Immunization with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) generates protective immunity against S. aureus abscess formation in mice. BALB/c mice were mock-immunized (A and B) or immunized with the combined vaccine (C and D) and challenged by retroorbital infection with S. aureus Newman. Four days after infection, animals were killed, and the kidneys were removed. Organ tissue was fixed in formalin, thin-sectioned, and stained with hematoxylin/eosin. Microscopic images of whole kidneys (A and C) or organ tissue at higher magnification (B and D) revealed abscess formation only in mock-immunized animals. Similar results were obtained for six organ tissues in each group. (Scale bars: A and C, 1 mm; B and D, 0.1 mm.) (B) Staphylococcal abscess (black arrow) with a central concentration of staphylococci (white arrow). (D) PMN infiltrate (black arrow).

Immunization with the combined vaccine generates protective immunity against lethal challenge with five different clinical S. aureus isolates. Mice (n = 10) were immunized with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) or with PBS, challenged by intraperitoneal injection with clinical S. aureus isolates (3–10 × 10⁹ cfu), and monitored for 7 days for survival. Compared with animals receiving mock-immunization (PBS), the significance of protective immunity generated by the combined vaccine was measured with Fisher's exact test: (A) N315, P = 0.06502; (B) NRS248, P = 0.00036; (C) NRS252, P = 0.03215; (D) USA100, P = 0.00542; and (E) USA400, P = 0.00542.

Antibodies against IsdA, IsdB, SdrD, and SdrE mediate complement-dependent opsonophagocytosis. Phagocytic assays were performed to determine the mechanism of protection afforded by surface-protein immunizations. Rabbit antibodies, rabbit complement, freshly isolated human PMNs, and S. aureus were incubated and plated on agar medium to measure bacterial survival as cfu; then the percent of killing was calculated. Antibodies were added either as individual antisera raised against one surface protein (α-IsdA, α-IsdB, α-SdrD, or α-SdrE) or as a mixture of antisera (α-IsdA + α-IsdB + α-SdrD + α-SdrE) to PMNs. Opsonsonophagocytic killing of staphylococci in the presence of complement was significantly increased for mixed antisera compared with individual surface-protein antisera (P < 0.03). Error bars represent the SEM.

Immunization with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) generates protective immunity against lethal S. aureus challenge. Mice (n = 8–10) were immunized with individual surface-protein antigens (IsdA, IsdB, SdrD, or SdrE), with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) or with PBS. Animals were challenged by intraperitoneal injection of S. aureus Newman (2 × 10¹⁰ cfu), and then they were monitored for 7 days. Compared with animals receiving mock-immunization (PBS), the significance of protective immunity generated by various antigens was measured with Fisher's exact test: IsdA, P = 0.34372; IsdB, P = 0.22049; SdrD, P = 0.24006; SdrE, P = 0.31508; and combined vaccine, P = 0.02941. Compared with animals receiving the combined vaccine, the significance of protective immunity was measured with Fisher's exact test: PBS, P = 0.02941; IsdA, P = 0.02941; IsdB, P = 0.05294; SdrD, P = 0.00377; and SdrE, P = 0.01131.