Research ArticleIMMUNOLOGY

Neutralization of pertussis toxin by a single antibody prevents clinical pertussis in neonatal baboons

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Science Advances  05 Feb 2020:
Vol. 6, no. 6, eaay9258
DOI: 10.1126/sciadv.aay9258
  • Fig. 1 Hu1B7 and aglycosylated hu1B7-N297A antibodies confer similar protection against B. pertussis challenge in mice.

    Four-week-old Balb/c mice were administered antibody or PBS intraperitoneally in a 200-μl volume 2 hours before infection with 107 CFU B. pertussis (Bp) strain D420. Infection severity was assessed after 7 days by (A) CD45+ leukocytosis (WBC) and (B) B. pertussis lung colonization (no colonies were recovered for PBS-treated, uninfected mice). Significance was determined by two-way analysis of variance (ANOVA) with Tukey post hoc test; *P < 0.05; ****P < 0.0001.

  • Fig. 2 Extended half-life antibody hu1B7-YTE persists longer than hu1B7 in neonatal baboons.

    Serum antibody concentrations were determined by PTx ELISA and fit to determine the β-decay rate and serum half-life of (A) hu1B7 and (B) hu1B7-YTE before infection. Data for each treated animal are shown with a unique symbol and line fit with the average decay for each antibody shown in a bold red line. The two datasets are significantly different (P < 0.01) on the basis of the two-tailed t test assuming unequal variances with α = 0.05. (C) Hu1B7 was detected in the nasopharyngeal wash (NPW) samples of animals 2 to 4 days after infection, which corresponds to 29 to 42 days after antibody infusion. Each group mean is shown as a red bar (n = 7 per group). Data for all animals are shown except for hu1B7-treated animal T2, which was excluded from this analysis on the basis of the Grubb’s test for outliers with α = 0.05.

  • Fig. 3 Anti-PTx prophylaxis did not affect initial colonization with B. pertussis or antibody response to Fha.

    (A) Nasopharyngeal washes collected from each baboon 3 to 4 days after infection were serially diluted and plated on selective media to quantify the B. pertussis CFU recovered (group means shown as a bar; n = 7 per group). No significant differences between treatment groups were observed (ANOVA; α = 0.05, P = 0.3). (B) Serum anti-Fha titers were determined by ELISA twice weekly throughout the experiment. The baboons had varied, but not significantly different, primary responses to Fha by treatment group; compared on day 27/28 by ANOVA (α = 0.05, P = 0.08; note that only three of seven control animals survived to this date).

  • Fig. 4 Hu1B7 and hu1B7-YTE administration reduces pertussis mortality, leukocytosis, and clinical symptoms in neonatal baboons.

    (A) All antibody-treated animals survived infection, while four of seven controls required euthanasia [95% confidence intervals (CI) in gray; P = 0.0023 for each treated group versus controls; log-rank test]. (B) The maximum WBC count was significantly reduced in antibody-treated baboons relative to controls (**P < 0.01; ANOVA, α = 0.05, P = 0.0003; post hoc comparison with Tukey post hoc test). Hu1B7-treated animal T2 was excluded from this analysis on the basis of the Grubb’s test for outliers with α = 0.05. (C) Leukocytosis was delayed and suppressed for antibody-treated animals (group averages ± SD shown; data for individual animals in fig. S2). The control group WBC was significantly higher than treated groups on days indicated (**P < 0.01), and the onset of leukocytosis was delayed from day 3 for control animals to days 10 to 14 for treated animals (P < 0.01). The dashed red line indicates the upper 95% CI for preinfection WBC and defines WBC elevation. (D) Clinical signs showed reduced maximum cough count (0, none; 1, occasional; 2, frequent; and 3, severe and frequent) with antibody treatment (*P < 0.05 control versus either treated group; ANOVA, α = 0.05, P = 0.005; post hoc comparison with Tukey post hoc test) and (E) improved activity scores (0, immobile and requires euthanasia; 1, very little activity; 2, reduced movement and no jumping/climbing; 3, movement but reduced jumping/climbing; and 4, normal activity) for treated versus control animals (**P < 0.01; ANOVA, α = 0.05, P = 0.005; post hoc comparison with Tukey post hoc test).

  • Fig. 5 Anti-PTx antibody concentrations correlate inversely with leukocytosis.

    (A) Control animals exhibited rapid onset of leukocytosis after infection. For control animals such as C1 that were ultimately able to control infection and did not require euthanasia, leukocytosis diminished rapidly upon appearance of an endogenous anti-PTx response. (B) Most antibody-treated animals exhibited typical antibody clearance kinetics with an early α-decay, followed by a slower β-decay phase, as seen in animal T1. Endogenous anti-PTx antibodies appeared ~12 days after infection but were not always distinguishable from the remaining hu1B7 background. (C) This is apparent in animal Y3, which cleared hu1B7-YTE more slowly than hu1B7. Other baboons had evidence of α, β, and additionally γ-decay kinetics, which correlate closely with the WBC rise and appearance of antidrug antibodies (fig. S4). This was especially pronounced and early in animals (D) T2 and (E) Y5, which shared the same mother. (F) Anti-PTx titers correlate most strongly with WBC counts when both are measured on days 9 to 11 after infection, just after disease is most severe, with control animals shown in circles, hu1B7-treated animals in triangles, and hu1B7-YTE–treated animals in diamonds. The day of infection is indicated by a red marker in both the anti-PTx and WBC data plots. Comparisons for all animals are shown in fig. S3.

  • Table 1 Summary of baboon enrollment and infection data.

    N/A, not applicable.

    Treatment
    group
    Age at
    infection
    (days)
    Weight at
    infection
    (kg)
    Anti-Fha
    titer at
    infection
    (IU/ml)
    Antibody
    β–half-life
    (days)
    Anti-PTx
    titer at
    infection
    (μg/ml)*
    B. pertussis
    CFU (×107/ml,
    day 3/4)
    Anti-Fha
    titer, day
    20/21
    (IU/ml)
    Max WBC
    (*1000/ μl)*
    Survival*
    Control (C)32.3 ± 3.91.2 ± 0.11.8 ± 1.5N/A0.2 ± 0.33.3 ± 3.366 ± 98 (n = 3)40.8 ± 14.93/7
    hu1B7 (T)34.3 ± 3.91.4 ± 0.21.9 ± 1.612 ± 241 ± 121.49 ± 0.6934 ± 2914.8 ± 4.77/7
    hu1B7-YTE (Y)32.9 ± 2.11.3 ± 0.21.4 ± 2.420 ± 594 ± 3215.6 ± 3130 ± 3616.5 ± 9.97/7

    *Only the following categories showed significant differences between treatment groups: antibody titer on day of infection (hu1B7 versus hu1B7-YTE, P < 0.01), survival (hu1B7 or hu1B7-YTE versus controls, P < 0.005), and maximum WBC (hu1B7 or hu1B7-YTE versus controls, P < 0.01).

    †Animal T2 had no detectable hu1B7 titer at the time of infection and was excluded from pharmacokinetic and efficacy analyses on the basis of the Grubb’s test for outliers with α = 0.05.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaay9258/DC1

      Fig. S1. Variants of antibody 1B7 bind PTx equivalently by ELISA.

      Fig. S2. Dosing hu1B7 in the mouse challenge model.

      Fig. S3. Temporal data for WBC count and bacterial colonization for each treatment group.

      Fig. S4. Temporal correlation of anti-PTx and WBC counts for each baboon.

      Fig. S5. Temporal correlation of anti-PTx and antidrug antibody titers for each treated baboon.

      Fig. S6. Pearson correlation of antidrug antibody titers and anti-PTx titers.

      Fig. S7. Presence of 1B7 does not interfere with anti-PTx responses due to vaccination.

      Table S1. Baboon raw data.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Variants of antibody 1B7 bind PTx equivalently by ELISA.
      • Fig. S2. Dosing hu1B7 in the mouse challenge model.
      • Fig. S3. Temporal data for WBC count and bacterial colonization for each treatment group.
      • Fig. S4. Temporal correlation of anti-PTx and WBC counts for each baboon.
      • Fig. S5. Temporal correlation of anti-PTx and antidrug antibody titers for each treated baboon.
      • Fig. S6. Pearson correlation of antidrug antibody titers and anti-PTx titers.
      • Fig. S7. Presence of 1B7 does not interfere with anti-PTx responses due to vaccination.
      • Table S1. Baboon raw data.

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