Protective Efficacy of Monoclonal Antibodies Neutralizing Alpha-Hemolysin and Bicomponent Leukocidins in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia

Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins.

ABSTRACT

Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins. We found that passive immunization with either a monoclonal antibody (MAb) neutralizing alpha-hemolysin or a broadly cross-reactive MAb neutralizing Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB conferred only partial protection, whereas the combination of those two MAbs conferred significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 methicillin-resistant S. aureus epidemic clone.

INTRODUCTION

Staphylococcus aureus is a major public health concern because of its notorious antibiotic-resistant capability (1). S. aureus employs a variety of secreted membrane-damaging toxins to counter-attack the host immune defenses and to disseminate from the primary site of infection, making its treatment difficult and complicated (2, 3). Loss of effectiveness of antibiotic therapies leads to the urgent need to develop alternative control agents, such as monoclonal antibodies (MAbs) (4–7). Engineering of human IgG1 MAbs, such as the triple-amino-acid YTE substitution in the Fc region, results in extended serum half-lives of 80 to 112 days, thus making MAbs ideal for prophylaxis (8).

Alpha-hemolysin (Hla) is a major virulence factor, and its neutralization by Hla-specific MAbs, including MEDI4893*, conferred significant protection in mouse and ferret pneumonia models (9–14) but partial protection in a phase 2 trial that showed 31.9% (90% confidence interval, −7.5% to 56.8%) relative risk reduction for pneumonia development in patients who were mechanically ventilated and colonized with S. aureus (15). MEDI4893* alone was not sufficient to confer full protection in a rabbit necrotizing pneumonia model (14), which may be due to the fact that rabbits are also susceptible to other lung-damaging toxins, including Panton-Valentine leukocidin (LukSF-PV) (16, 17). Rabbit is a more relevant animal species for evaluating the effects of neutralizing MAbs against staphylococcal bicomponent leukocidins, because rabbit and human neutrophils are similarly susceptible to these toxins (16, 18, 19), whereas mouse neutrophils are resistant to LukSF-PV, HlgAB, and HlgCB and are lysed by leukocidin ED (LukED) but to a lesser extent (16, 18).

Here, we report protective efficacy of MEDI4893* and two cross-neutralizing leukocidin MAbs, SAN177 and SAN481, in the rabbit model of necrotizing pneumonia caused by USA300/SF8300, a community-associated methicillin-resistant S. aureus (MRSA) epidemic clone producing multiple leukocidins. The rabbit experimental pneumonia protocol was reviewed and approved by the University of California, San Francisco, Institutional Animal Care and Use Committee and was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. SAN177 and SAN481 (20) were selected from human tonsillar memory B cells, as described previously (21), for their ability to cross-neutralize HlgAB, HlgBC, LukSF-PV, and LukED by binding the F subunits of the leukocidins (C. Tkaczyk and B. R. Sellman, unpublished data). Two animal efficacy studies were performed. In the first study, rabbits were randomly assigned to prophylaxis with 30 mg/kg isotype-matched control MAb (c-IgG), 30 mg/kg MEDI4893*, 30 mg/kg SAN177, or a combination of 15 mg/kg SAN177 and 15 mg/kg MEDI4893*, which were administered intravenously at 24 h before endobronchial challenge with 5.0 × 10⁹ CFU of SF8300 (in 1.8 ml of saline solution), as described previously (17, 19). All rabbits (15 of 15 rabbits) that were pretreated with c-IgG had lethal pneumonia between 6 and 20 h after infection (Fig. 1A). Mortality rates were 67% (10/15 rabbits; P < 0.0001 versus c-IgG by log-rank test) for rabbits pretreated with MEDI4893* and 27% (4/15 rabbits; P < 0.0001 versus c-IgG by log-rank test) for those pretreated with SAN177. Rabbits pretreated with SAN177 plus MEDI4893* had the lowest mortality rate of 7% (1/15 rabbits; P < 0.0001 versus c-IgG by log-rank test), indicating the importance of neutralization of both Hla and bicomponent leukocidins. Lung weight (LW)/body weight (BW) ratios, a quantitative marker of pulmonary edema, were not significantly different among the four experimental groups (Fig. 1B). Lung bacterial counts were significantly lower in rabbits pretreated with SAN177 or SAN177 plus MEDI4893* than in those pretreated with MEDI4893* or c-IgG (Fig. 1C), and spleen and kidney bacterial counts were significantly reduced only in rabbits pretreated with SAN177 plus MEDI4893* (Fig. 1D and E). Gross images of lungs harvested at the time of death or euthanasia at 96 h postinfection demonstrated areas of severe lung necrosis in rabbits pretreated with c-IgG, but areas were reduced in rabbits pretreated with MEDI4893*, SAN177, or SAN177 plus MEDI4893* (Fig. 1F).

FIG 1.

Passive immunization with an anti-Hla MAb or an anti-F subunit broadly cross-reactive antibody (SAN177) that neutralizes LukSF-PV, LukED, and gamma-hemolysin, in a rabbit model of necrotizing pneumonia. (A to E) Kaplan-Meier survival curves (A), LW/BW ratios (B), log₁₀CFU/lung (C), log₁₀CFU/spleen (D), and log₁₀CFU/kidneys (E) were compared for rabbits pretreated intravenously with 30 mg/kg c-IgG (n = 15), 30 mg/kg MEDI4893* (n = 15), 30 mg/kg SAN177 (n = 15), or the combination of 15 mg/kg SAN177 and 15 mg/kg MEDI4893* (n = 15) at 24 h before infection with 5.0 × 10⁹ CFU of USA300/SF8300 in the rabbit model of necrotizing pneumonia. The two-sided log-rank (Mantel-Cox) test was used to evaluate differences in survival for rabbits pretreated with c-IgG versus MEDI4893* (P < 0.0001), c-IgG versus SAN177 (P < 0.0001), c-IgG versus SAN177 plus MEDI4893* (P < 0.0001), SAN177 versus MEDI4893* (P = 0.0142), SAN177 versus SAN177 plus MEDI4893* (P = 0.099), and MEDI4893* versus SAN177 plus MEDI4893* (P = 0.0006), with a P value of <0.0083 (significance level of 0.05 divided by 6 different comparisons) being considered statistically significant, to account for multiple comparisons using the Bonferroni method. Open symbols represent surviving animals, while closed symbols represent animals that were found dead or were euthanized after becoming moribund and were recorded as nonsurviving (B to E). For LW/BW ratios and bacterial counts, all 6 pairwise comparisons were analyzed by nonparametric one-way analysis of variance with the Kruskal-Wallis test followed by Dunn’s multiple-comparison test. n.s., not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (F) Photographs depict gross pathology of representative lungs; these lungs were selected because they had median LW/BW ratios for their respective experimental groups, as shown in panel B.

Although SAN177 conferred significant protection in the rabbit model, there were developability issues associated with this molecule, due to aggregation at high concentrations (unpublished data). Therefore, we identified SAN481 (20), another candidate MAb that binds to LukF, LukD, and HlgB, and evaluated its protective efficacy, with and without MEDI4893*, in the rabbit model of necrotizing pneumonia. In the second study, rabbits were randomly assigned to prophylaxis with 30 mg/kg isotype-matched c-IgG, 30 mg/kg SAN481, or a combination of 15 mg/kg SAN481 plus 15 mg/kg of MEDI4893*, which were administered intravenously 24 h before endobronchial challenge with 7.0 × 10⁹ CFU of USA300 (in 1.8 ml of saline solution), as described previously (17, 19). Eighty percent of rabbits (8/10 rabbits) pretreated with c-IgG had lethal pneumonia between 10 and 25 h after infection (Fig. 2A). Mortality rates were 22% (2/9 rabbits; P = 0.008 versus c-IgG by log-rank test) for rabbits pretreated with SAN481 and 0% (0/10 rabbits; P = 0.0002 versus c-IgG by log-rank test) for those pretreated with SAN481 plus MEDI4893*. The groups pretreated with SAN481 and SAN481 plus MEDI4893* showed significantly reduced LW/BW ratios of 8.24 and 8.33, respectively, compared to 11.22 for the c-IgG-pretreated rabbits, suggesting that SAN481 is potent in reducing acute lung injury. Interestingly, rabbits pretreated with Hla-F#5 MAb, which cross-neutralizes Hla, HlgAB, HlgCB, LukSF-PV, and LukED, showed an increased LW/BW ratio, compared to those pretreated with a control MAb, although it should be noted that Hla-F#5 protected all rabbits from lethal pneumonia (16). Lung, spleen, and kidney bacterial counts were significantly lower in rabbits pretreated with SAN481 or SAN481 plus MEDI4893* than in those pretreated with c-IgG (Fig. 2C to E). Gross images of lungs harvested at the time of death or euthanasia at 96 h postinfection demonstrated areas of severe necrosis in rabbits pretreated with c-IgG, but areas were reduced in rabbits pretreated with SAN481 or SAN481 plus MEDI4893* (Fig. 2F).

FIG 2.

Passive immunization with an anti-Hla MAb or an anti-F subunit broadly cross-reactive antibody (SAN481) that neutralizes LukSF-PV, LukED, and gamma-hemolysin, in a rabbit model of necrotizing pneumonia. (A to E) Kaplan-Meier survival curves (A), LW/BW ratios (B), log₁₀CFU/lung (C), log₁₀CFU/spleen (D), and log₁₀CFU/kidneys (E) were compared for rabbits pretreated intravenously with 30 mg/kg c-IgG (n = 10), 30 mg/kg SAN481 (n = 10), or the combination of 15 mg/kg SAN481 and 15 mg/kg MEDI4893* (n = 10) at 24 h before infection with 6.5 × 10⁹ CFU of USA300/SF8300 in the rabbit model of necrotizing pneumonia. One rabbit pretreated with SAN481 experienced anesthesia-related death immediately before bacterial inoculation; therefore, the rabbit was excluded from subsequent analysis. The two-sided log-rank (Mantel-Cox) test was used to evaluate differences in survival for rabbits pretreated with c-IgG versus SAN481 (P = 0.008), c-IgG versus SAN481 plus MEDI4893* (P = 0.0002), and SAN481 versus SAN481 plus MEDI4893* (P = 0.1246), with a P value of <0.0167 (significance level of 0.05 divided by 3 different comparisons) being considered statistically significant, to account for multiple comparisons using the Bonferroni method. Open symbols represent surviving animals, while closed symbols represent animals that were found dead or were euthanized after becoming moribund and were recorded as nonsurviving (B to E). For LW/BW ratios and bacterial counts, all 3 pairwise comparisons were analyzed by nonparametric one-way analysis of variance with the Kruskal-Wallis test followed by Dunn’s multiple-comparison test. n.s., not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (F) Photographs depict gross pathology of representative lungs; these lungs were selected because they had median LW/BW ratios for their respective experimental groups, as shown in panel B.

Taking these data together, we found that addition of SAN177 or SAN481 to MEDI4893* resulted in enhanced efficacy in the rabbit model of USA300 necrotizing pneumonia. These preclinical data support the continued development of antileukocidin MAbs in combination with an anti-Hla MAb to extend strain coverage to S. aureus strains expressing leukocidins and to prevent life-threatening pneumonia. Future rabbit studies will further evaluate the efficacy of MEDI4893* and SAN481 MAbs for the prevention and adjunctive treatment of MRSA ventilator-associated pneumonia, which are primary indications for these antitoxin MAbs.