ELISpot detection of anti-IsdB antibody-secreting cells in S. aureus infected bone marrow: Case Reports of Total Hip Arthroplasty Patients

Abstract

Staphylococcus aureus prosthetic joint infection (PJI) is notoriously difficult to treat and is often associated with septic death. Results from the V710 Phase IIB/III clinical trial and subsequent research have demonstrated that non-neutralizing antibodies against the iron-regulated surface determinant protein B (IsdB) facilitate bacterial entry into leukocytes, generating "Trojan horse" leukocytes and the dissemination of surgical-site infections. In contrast, anti-glucosaminidase (Gmd) antibodies, which mediate opsonophagocytosis of bacterial clusters, are associated with protection from S. aureus osteomyelitis in both mice and humans. To further test whether anti-IsdB antibodies are pathogenic and anti-Gmd antibodies are protective in PJI patients, we performed a clinical pilot study of three healthy patients undergoing primary total hip arthroplasty (THA) and a patient undergoing revision THA for S. aureus PJI to assess the feasibility of quantifying anti-IsdB and anti-Gmd antibody-secreting cells in blood and periprosthetic bone marrow. We also assessed anti-IsdB and anti-Gmd antibody levels in serum, blood plasma, bone marrow plasma, and cell culture supernatant collected after six days with and without a previously established memory B-cell (MBC) stimulation cocktail. The frequencies of MBC and antibody-secreting plasmablasts were measured using IgG ELISpot. Consistent with our hypothesis, we found large numbers of anti-IsdB antibody-secreting cells in unstimulated periprosthetic bone marrow from the PJI patient, which increased with stimulation. However, these cells were only detectable in peripheral blood mononuclear cells following stimulation. Furthermore, anti-Gmd antibody-secreting cells were undetectable in the bone marrow of this PJI patient at the time of revision surgery. This patient also had very high titers of anti-IsdB antibodies in serum, blood plasma, and bone marrow plasma. Anti-IsdB and anti-Gmd antibody-secreting cells were only detected in bone marrow for one of the uninfected patients. Collectively, the results are consistent with the hypothesis that anti-IsdB antibodies are associated with disease and related to the presence of IsdB-specific MBC and plasmablasts. These case reports demonstrate the utility of ELISpot to assess local vs. systemic antigen-specific humoral responses in THA patients and support a larger study to test the hypothesis that anti-IsdB antibody-secreting cells proximal to S. aureus infected implants are associated with recalcitrant infections and septic death.