Evidence of Neutralizing and Non-Neutralizing Anti-Glucosaminidase Antibodies in Patients With S. Aureus Osteomyelitis and Their Association With Clinical Outcome Following Surgery in a Clinical Pilot

Abstract

Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC₅₀) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 μg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/- neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted.

Keywords: Staphylococcus aureus; antibodies; glucosaminidase; immunoassay; osteomyelitis.

Figure 1

Optimization of in vitro assay assessing Gmd digestion of M. luteus cell wall. The indicated concentration (μg/ml) of purified recombinant S. aureus Gmd was incubated with heat-killed M. luteus in the presence of the indicated amount of Triton X-100 at 37°C for 5 min (A), 60 min (B), or 120 min (C), and the percentage of lysis of M. luteus cell wall extract was determined by optical density as described in Materials and Methods. Note that the peak percentage of lysis (~80%) was achieved with a concentration of 10 μg/ml Gmd, 0.5% Triton X-100, and incubation time of 60 min.

Figure 2

Quantification of the neutralizing activity of humanized anti-Gmd mAb (TPH-101) in vitro. The indicated concentration (μg/ml) of purified anti-Gmd TPH-101 mAb or irrelevant control mAb (c21D10) was added to 10 μg/ml of recombinant Gmd prior to incubation with 0.075% heat-killed M. luteus cell wall extract in the presence of 0.5% Triton X-100 at 37°C for 30 min (A) or 60 min (B), and the percentage of lysis was determined as described in Figure 1 . These data were reanalyzed using Sigmoidal, 4PL, X is concentration least squares fit to quantify the 50% neutralizing concentration (NC₅₀) of TPH-101, which is 14.1 μg/ml in the 30-min incubation (A) and 17.0 μg/ml in the 60-min incubation (B), respectively. Dotted red and green lines are ± SD.

Figure 3

Quantification of the neutralizing activity of humanized anti-Gmd mAb (TPH-101) via M. luteus killing assay. The indicated concentration (μg/ml) of purified anti-Gmd TPH-101 mAb or irrelevant control mAb (c21D10) was added to 10 μg/ml of recombinant Gmd prior to incubation with live M. luteus in the presence of 0.5% Triton X-100 at 37°C for 30 min (A) or 60 min (B). The percentage of neutralization was determined as described in Material and Methods. Sigmoidal, 4PL, X is concentration least squares fit to quantify the 50% neutralizing concentration (NC₅₀) of TPH-101, which is 12.70 μg/ml in the 30-min incubation (A) and 12.25 μg/ml in the 60-min incubation (B), respectively. Dotted red and green lines are ± SD.

Figure 4

TPH-101 complementation of anti-Gmd antibody-deficient human serum in vitro. The indicated concentration of purified anti-Gmd TPH-101 mAb, irrelevant control mAb (c21D10), or twofold serial dilutions of human sera that do not contain neutralizing anti-Gmd antibodies (Patient 4 in the AO-CPP cohort) was added to 10 μg/ml of recombinant Gmd prior to incubation with 0.075% heat-killed M. luteus cell wall extract in the presence of 0.5% Triton X-100 at 37°C for 30 min (A) or 60 min (B), and the percentage of neutralization was determined as described in Figure 2 . Complementation of the patient 4 serum was also assessed by addition of TPH-101 or c21D10 at the indicated concentration. No differences in the percentage of neutralization of purified TPH-101 vs. TPH-101 in the human serum were detected at any concentration of antibody.