Natural antibodies in normal human serum inhibit Staphylococcus aureus capsular polysaccharide vaccine efficacy

Abstract

Background: Vaccines against Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae type b induce functional opsonic or bactericidal antibodies to surface capsular polysaccharides (CP). Targeting the comparable Staphylococcus aureus CP seems logical, but to date such efforts have failed in human trials. Studies using immunization-induced animal antibodies have documented interference in opsonic and protective activities of antibodies to CP by antibodies to another S. aureus cell surface polysaccharide, poly-N-acetyl glucosamine (PNAG). Here we evaluated whether natural antibody to PNAG in normal human serum (NHS) had a similar deleterious effect.

Methods: Functional and/or protective activities of antibody to S. aureus CP and PNAG antigens in patients with bacteremia, in mice immunized with combinations of CP and PNAG conjugate vaccines, and in serum samples of healthy subjects with natural antibody to PNAG, to which immunization-induced animal antibodies to CP antigens were added, were evaluated.

Results: Antibodies to PNAG and CP that mutually interfered with opsonic killing of S. aureus were detected in 9 of 15 bacteremic patients. Active immunization of mice with combinations of PNAG and CP conjugate antigens always induced antibodies that interfered with each other's functional activity. Non-opsonic natural antibodies to PNAG found in NHS interfered with the functional and protective activities of immunization-induced antibody to CP antigens during experimental infection with S. aureus.

Conclusions: Both immunization-induced animal antibodies and natural antibodies to PNAG in NHS interfere with the protective activities of immunization-induced antibody to S. aureus CP5 and CP8 antigens, representing potential barriers to successful use of CP-specific vaccines.

Figure 1.

Opsonophagocytic killing activity (OPKA) of 15 human serum samples from patients with Staphylococcus aureus bacteremia. A, Serum tested without absorption. Killing of <30%, indicative of a lack of specific OPKA, was measured in all but 1 sample (patient 8) of the unabsorbed serum containing interfering antibodies. Bars indicate the means of 4 replicates per assay. B, Serum tested after absorption with S. aureus MN8 Δcap8, MN8 Δica, or MN8 Δcap8 + Δica (leaving behind, respectively, antibodies to capsular polysaccharides [CP], to poly-N-acetyl glucosamine [PNAG], or to both PNAG and CP). Stars indicate a decrease of >30% in killing, observed in 9 of 15 of the serum samples when anti-CP and anti-PNAG activities were combined together (patients 7–15), indicating the presence of interfering antibodies. Abbreviations: CP, capsular polysaccharides; PNAG, poly-N-acetyl glucosamine.

Figure 2.

Opsonophagocytic killing activity in antiserum from mice immunized by antigens injected individually or in combination. A and B, Killing of Staphylococcus aureus PS80 (CP8) or S. aureus Newman (CP5), respectively, by indicated antiserum at the dilution indicated on the x-axis from mice immunized with dPNAG-TT, CP5-TT, or CP8-TT antigens alone. C and D, Killing of CP5 or CP8, respectively, by antibody to dPNAG-TT or CP injected individually or mixed together (decreasing amounts of antibody to dPNAG-TT added to constant amount of antibody to CP). Bars indicate the means of 4 replicates per assay. Controls were pre-immune serum (A and B) or tubes lacking complement (C and D). Abbreviations: CP, capsular polysaccharide; dPNAG, deacetylated poly-N-acetyl glucosamine; TT, tetanus toxoid.

Figure 3.

Binding of immunoglobulin G (IgG) in normal human serum to Staphylococcus aureus antigens poly-N-acetyl glucosamine (PNAG), capsular polysaccharide 5 (CP5), and capsular polysaccharide 8 (CP8). A–C, Binding of IgG in 15 normal human serum (NHS) samples and a pool of NHS to S. aureus antigens: PNAG (0.06 µg/well), CP5 (3 µg/well) or CP8 (3 µg/well), respectively. Dashed lines indicate NHS with high level of IgG to CP5 (B) or CP8 (C). D and E, Binding of rabbit antibody raised to CP5–tetanus toxoid (TT) to CP5 or rabbit antibody raised to CP8-TT to CP8, respectively, in the presence of 1:10 dilutions of 15 NHS samples. F, Positive controls: rabbit antiserum raised to PNAG (top), rabbit antiserum raised to CP5-TT (center), or rabbit antiserum raised to CP8-TT (bottom). Abbreviations: CP, capsular polysaccharide; OD, optical density; PNAG, poly-N-acetyl glucosamine; TT, tetanus toxoid.

Figure 4.

Opsonophagocytic killing activity (OPKA) of 15 normal human serum (NHS) samples. A, OPKA of Staphylococcus aureus Newman or PS80 strains. Controls lacked functional complement; test samples had all OPK components present. Symbols indicate OPK in 1 NHS sample, and bars indicate the group medians. Control rabbit antibody to CP5 or CP8 mediated 62%–70% killing at a 1:10 serum dilution. B, OPKA of S. aureus Newman or PS80 strains mediated by rabbit antibody to CP5-TT or to CP8-TT, respectively, when added to the indicated dilution of 15 unabsorbed NHS samples. Three NHS samples showed a greater decrease in OPKA of the rabbit antibody to capsular polysaccharide (CP) at a 1:40 dilution compared with that at a 1:10 dilution (1 for CP5, 2 for CP8), and 1 NHS sample had no reduction in the OPKA of rabbit antibody to CP5. Symbols indicate OPKA of rabbit immune serum mixed with 1 NHS sample at the indicated dilution, and bars indicate the group medians. Control rabbit antibody to CP5 and CP8 in the absence of added NHS mediated 60%–62% killing at a 1:10 serum dilution. Controls lacking complement had <1% killing. C, OPKA of S. aureus Newman or PS80 strains mediated by rabbit antibody to CP5-TT or CP8-TT, respectively, when added to indicated dilutions of 15 NHS samples that had been absorbed to remove natural antibody to poly-N-acetyl glucosamine. Results shown are in the presence of all OPK components. Symbol indicate activity of the rabbit antiserum in 1 NHS sample at the indicated dilution, and bars indicate the group medians. Control rabbit antibody to CP5 and CP8 in the absence of added NHS mediated 65%–70% killing at a 1:10 serum dilution. Controls lacking complement had <1% killing. Abbreviations: NHS, normal human serum; TT, tetanus toxoid.

Figure 5.

Opsonophagocytic killing activity (OPKA) in a pool of normal human serum (NHS) samples. A and B, OPK of Staphylococcus aureus strain Newman or PS80, respectively, mediated by rabbit antibody raised to dPNAG-TT or rabbit antibody raised to CP5-TT or CP8-TT or mediated by the NHS pool. Controls had no complement. Rabbit antiserum raised to dPNAG-TT or CP-TT antigens individually had good killing. No killing (>30%) was mediated by the pool of NHS, unabsorbed or absorbed with S. aureus mutants to leave behind only antibodies to poly-N-acetyl glucosamine (PNAG; absorption with S. aureus Δica) or to capsular polysaccharide (CP; absorption with S. aureus Δcap). C and D, Effect on OPKA of the addition of indicated dilutions of pooled NHS to a constant amount of rabbit antibody to either CP5-TT or CP8-TT, respectively. NHS samples were either unabsorbed or absorbed to remove antibody to CP antigens (absorption with Δica mutant) or PNAG (absorption with Δcap mutant). Bars indicate the means of 4 replicates per assay. Controls lacking complement had killing of <1%. Abbreviations: CP, capsular polysaccharide; dPNAG, deacetylated poly-N-acetyl glucosamine; NHS, normal human serum; TT, tetanus toxoid.

Figure 6.

Effect of pooled normal human serum (NHS) samples on the number of colony-forming units (CFUs) of Staphylococcus aureus per abscess detected in mice given rabbit antibody to deacetylated poly-N-acetyl glucosamine (dPNAG)–tetanus toxoid (TT) or to CP5-TT or CP8-TT. A and B, Antibody raised to dPNAG-TT without or with NHS (equal volume) significantly reduced the CFUs of S. aureus per abscess compared with mice injected with NHS. Challenge was S. aureus PS80 (A) or Newman (B). Overall analysis of variance (ANOVA); P < .0005; pairwise comparisons shown on figure. C, CFUs of S. aureus per abscess in mice injected with rabbit antibody to CP8-TT, CP8-TT plus NHS, or NHS alone. Overall ANOVA; P < .006; pairwise comparisons shown on figure. Challenge was S. aureus PS80 (10⁶ CFUs). D, CFUs of S. aureus per abscess in mice injected with rabbit antibody to CP5-TT, CP5-TT plus NHS, or NHS alone. Overall ANOVA; P < .0001; pairwise comparisons shown on figure. Challenge was S. aureus Newman (10⁶ CFUs). Abbreviations: CP, capsular polysaccharide; NHS, normal human serum; NS, not significant; PNAG, poly-N-acetyl glucosamine; TT, tetanus toxoid.