP-1 macrophages were infected with nonopsonized S. Typhi, S. Paratyphi A (Para A), or S. Paratyphi B (Para B) at a multiplicity of infection of 10:1 (bacteria/cell) (filled bars). Parallel cultures were pretreated with cytochalasin D (Cyto D) to prevent phagocytosis. Numbers of internalized bacteria after 30 min of infection were quantified as CFU/ml as described in Materials and Methods. (B) Fold increases of phagocytosis in differentiated THP-1 macrophages following opsonization with 5% prevaccination sera (phagocytosis index) were calculated relative to the corresponding values for nonopsonized bacteria. ***, P < 0.001 compared to the corresponding untreated group (two-tailed nonparametric Mann-Whitney test).

FIG 2

Fold increases in phagocytosis of bacteria following opsonization with postvaccination sera. Phagocytosis assays were performed in differentiated THP-1 macrophages with Salmonella strains opsonized with 5% prevaccination (day 0) or postvaccination (day 10 and/or 14) sera (see Materials and Methods). Fold increases in phagocytosis were calculated as the peak of phagocytosed bacteria (CFU/ml) opsonized with day 10 or day 14 sera/phagocytosed bacteria (CFU/ml) opsonized with corresponding day 0 sera. No changes with regard to preimmunization levels are denoted by the dotted line (1-fold). Data are presented as the mean fold increases ± the standard errors (SE) of volunteers who were immunized with either four doses of Ty21a (n = 8) or a single dose of CVD 909 (n = 7) separately (A) or together (B). The P values were determined by two-tailed Wilcoxon matched-pair tests.

FIG 3

Survival of bacteria incubated with prevaccination sera within differentiated THP-1 macrophages. The 3 Salmonella strains were opsonized with prevaccination (day 0) sera (n = 14 [7 Ty21a and 7 CVD 909]), and survival assays were performed as described in Materials and Methods. Rate of survival was calculated as (bacteria recovered [CFU/ml] after 24 h/corresponding opsonophagocytosed bacteria [CFU/ml] after 30 min of infection) × 100. The P values were determined by a two-tailed Mann-Whitney test. ***, P < 0.001.

FIG 4

Fold changes in survival of opsonophagocytosed bacteria within differentiated THP-1 macrophages. The 3 strains of Salmonella were opsonized with prevaccination (day 0) and postvaccination (day 10 and/or 14) sera, and survival assays were performed as described in Materials and Methods. The rates of survival were calculated as (bacteria recovered [CFU/ml] after 24 h/corresponding opsonophagocytosed bacteria [CFU/ml] after 30 min of infection] × 100. Postvaccination fold changes in rate of survival were determined by comparing postvaccination rates of survival with their corresponding day 0 values. No changes with regard to preimmunization levels are denoted by the dotted line (1-fold). Data are presented as means ± SE of the peak postvaccination changes in survival (lowest fold changes at either day 10 or 14) in volunteers (n = 14) vaccinated with either four doses of Ty21a (n = 7) or a single dose of CVD 909 (n = 7). The P values were determined by a two-tailed Wilcoxon matched-pair test.

FIG 5

Correlation of opsonophagocytosis activity and bacterial survival rates with serum IgG anti-S. Typhi lipopolysaccharide (LPS) antibody titers. Peak postvaccination fold changes (day 10 and/or 14) were determined by comparison to the corresponding prevaccination levels. Peak postvaccination increases in serum IgG anti-LPS antibody titers in volunteers (n = 14) following immunization with either Ty21a (n = 7) or CVD 909 (n = 7) were correlated with the corresponding peak postvaccination fold increases in opsonophagocytosis (A, B, C) or peak bacterial killing (D, E, F) for S. Typhi (A, D), S. Paratyphi A (B, E), and S. Paratyphi B (C, F). The broken lines represent 95% confidence intervals. p, P value; r, Spearman's rho.