Research ArticleInfectious Disease

IVIG-mediated protection against necrotizing pneumonia caused by MRSA

See allHide authors and affiliations

Science Translational Medicine  21 Sep 2016:
Vol. 8, Issue 357, pp. 357ra124
DOI: 10.1126/scitranslmed.aag1153
  • Fig. 1. Human IVIG contains neutralizing antibodies to PVL and Hla.

    (A) Kinetics of pore formation in human PMNs determined by uptake of propidium iodide (PI) in response to the S. aureus toxins LukS-PV and LukF-PV (PVL) at biologically relevant concentrations (see also Fig. 5G). (B and C) Two different lots of IVIG {ClairYg lot nos. 11L00258 [IVIG(L8)] and 11L00443 [IVIG(L3)]} blocked PVL-induced pore formation in human PMNs. (D) Kinetics of neutralization of PVL-induced membrane pore formation in response to human IVIG, which was added at the indicated times after PVL was mixed with human PMNs. (E) Two different lots of IVIG exhibited similar inhibition of rabbit red blood cell lysis in response to varying concentrations of Hla. Results in (A) to (D) are representative of two independent experiments with three human blood donors, and those in (E) are representative of three independent experiments with a single batch of rabbit erythrocytes.

  • Fig. 2. Human IVIG, either alone or in combination with vancomycin or linezolid, confers protection in a rabbit model of necrotizing pneumonia.

    (A) Total human IgG, (B) anti-PVL human IgG, and (C) anti-Hla human IgG in the serum of rabbits taken at the indicated time after intravenous administration of human IVIG (200 mg/kg). Error bars indicate SEM. AU, arbitrary units. (D) Kaplan-Meier survival curves, (E) LW/BW (×103) ratio, and (F) bacterial densities, log10 (CFU per lung) for animals treated intravenously with IVIG (200 mg/kg) once at 1.5 hpi (n = 14 rabbits); vancomycin (30 mg/kg) four times at 1.5, 13, 25, and 37 hpi (n = 15 rabbits); IVIG + vancomycin (n = 13 rabbits); or saline at 1.5 hpi (n = 14 rabbits) with 5.4 × 109 CFU of the SF8300 WT strain. One-sided log-rank (Mantel-Cox) test was used to test hypothesis that survival of animals treated with saline is shorter than survival of those treated with IVIG, vancomycin, or the combination of IVIG + vancomycin, as well as the hypothesis that survival of animals treated with IVIG alone or with vancomycin alone is shorter than those treated with the combination of IVIG + vancomycin, with P < 0.010 (significance level of 0.05 divided by five different comparisons) being considered statistically significant to account for multiple comparisons using Bonferroni method. (G) Kaplan-Meier survival curves, (H) LW/BW (×103) ratio, and (I) bacterial densities, log10 (CFU/lung) for animals treated with IVIG(L3) (200 mg/kg) intravenously once at 1.5 hpi (n = 15 rabbits), linezolid (50 mg/kg) subcutaneously four times at 1.5, 10, 18, and 26 hpi (n = 15 rabbits), IVIG + linezolid (n = 15 rabbits), or IVIG (200 mg/kg) depleted of anti-Hla IgG and anti-LukS/LukF IgG intravenously once at 1.5 hpi (n = 15 rabbits) with 5.3 × 109 CFU of the SF8300 WT strain. One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals treated with saline is shorter than survival of those treated with IVIG, linezolid, or the combination of IVIG + linezolid, as well as the hypothesis that survival of animals treated with IVIG alone or with linezolid alone is shorter than those treated with the combination of IVIG + linezolid, with P < 0.010 (significance level of 0.05 divided by five different comparisons) being considered statistically significant to account for multiple comparisons using Bonferroni method. (E and H) LW/BW (×103) ratio and (F and I) bacterial densities, log10 (CFU per lung), for saline-treated animals were compared to those of each of the other three treatment groups by nonparametric one-way analysis of variance (ANOVA) with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 48 hpi (E and F) or 96 hpi (H and I).

  • Fig. 3. Prophylaxis with IVIG protects against lethal necrotizing pneumonia caused by five clinical community-associated MRSA strains.

    Eight rabbits per experimental group were randomized to receive either saline or IVIG(L8)/IVIG(L3) (200 mg/kg) at 24 hours before infection (hbi) with 5.8 × 109 CFU of USA300/SF8300 (A to C), 5.7 × 109 CFU of USA400 (D to F), 1.5 × 109 CFU of USA1000 (G to I), 6.3 × 109 CFU of USA1100 (J to L), and 6.8 × 109 CFU of ST80 (M to O). One rabbit randomized for prophylaxis with IVIG(L3) had anesthesia-related death before challenge with the ST80 strain (M to O). Kaplan-Meier survival curves were compared with one-sided log-rank (Mantel-Cox) test to evaluate the hypothesis that survival of animals pretreated with saline is shorter than survival of those pretreated with IVIG, with P < 0.05 being considered statistically significant. LW/BW ratio and log10 (CFU per lung) for saline-pretreated animals were compared to those pretreated with IVIG by nonparametric Mann-Whitney U test. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 48 hpi.

  • Fig. 4. Effects of major staphylococcal exotoxins in acute lung injury.

    Comparison of (A to C) LW/BW (×103) and (D to F) log10 CFU per lung for rabbits (n = 9 animals per experimental group) euthanized at 9 hpi with SF8300 WT or isogenic mutants containing deletion of genes encoding PVL (ΔlukSF), Δpsm-α, Δhla, ΔhlgABC, ΔlukED, ΔlukGH (also known as leukocidin A and B), Δseq/sek, and Δselx. (G to J) Concentrations of IL-8 and MCP-1 in the lungs and plasma of rabbits euthanized at 9 hpi with SF8300 WT, or Δpsm-α, Δhla, and ΔlukSF mutant strains. Nonparametric one-way ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used to evaluate differences between WT and each of the mutant strains. (K) Photographs depict gross pathology of representative lungs harvested from rabbits at 9 hpi with the four isogenic SF8300 strains: WT, Δpsm-α, Δhla, and ΔlukSF mutant strains.

  • Fig. 5. Staphylococcal necrotizing pneumonia is mediated principally by two toxins, PVL and Hla.

    Comparison of Kaplan-Meier survival curves (A), LW/BW ratio (×103) (B), and log10 CFU per lung (C) for rabbits challenged with USA300/SF8300 WT strain, or isogenic mutant strains Δpsm-α, Δhla, ΔlukSF, or the double mutant strain ΔhlaΔlukSF (n = 12 animals per experimental group). One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals challenged with SF8300 WT is shorter than survival of those challenged with each of the four mutant strains Δpsm-α, Δhla, ΔlukSF, or ΔhlaΔlukSF with P < 0.0125 (significance level of 0.05 divided by four different comparisons) being considered statistically significant to account for multiple comparisons using the Bonferroni method. Comparison of Kaplan-Meier survival curves (D), LW/BW ratio (×103) (E), and log10 CFU per lung (F) of rabbits challenged with WT strains belonging to the epidemic community-associated MRSA strains USA300/SF8300 (ST8), USA400/MW2 (ST1), USA1000 (ST59), USA1100 (ST30), and ST80 (n = 7 animals per experimental group). Amounts of LukS-PV (G) and Hla (H) produced in the lungs of infected animals. Two-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals challenged with SF8300 WT is not different from survival of those challenged with each of the other four community-associated MRSA strains, with P < 0.0125 (significance level of 0.05 divided by four different comparisons) being considered statistically significant to account for multiple comparisons using the Bonferroni method. The following log-rank test P values are for comparison of SF8300 WT versus each of the other community-associated MRSA strains: P = 0.20 for SF8300 versus USA400, P = 0.19 for SF8300 versus USA1000, P = 0.46 for SF8300 versus USA1100, and P = 0.006 for SF8300 versus ST80. For both studies, the LW/BW ratio (×103), bacterial densities in lungs, and toxin concentrations in lungs for animals challenged with SF8300 were compared to each of the four other strains by nonparametric one-way ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 36 hpi.

  • Fig. 6. Anti-Hla and anti-PVL antibodies affinity purified from human IVIG protect against USA300 community-associated MRSA necrotizing pneumonia.

    Comparison of Kaplan-Meier survival curves (A), LW/BW ratio (×103) (B), and log10 CFU per lung (C) of rabbits pretreated at 1.5 hours before infection with 6.4 × 109 CFU of SF8300 WT strain with the following regimens: saline (n = 10 rabbits), IVIG(L8) (200 mg/kg; n = 12 rabbits), IVIG(L8) (200 mg/kg) depleted of anti-Hla IgG and anti-LukS/LukF IgG (n = 9 rabbits), anti-Hla IgG (1 mg/kg) affinity-purified from IVIG (n = 8 rabbits), anti-LukS IgG (1 mg/kg), and anti-LukF IgG (1 mg/kg) affinity-purified from IVIG (n = 8 rabbits). One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals pretreated with saline is shorter than survival of those pretreated with each of four antibody preparations, with P < 0.0125 (significance level of 0.05 divided by four different comparisons) being considered statistically significant to account for multiple comparisons using the Bonferroni method. LW/BW ratio (×103) and log10 CFU per lung for animals challenged with SF8300 were compared to each of the four other strains by nonparametric one-way ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Comparison of Kaplan-Meier survival curves (D), LW/BW ratio (×103) (E), and log10 CFU per lung (F) of rabbits pretreated at 1.5 hours before infection with 5.1 × 109 CFU of SF8300 WT with depleted IVIG(L3) (200 mg/kg) or IVIG(L3) (200 mg/kg) (eight rabbits per experimental group). Comparison of Kaplan-Meier survival curves (G), LW/BW ratio (×103) (H), and log10 CFU per lung (I) of rabbits pretreated at 1.5 hours before infection with 5.3 × 109 CFU of SF8300 WT with depleted IVIG(L3) (200 mg/kg) or anti-Hla IgG (4 mg/kg) (eight rabbits per experimental group). Comparison of Kaplan-Meier survival curves (J), LW/BW ratio (×103) (K), and log10 CFU per lung (L) of rabbits pretreated at 1.5 hours before infection with 5.4 × 109 CFU of SF8300 WT with depleted IVIG(L3) (200 mg/kg) or anti-LukS/LukF IgG (4 mg/kg) (eight rabbits per experimental group). One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals pretreated with depleted IVIG is shorter than survival of those pretreated with the other antibody preparations, with P < 0.05 being considered statistically significant. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 48 hpi. LW/BW ratio (×103) and bacterial densities in rabbit lung for the different pairwise comparisons were evaluated with the nonparametric Mann-Whitney U test.

  • Fig. 7. Human IVIG protects against pneumonia caused by community-associated MRSA strain USA400 and hospital-associated MRSA strain USA100.

    Comparison of Kaplan-Meier survival curves (A), LW/BW ratio (×103) (B), and log10 CFU per lung (C) of rabbits pretreated at 1.5 hours before infection with 5.5 × 109 CFU of USA400 WT strain with IVIG(L3) (200 mg/kg) depleted of anti-Hla IgG and anti-LukS/LukF IgG or complete IVIG(L3) (200 mg/kg) (eight rabbits per experimental group). Comparison of Kaplan-Meier survival curves (D), LW/BW ratio (×103) (E), and log10 CFU per lung (F) of rabbits pretreated at 1.5 hours before infection with 5.6 × 109 CFU of USA100/NRS382 WT strain with depleted IVIG(L3) (200 mg/kg) or IVIG(L3) (200 mg/kg) (eight rabbits per experimental group). One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals pretreated with depleted IVIG is shorter than survival of those pretreated with the other antibody preparations, with P < 0.05 being considered statistically significant. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 96 hpi. LW/BW ratio (×103) and bacterial densities in rabbit lung for the different pairwise comparisons were evaluated with the nonparametric Mann-Whitney U test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/357/357ra124/DC1

    Materials and Methods

    Fig. S1. SElX does not contribute to lethal infection with USA300/LAC strain in the New Zealand white rabbit model of necrotizing pneumonia.

    Fig. S2. Anti-Hla IgG and anti-LukF/S IgG affinity-purified from IVIG have potent neutralization activities.

    Table S1. Bacterial strains used in the present study.

    Table S2. Oligonucleotides used for construction of in-frame gene deletions using pKOR1 allelic replacement system.

    References (3436)

  • Supplementary Material for:

    IVIG-mediated protection against necrotizing pneumonia caused by MRSA

    Binh An Diep,* Vien T. M. Le, Cedric Badiou, Hoan N. Le, Marcos Gabriel Pinheiro, Au H. Duong, Xing Wang, Etyene Castro Dip, Fábio Aguiar-Alves, Li Basuino, Helene Marbach, Thuy T. Mai, Marie N. Sarda, Osamu Kajikawa, Gustavo Matute-Bello, Christine Tkaczyk, Jean-Philippe Rasigade, Bret R. Sellman, Henry F. Chambers, Gerard Lina*

    *Corresponding author. Email: binh.diep{at}ucsf.edu (B.A.D.); gerard.lina{at}univ-lyon1.fr (G.L.)

    Published 21 September 2016, Sci. Transl. Med. 8, 357ra124 (2016)
    DOI: 10.1126/scitranslmed.aag1153

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. SElX does not contribute to lethal infection with USA300/LAC strain in the New Zealand white rabbit model of necrotizing pneumonia.
    • Fig. S2. Anti-Hla IgG and anti-LukF/S IgG affinity-purified from IVIG have potent neutralization activities.
    • Table S1. Bacterial strains used in the present study.
    • Table S2. Oligonucleotides used for construction of in-frame gene deletions using pKOR1 allelic replacement system.
    • References (3436)

    [Download PDF]

Navigate This Article