Comparison of Antibody Repertoires against Staphylococcus aureus in Healthy Individuals and in Acutely Infected Patients

Human serum samples.Sera were collected from healthy individuals without evidence of S. aureus infection and from patients in the acute phase of diseases caused by S. aureus. Samples were obtained from 70 adults (between 20 and 60 years old; average age, ∼30 years) and 89 children (from 0 to 18 years old). We performed serial sampling (two to four samples from 13 donors) from the adult donor group for studies of the stability of antibody levels and nasal carriage; thus, a total of 100 serum samples were analyzed. A total of 42 samples were obtained from patients with documented S. aureus infections such as wound infection (17 subjects), bacteremia and sepsis (15 subjects), and other diseases such as pneumonia, arthritis, urinary tract infection, catheter-related infections, and peritonitis (10 subjects) within 2 to 8 days after disease onset. The patients were between 25 and 92 years of age, with the majority (23 out of 44) above 70 years of age. Sera with serial sampling (acute, early, and late convalescent phases) were collected from patients suffering from orthopedic prosthetic device-related infections. The serum samples used for the enzyme-linked immunosorbent assay (ELISA) were stored at 4°C with 0.02% sodium azide as preservative; those used for functional assays were stored at −80°C without preservative.

ELISA.ELISA was performed according to standard protocols (using a 50-μl volume in each step except for blocking [100 μl]). Briefly, 96-well plates (Maxisorp; NUNC, Roskilde, Denmark) were coated with antigens (50 μl) diluted in 0.1 M NaHCO₃ buffer (pH 9.3) to a concentration of 10 μg/ml for total lysate and culture supernatant proteins prepared from a S. aureus spa (protein A-deficient) strain or 2 to 5 μg/ml for recombinant proteins. The plates were incubated overnight at 4°C. Peptide ELISA was performed with biotin-labeled peptides and coated on streptavidin ELISA plates (EXIQON, Vedbaek, Denmark) at 10 μg/ml, according to the manufacturer's instructions. After blocking of nonspecific sites with 1% bovine serum albumin (BSA)-phosphate-buffered saline (PBS), human sera were added at various dilutions. The optimal dilutions of sera were 200-, 1,000-, and 5,000-fold for recombinant proteins and peptides and 5,000- to 50,000-fold for total lysate, supernatant proteins, lipoteichoic acid (LTA), and peptidoglycan (PG). The plates were incubated for 1.5 h at 37°C. After washing with PBS-0.1% Tween 20 (PBS-T), horseradish peroxidase-conjugated goat anti-human antibodies (Southern Biotechnology Associates, Birmingham, Ala.) were added to each well at a 1,000-fold dilution and plates were incubated for 1 h at 37°C. After the last washing step with PBS-0.1% Tween 20, incubation with the substrate 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) was performed for 30 min at 37°C, and titers were measured at 405 nm on a Sunrise ELISA reader (Tecan, Maennedorf, Switzerland). All serum samples were analyzed in duplicate, and mean values were calculated. Internal positive- and negative-control sera were included on each plate. The results were expressed as ELISA units calculated from optical density (OD) readings, serum dilutions in the linear range of response, and total IgG and IgA concentrations of individual serum samples.

Avidity measurements were performed by including Na-isothiocyanate (in 1, 2, 3, or 4 M final concentrations) during the incubation of the antigens with serum samples in the 1,000- to 25,000× dilution range according to the method of Romero-Steiner et al. (46).

Serum IgG and IgA concentrations were determined by ELISA. Affinity-purified goat anti-human IgG or anti-human IgA capture antibodies (Bethyl Laboratories, Montgomery, Tex.) were used at a 10 μg/ml concentration for coating 96-well plates (Maxisorp; NUNC). Serial dilutions of sera (1:10,000 to 1:600,000) were made in PBS-BSA, and human reference serum (Bethyl Laboratories) (in the range of 7.8 to 1,000 ng/ml) was used as a standard. Highly specific horseradish peroxidase-labeled goat anti-human IgG or anti-human IgA secondary antibodies (Bethyl Laboratories) were used as detecting reagents according to the recommendations of the manufacturers (dilution, ∼1:50,000).

Staphylococcal antigens.Total bacterial lysate was prepared using the lysostaphin digestion method. Briefly, S. aureus was cultivated overnight at 37°C with 150-rpm shaking in brain heart infusion (BHI) medium. Approximately 10¹⁰ bacteria were harvested and washed with PBS. The pellet was resuspended in 200 μl of PBS buffer containing protease inhibitor cocktail Complete EDTA-free tablets (Roche Diagnostics, Mannheim, Germany) and 20 μg of lysostaphin (Sigma-Aldrich, Steinheim, Germany). After enzymatic digestion at 37°C for 30 min, the cells were disrupted by sonication using a microsonicator (Sonoplus HD 2200; Bandelin Electronics, Berlin, Germany) and the soluble fractions were recovered by centrifugation. Culture supernatant fraction was prepared by ethanol precipitation. Briefly, culture supernatants of overnight S. aureus cultures grown in BHI medium were incubated with 3 volumes of ice-cold ethanol at −20°C for at least 12 h and precipitated proteins were collected by centrifugation. The pellet was dried and resuspended in PBS. Protein concentrations were determined by the Bradford method (protein assay; Bio-Rad, Munich, Germany).

The staphylococcal recombinant proteins were expressed as fusion proteins with a StrepII tag (IBA, Göttingen, Germany), six-His tag, or glutathione S-transferase (GST) tag. All genes were cloned using genomic DNA from the S. aureus COL strain. DNA was amplified by gene-specific oligonucleotides with incorporated BsaI and SalI sites for StrepII- and GST-tagged proteins (IsdB and StbA), respectively. Restriction enzyme-digested PCR products were cloned into BsaI-cleaved pASK-IBA4 vector (IBA) or into SalI-cleaved pGEX-4T vector. The recombinant genes encoding the gram-positive signal peptides (according to the SignalP prediction program) and C-terminal sequences downstream from the sortase cleavage site (LPXT↑G) (where “X” represents any amino acid) were removed. Sequences corresponding to SD repeats (serine, aspartic acid) of ClfB and the Sdr proteins were also omitted. ClfA was produced as an N-terminal six-His-tagged protein, and the corresponding DNA was amplified from pCF4, a multicopy plasmid carrying a clfA gene from the S. aureus Newman strain (a gift from Tim Foster, Trinity College, Dublin, Ireland) (33), by gene-specific oligonucleotides with incorporated XhoI/NdeI sites and cloned into the pFS23 vector. The recombinant proteins were purified from bacterial extracts of Escherichia coli BL21 induced by anhydrotetracyclin (pASK-IBA4) or IPTG (isopropyl-ā-d-thiogalactopyranoside) (pGEX-4T) by affinity chromatography with StrepTactin Sepharose (IBA), glutathione-Sepharose (Amersham Biosciences, Uppsala, Sweden) or Ni-nitrilotriacetic acid agarose (QIAGEN, Hilden, Germany) (according to the manufacturer's instructions).

LTA and PG were purified from S. aureus and purchased from Sigma-Aldrich.

Peptides were synthesized in small scale by use of standard F-moc chemistry on a Rink amide resin (PepChem, Tübingen, Germany) by the use of a SyroII synthesizer (Multisyntech, Witten, Germany). After the sequence was assembled, peptides were elongated with Fmoc-epsilon-aminohexanoic acid and biotin (Sigma-Aldrich). Peptides were cleaved off the resin with 93% TFA-5% triethylsilane-2% water for 1 h, dried under vacuum, and freeze-dried three times using acetonitrile-water (1:1). The presence of the correct mass was verified by mass spectrometry on a Reflex III matrix-assisted laser desorption ionization-time of flight (Bruker, Bremen, Germany).

In vitro opsonophagocytosis assay. S. aureus Wood strain cells were labeled with the fluorescent dye Alexa 488 (Molecular Probes, Leiden, The Netherlands), and following preopsonization with 10% human serum for 15 min at 37°C, 2% complement (guinea pig serum; Institute for Immunology, Johannes Gutenberg-University, Mainz, Germany) and 2 × 10⁶ macrophages of the cell line P388.D1 were added to 2 × 10⁷S. aureus cells and incubation was continued for 15 min at 37°C. The phagocytic cells were washed three times to remove loosely attached bacteria before fixation with 2% paraformaldehyde. Opsonization was monitored as an increase in mean fluorescence intensity of the phagocytic cells measured with a fluorescence-activated cell sorter (FACS) (BD Biosciences, Bedford, Mass.).

In vitro bactericidal assay.Phagocytic cells and bacteria were incubated in the presence of complement to measure antibody-dependent killing on the basis of the loss of viable bacteria as determined by colony counting. In brief, S. aureus 8325-4 cells were washed twice in Hanks balanced salt solution and the cell density was adjusted to 10⁵ CFU in 50 μl of Hanks balanced salt solution. Bacteria were incubated with human serum IgGs (0.25 to 4 μg) and guinea pig complement (up to 5%) in a total volume of 100 μl for 60 min at 4°C. Preopsonized bacteria were mixed with macrophages (murine cell line RAW264.7; 2 × 10⁶ cells per 100 μl) at a 1:20 ratio and were incubated at 37°C on a rotating shaker at 500 rpm. An aliquot of each sample was diluted in sterile water and incubated for 5 min at room temperature to lyse macrophages. Triplicates were then plated onto BHI agar plates, and colonies were counted with a Countermat flash colony counter (IUL Instruments, Königswinter, Germany).

Toxin neutralization assay.During the assay setup it was established that 250 ng of recombinant S. aureus alpha-toxin (Toxin Technology, Sarasota, Fla.) resulted in about 90% hemolysis of rabbit red blood cells as quantitated by measuring released hemoglobin. To compare the characteristics of neutralizing activity of human sera, individual samples in the 20- to 320-fold dilution range were preincubated with 250 ng of alpha-toxin before being added to rabbit blood (30-fold diluted with PBS) for 30 min at 37°C. Hyperimmune anti-alpha-toxin rabbit serum was used in the assay as a positive control, and toxin-neutralizing activity expressed as a percentage of inhibition of hemolysis-hemoglobin release was quantified by spectophotometric measurements at 545 nm and expressed as a percentage of lysis (100% lysis was achieved by incubation of rabbit red blood cells in distilled water).

Western blotting.Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a mini-Protean electrophoresis system (Bio-Rad, Austria) and transferred to a nitrocellulose membrane (ECL; Amersham Biosciences, Buckinghamshire, United Kingdom) by a semidry transfer system (Bio-Rad, Vienna, Austria). After overnight blocking in 5% milk, human sera at 1:10,000 dilutions were added, and horseradish peroxidase-labeled goat anti-human IgGs (Southern Biotechnology Associates) were used for specific detection of immune complexes. The signal was developed using an ECL detection system (Amersham Biosciences, Buckinghamshire, United Kingdom).

Purification of IgGs.Highly enriched preparations of IgGs were generated by protein G affinity chromatography according to the manufacturer's instructions (UltraLink immobilized protein G; Pierce, Rockford, Ill.). Antibody concentrations were measured at an OD of 280 nm (OD₂₈₀) and were also estimated by SDS-PAGE analysis (using BSA as a standard).

Statistical analysis.An unpaired Student's t test was used to compare ELISA values between two groups. Differences were considered significant when the P value was equal to or less than 0.05.

A wide range of antistaphylococcal antibody levels in healthy individuals.It has been reported previously that serum levels of antibodies recognizing S. aureus components can be quite high not only in infected patients but also in clinically healthy individuals. We therefore measured antistaphylococcal antibodies in serum samples obtained from healthy adults who did not suffer from any apparent infectious diseases at the time of sampling. The sera obtained from 54 different donors showed great variability in total levels of antibodies as measured by ELISA using total bacterial lysates or culture supernatants of in vitro-grown S. aureus 8325-4 spa (lacking Protein A) as coating antigens (Fig. 1). We determined the contribution of antistaphylococcal antibodies by analyzing samples characterized with the highest and lowest antistaphylococcal titers for total and specific IgG content and found that approximately 2.5 to 3.0% of the total serum IgG antibodies reacted with the staphylococcal lysate in high-titer sera whereas the lowest-titer samples resulted in reactions with only 0.1% (data not shown). We also detected a wide range of IgA levels that were lower than those of IgG (Fig. 1). However, there was no correlation between the two antibody classes of individual samples, since we detected a significant number of sera with high IgA and low IgG titers as well as sera with low IgA and high IgG titers.

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FIG. 1.

Antistaphylococcal IgG and IgA antibody levels show great variability in healthy individuals. Antibody levels were determined by ELISA using total bacterial lysate (upper panel) or culture supernatant (lower panel) from S. aureus 8325-4 (spa) (grown in BHI overnight) as a coating antigen and serum samples from 54 healthy adults. Antigen-antibody interactions were detected with highly specific anti-human IgG and IgA antibodies. Results are shown as OD₄₀₅ values for serum dilutions in the linear range (1:50,000 for lysate and 1:10,000 for culture supernatant).

Induction of antistaphylococcal IgM, IgA, and IgG antibodies during childhood.The analysis of sera obtained from 89 healthy children ranging in age from 2 months to 18 years showed that antistaphylococcal antibody levels increased with age (Fig. 2). However, the three main antibody classes were induced with different kinetics. The first antibodies to appear were of the IgM class and reached a plateau by the age of 2 years. Antistaphylococcal IgAs reached the highest level in 4- to 6-year-old children and did not show an increase in older age groups. In contrast to IgM and IgA levels, mean IgG levels increased continuously until adolescence (13 to 15 years). The pronounced heterogeneity in IgG and IgA antibody levels also seen in adults (Fig. 1) was already apparent in 7- to 9-year-old children.

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FIG. 2.

Kinetics of induction of antistaphylococcal antibodies during childhood. IgM, IgA, and IgG antibodies were detected by ELISA using total bacterial lysates prepared from S. aureus 8325-4 (spa) as a coating antigen and serum samples from 89 healthy children in different age groups. Results are shown as OD₄₀₅ values for serum dilution in the linear range (1:10,000 for IgG and 1:200 for IgA and IgM). For each of the eight different age categories, 9 to 16 sera were analyzed for antibody levels.

Antistaphylococcal antibodies react mainly with surface components.We observed that levels of antistaphylococcal IgG antibodies were very similar when total bacterial lysate or whole S. aureus cells were used as coating antigens (data not shown). This result suggested that the majority of antistaphylococcal IgGs reacted with the surface of the pathogen. LTA and PG, the two most abundant cell wall components, have been known to be highly immunogenic in humans. Consistent with this notion, we found a strong correlation between IgG levels seen with these cell wall components and those seen with total lysate (Fig. 3A). In competitive ELISAs using LTA and PG as soluble antigens and total S. aureus lysate as coating antigen, we determined that LTA and PG contributed to approximately 60 to 85% and 15 to 25% of total antistaphylococcal IgG reactivity, respectively (Fig. 3B and data not shown). However, the presence of LTA and PG did not contribute to IgA level changes (Fig. 3C).

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FIG. 3.

The majority of antistaphylococcal IgG antibodies are induced by lipoteichoic acid and peptidoglycan. (A) Correlation between serum antibody levels against LTA (upper panel) or PG (lower panel) and total S. aureus 8325-4 (spa) lysate. Sera from 40 healthy donors were diluted 10,000-fold for LTA and bacterial lysate measurements and 5,000-fold for PG. Results are shown as OD₄₀₅ values. (B) Determination of the contribution of LTA to total antistaphylococcal reactivity with competition ELISA using soluble LTA as a competitor at the indicated amounts and total S. aureus lysate as a coating antigen. (C) IgG and IgA anti-LTA and anti-PG antibody levels were determined in standard ELISAs using five different human serum samples from healthy adults determined to have high total antistaphylococcal antibody titers.

Staphylococcal cell surface and secreted proteins induce IgG and IgA antibodies in healthy adults and acutely infected patients.To determine the antigenic specificity of circulating antistaphylococcal antibodies present in sera of healthy individuals, a collection of recombinant S. aureus proteins was analyzed. The selection of proteins was made on the basis of the predicted surface localization and/or determined function; the selected proteins included 11 members of the LPXTG motif-containing cell wall-anchored protein family, four lipoproteins, and two surface and two secreted proteins (Table 1). All 19 recombinant proteins were expressed in E. coli and purified by affinity chromatography using affinity tags fused to the N or C terminus of each protein. The integrity and purity of the proteins was evaluated by SDS-PAGE analysis (Fig. 4A) or tag-specific immunodetection (data not shown). Antibodies against these extracellular proteins were measured in sera from 40 healthy adults and 42 patients in the early phase of staphylococcal diseases to identify antibodies that were induced by the onset of disease or were missing and underrepresented in acute-phase patients.

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FIG. 4.

Comparison of IgG and IgA antibody levels against staphylococcal surface proteins. (A) Purified recombinant proteins were visualized by Coomassie blue staining of SDS-12% PAGE to assess integrity and purity. Proteins are indicated by common names; predicted molecular weight values are shown in parentheses. All proteins except Stbp and IsdB (GST negative) were expressed with a six-His tag. (B) IgG and IgA antibody levels in sera from healthy and infected individuals were determined by ELISA with recombinant proteins. Antibody reactivity was expressed in ELISA units calculated from OD readings obtained from measurements with 1,000-fold serum dilutions. Representative examples of antigens showing higher, equal, or lower IgG (upper panel) and IgA (lower panel) antibody levels in healthy donors (open circle) relative to infected donors (filled circle) are shown. Median values are shown for both groups, and statistically significant differences are indicated by asterisks.

Standard ELISA measurements were performed with saturating amounts of recombinant proteins to quantitatively analyze antibody levels. We detected diverse levels of both IgG and IgA serum antibodies with all of the recombinant proteins tested (Table 2 and Fig. 4B). Some antigens, such as the Sdr proteins, seemed to be very potent at inducing both antibody classes, while the majority of antigens preferentially induced IgGs. Both donor groups showed a wide range of antibody reactivities when tested with individual recombinant proteins; however, for the majority of proteins, no significant differences in median values of ELISA units were seen. Significantly higher IgG levels (P < 0.05) were measured in the patient group compared to the results seen with the group of healthy adults for SdrD, HarA, FnbpA, Enolase, EbpS, and SA0688 (ABC iron transporter), whereas IgA levels were higher only for HarA. Most importantly, IgG antibodies against three proteins—ClfB, coagulase, and Efb—and the IgA levels for ClfB were significantly underrepresented in the sera of infected patients relative to those of healthy individuals (Table 2 and Fig. 4B). In contrast, total antistaphylococcal IgG and IgA levels for the same serum sample (as measured using total bacterial lysates) were comparable, although infected patients displayed a wider range of antibody levels than healthy adults (Fig. 4B, leftmost panels).

Carriage and antistaphylococcal antibody levels.To correlate antibody levels with bacterial carriage, we determined levels of nasal and nasopharyngeal colonization of adults at the time of serum sampling with routine clinical microbiological tests. A total of 30% of individuals tested positive for at least one time point for nasal carriage; 10% of them also tested positive for nasopharyngeal colonization. Although we could not find a strong correlation between carriage and antibody levels, there was a tendency for higher IgG and lower IgA levels in permanent carriers compared to the results seen with individuals for whom carriage was not detected at any time point (Fig. 5). By analyzing the relationship between carriage and antibody levels against individual recombinant S. aureus proteins, we observed higher levels of IgG against ClfB, HarA, and Map-w and of IgAs against ClfB in healthy individuals who repeatedly tested negative for S. aureus according to the results obtained with nasal and pharyngeal swab cultures relative to results for those who were found to be intermittent or permanent carriers (Table 2).

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FIG. 5.

Tendency for higher IgG and lower IgA levels in S. aureus nasal carriers. IgG and IgA levels for total S. aureus 8325-4 (spa) bacterial lysates were measured by ELISA and individual data points are shown for noncarriers (open circle) (n = 17), transient carriers (gray-shaded circle) (n = 13), and permanent carriers (filled circle) (n = 9). Median values are indicated for all groups.

Immunodominant peptide epitopes of S. aureus identified using high-titer sera from healthy individuals react with sera from seroconverted patients.In our previous work we used serum samples with high antistaphylococcal titers to identify novel antigens by a genome-wide method (14). In these experiments S. aureus genomic peptide expression libraries were exposed to selection by antibodies derived from high-titer sera also included in this study. The majority of the identified epitopes and almost all of the most frequently selected epitopes belonged to surface located or secreted proteins of S. aureus (14). The immunogenicity of these antigens was further evaluated by ELISA using synthetic peptides corresponding to these epitopes. In a series of automated ELISA measurements 20 human serum samples—10 from infected patients and 10 from healthy individuals—determined to have high-titer antibodies against S. aureus were analyzed for their epitope-specific antibody levels (Fig. 6). Peptide epitopes inducing high levels of antibodies in many individuals belonged to surface and secreted proteins, such as the secretory extracellular matrix and plasma binding protein (Empbp), aerolysin-leukocidin family protein, exotoxin, staphylococcal IgG binding proteins protein A and Sbi, SdrH, LysM domain proteins, Cell wall associated fibronectin binding protein (Ebh), bifunctional autolysin, LPXTG protein, and membrane proteins. The majority of these peptide epitopes reacted with sera derived from infected patients as well as sera from healthy individuals. Moreover, we observed seroconversion against immunogenic epitopes when we compared serum samples obtained from the acute- and convalescent-phase patients with prosthetic-device-related S. aureus infections (Fig. 7; patients P60 and P63). In contrast, in patients suffering from chronic infections epitope-specific antibody levels did not change during convalescence from an acute exacerbation (Fig. 7; patient P66). (In total, 12 patients [5 acutely infected and 7 chronically infected] were included in this analysis, and the data shown for these three patients are representative).

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FIG. 6.

Biotin-labeled peptides (e.g., SA0858.3) representing selected epitopes were used as coating antigens in ELISAs to measure specific IgG levels from human sera. Lanes P1 to P10, sera from infected patients; lanes H1 to H10, sera from healthy individuals. Black panels represent high reactivity; dark grey panels represent intermediate reactivity; grey panels represent low reactivity; light grey panels represent very low reactivity. Each score was calculated from the number of positive serum results and the extent of reactivity. SA0095, IgG binding protein A (Spa); SA0164, gramicidin S synthetase 2-related protein; SA0177, glucokinase regulator-related protein; SA0178, PTS system, IIBC components; SA0193, maltose ABC transporter, maltose binding protein; SA0316, conserved hypothetical protein; SA0470, exotoxin; SA0507, LysM domain protein; SA0632, membrane protein; SA0723, LysM domain protein; SA0858, secretory extracellular matrix and plasma binding protein (Empbp); SA1062, bifunctional autolysin; SA1472, cell wall-associated fibronectin binding protein (Ebh); SA1489, recombinant protein U; SA1791, FtsK/SpoIIIE family protein; SA2006, aerolysin/leukocidin family protein; SA2019, sdrH protein, putative; SA2291, staphyloxanthin biosynthesis protein; SA2236, ribosomal protein L2; SA2418, IgG binding protein SBI; SA2505, cell wall surface anchor family protein (LPXTG); SA2581, staphyloxanthin biosynthesis protein.

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FIG. 7.

Novel peptide epitopes identified with high-titer staphylococcal sera induce antibodies in infected patients during convalescence. A total of 14 biotin-labeled peptides representing epitopes with high antibody reactivities with sera from healthy adults were used as capture antigens on streptavidin-coated ELISA plates. Two serum samples were taken from patients with osteomyelitis in the acute phase (open bar) and of the convalescent phase (filled bar) and used to measure IgG antibodies induced during disease. Patients P60 and P63 had acute osteomyelitis, while patient P66 suffered from chronic infection. Serum samples were taken 3 to 4 weeks apart.

Stability and avidity of antistaphylococcal antibody levels in healthy adults.To investigate the stability of antistaphylococcal antibodies in high-titer healthy individuals, we collected and analyzed serum samples at different time points during a 1-year time period. By comparing the ELISA results obtained at 0, 4, 9, 11, or 12 months, we observed that IgG and IgA levels of antibody against the different staphylococcal components were stable over a year without changing profile and that no fluctuation in the abundance was detected whether single antigenic components, such as recombinant proteins (Fig. 8), LTA, or peptidoglycan, or a complex mixture of antigens (total lysate, culture supernatant) were tested (data not shown).

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FIG. 8.

Stable levels of circulating antistaphylococcal serum antibodies in healthy individuals. Serial serum samples were taken from eight healthy adults (patients H1 to H8) at different time points (as indicated) and analyzed by ELISA for antigen-specific IgG antibody levels by the use of four different recombinant proteins. Results are expressed as ELISA units calculated from OD readings at 1,000-fold serum dilutions.

Because avidity is an important parameter of antibody quality, we measured the level of antibodies capable of binding to complex staphylococcal antigens (bacterial lysate) or to a highly immunogenic surface protein (IsdB) in the presence of different concentrations of the chaotropic reagent sodium isothiocyanate. The total absorbance in individual sera with similar initial titers was equally reduced, indicating that levels of antibody avidity were very comparable between the healthy and patient donor groups (data not shown).

Antistaphylococcal antibody levels correlate with functionality.We reported previously that pools of high-titer sera from healthy individuals and from infected patients were equally effective in inducing complement-dependent opsonization (14). We now investigated whether a correlation between antistaphylococcal ELISA titers and functional activity could be established. In a FACS-based in vitro opsonophagocytosis assay using fluorescently labeled S. aureus, we compared several human serum samples characterized with low, intermediate, or high antistaphylococcal antibody levels for their activity to induce the uptake of S. aureus by cultured murine monocytic cells (p388/D1) (Fig. 9A) and freshly isolated human polymorphonuclear leukocytes (data not shown). The enhancement of opsonophagocytosis correlated well with IgG levels, but such a correlation was not observed with antistaphylococcal IgA levels.

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FIG. 9.

Antistaphylococcal antibody levels correlate with functionality. (A) A FACS-based opsonophagocytosis assay was performed with fluorescently labeled S. aureus (Wood strain) and P388.D1 mouse macrophage cells in the presence of 10% human sera with different total antistaphylococcal titers. Sera were from low (H_L1)-, intermediate (H_M1)-, and high (H_H1 and H_H2)-titer sera, all from healthy (H) donors. C, control (no serum or complement added); C′, only complement (no serum added); H_L1, H_M1, and H_H1 and H_H2, 10% serum added in the presence of complement. (B) Levels of bactericidal killing activity of total serum IgG preparations were determined by a plating assay. IgG (1 μg) was preincubated with S. aureus 8325-4 cells before addition to cultured mouse RAW264.7 monocytic cells in the presenceof complement. Low (H_L1 and H_L2)-, intermediate (P_M1 and P_M2)-, and high (H_H2)-titer sera were from healthy donors (H) or infected patients (P). Bactericidal activity is expressed as the percentage of decrease in CFU levels relative to the results seen with control samples (no IgG). (C) Toxin neutralization activity was measured as the level of hemolysis of rabbit red blood cells by alpha-toxin from S. aureus in the presence of human sera used at 20- to 320-fold dilutions. Low (H_L1 and H_L2)-, intermediate (H_M1 and H_M2)-, and high (H_H1)-titer sera were from healthy (H) donors. Toxin-neutralizing activity was expressed as a percentage of inhibition of hemolysis by alpha-toxin at different serum dilutions. Hemolysis was measured as a release of hemoglobin that was quantified by spectrophotometric measurements at 545 nm. Rabbit anti-alpha-toxin serum was used as a positive control in the assay.

To provide further evidence about the functionality of circulating antibodies, we performed an in vitro bactericidal assay using purified IgG preparations and the cell line RAW264.7 as a source of phagocytic cells. While IgGs from high-titer sera induced bacterial killing efficiently, the same amount of IgGs from low-titer sera was much less effective (Fig. 9B). The bactericidal activities of patient sera with high levels of antistaphylococcal antibodies were comparable to those of high-titer sera from healthy individuals.

A sensitive in vitro toxin neutralization assay using recombinant alpha-toxin of S. aureus and rabbit blood was performed as a third assay to correlate antibody levels with functionality. Five selected sera from healthy donors with different levels of total antistaphylococcal antibodies were analyzed. We observed that serum samples characterized with the lowest total antistaphylococcal IgG levels displayed the lowest toxin-neutralizing activity, while a high antistaphylococcal titer correlated with efficient toxin neutralization activity (Fig. 9C).