Clearance of Staphylococcus aureus from In Vivo Models of Chronic Infection by Immunization Requires Both Planktonic and Biofilm Antigens

Abstract

Staphylococcus aureus is a causative agent of chronic biofilm-associated infections that are recalcitrant to resolution by the immune system or antibiotics. To combat these infections, an antistaphylococcal, biofilm-specific quadrivalent vaccine against an osteomyelitis model in rabbits has previously been developed and shown to be effective at eliminating biofilm-embedded bacterial populations. However, the addition of antibiotics was required to eradicate remaining planktonic populations. In this study, a planktonic upregulated antigen was combined with the quadrivalent vaccine to remove the need for antibiotic therapy. Immunization with this pentavalent vaccine followed by intraperitoneal challenge of BALB/c mice with S. aureus resulted in 16.7% and 91.7% mortality in pentavalent vaccine and control groups, respectively (P < 0.001). Complete bacterial elimination was found in 66.7% of the pentavalent cohort, while only 8.3% of the control animals cleared the infection (P < 0.05). Further protective efficacy was observed in immunized rabbits following intramedullary challenge with S. aureus, where 62.5% of the pentavalent cohort completely cleared the infection, versus none of the control animals (P < 0.05). Passive immunization of BALB/c mice with serum IgG against the vaccine antigens prior to intraperitoneal challenge with S. aureus prevented mortality in 100% of mice and eliminated bacteria in 33.3% of the challenged mice. These results demonstrate that targeting both the planktonic and biofilm stages with the pentavalent vaccine or the IgG elicited by immunization can effectively protect against S. aureus infection.

Keywords: Staphylococcus aureus; animal model; biofilm; vaccine.

FIG 1

Survival rates over 21 days (A) and percent bacterial clearance (B) for mice immunized with the pentavalent vaccine (n = 12) and nonimmunized mice (n = 12) after challenge by intraperitoneal injection with a 90% lethal dose (LD₉₀) standard infectious dose (3 × 10⁸ to 4 × 10⁸ CFU) of S. aureus. Survival rates are illustrated with a Kaplan-Meier curve, and statistical significance is calculated by the log rank test between mice immunized with the pentavalent vaccine and nonimmunized mice (survival of 83.3% versus 8.3%; P < 0.001). Bacterial clearance is defined by the absence of S. aureus in the kidneys and peritoneal abscess(es). Animals are considered infected if death occurred prior to the experimental endpoint (day 21). Increased clearance of S. aureus is observed in the pentavalent vaccine cohort (66.7%) compared to the control group (8.3%) (P < 0.01), which is statistically significant by a two-tailed Fisher exact test. CFU in surviving mice could not be statistically compared between vaccinated and control animals since only a single mouse survived in the control infected group.

FIG 2

Survival rates over 21 days for mice immunized with the pentavalent vaccine (n = 8), quadrivalent vaccine (n = 8), or SACOL0119 (n = 8) versus nonimmunized mice (n = 8) after challenge by intraperitoneal injection with a high infectious dose (1 × 10⁹ CFU) of S. aureus. Survival rates are illustrated with a Kaplan-Meier curve, and statistical significance is calculated by the log rank test between mice immunized with the pentavalent vaccine and nonimmunized mice (survival of 62.5% versus 12.5%; P < 0.05).

FIG 3

Bacterial clearance of rabbits immunized with the pentavalent antigens (n = 8) versus the control (n = 7) after challenge by intramedullary infection with 1.3 × 10⁶ CFU of S. aureus. Bacterial clearance is defined by the absence of S. aureus in the total bone homogenate. Increased clearance of S. aureus is observed in the pentavalent vaccine cohort (62.5%) compared to the control group (0%), which is statistically significant by a two-tailed Fisher exact test (P < 0.05).

FIG 4

Survival rates over 21 days (A) and percent bacterial clearance (B) for mice passively immunized with IgG against the pentavalent antigens (n = 12), quadrivalent antigens (n = 6), or SACOL0119 (n = 11) versus control mice (n = 11) after challenge by intraperitoneal injection with 5 × 10⁸ to 8 × 10⁸ CFU of S. aureus. Survival rates are illustrated with a Kaplan-Meier curve, and statistical significance is calculated by the log rank test between mice immunized with IgG against the pentavalent vaccine and control mice (survival of 100% versus 36.4%; P = 0.001). Bacterial clearance is defined by the absence of S. aureus in the kidneys and peritoneal abscess(es). Animals are considered infected if death occurred prior to the experimental endpoint (day 21). Increased clearance of S. aureus is observed in the pentavalent vaccine cohort (33.3%) compared to the control group (0%), which is statistically significant by a one-tailed Fisher exact test (P < 0.05).

FIG 5

Bacterial clearance of mice passively immunized with IgG against the pentavalent antigens (n = 5) or quadrivalent antigens (n = 5) versus control mice (n = 5) after challenge by implant infection with 1,000 CFU of S. aureus. Bacterial clearance is defined by the absence of S. aureus in the total bone homogenate. Increased clearance of S. aureus is observed in the pentavalent vaccine cohort (60%) compared to the control group (0%), which is not statistically significant by the Fisher exact test.