Research ArticleInfectious Disease

Human polyclonal immunoglobulin G from transchromosomic bovines inhibits MERS-CoV in vivo

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Science Translational Medicine  17 Feb 2016:
Vol. 8, Issue 326, pp. 326ra21
DOI: 10.1126/scitranslmed.aaf1061
  • Fig. 1. MERS-CoV Tc hIgG production and ELISA titers.

    (A) Study design diagram shows events. Large ticks denote vaccine administration (V1 to V5) to Tc bovines (n = 5). Arrows denote collection of serum and/or plasma. Double arrows on days 10 and 14 after V2 to V5 indicate high-volume plasma collection. (B) Mean MERS-CoV anti-spike ELISA antibody titers (U/ml) plus SD in Tc bovine serum samples resulting in an optical density at 450 nm (OD450) reading 2.5-fold higher than blank (see table S1A). *P ≤ 0.0003, significant difference between V2 to V5 versus pre-V1D0 serum (Dunnett’s test following ANOVA; see table S1B). Tc bovine #2254 had a single measurement at V5 that precluded statistical testing. (C) Mean MERS-CoV ELISA antibody (Ab) titers of SAB-300 and SAB-301 plus SD versus negative control Tc hIgG. *P < 0.0001, significant difference between SAB-300 or SAB-301 versus negative control Tc hIgG (Dunnett’s test following ANOVA; see table S1, C and D). For (B) and (C), all samples but #2254 V5 serum were independently tested in duplicate or more (n ≥ 2).

  • Fig. 2. MERS-CoV neutralization assays.

    (A) Sera from vaccinated (V2 to V5) bovines (n = 5) were evaluated for the quantity of neutralizing antibody (μg) that inhibited MERS-CoV infection in Vero E6 cells as measured by FRNA50. Anti-spike rabbit Ig served as a positive control (see table S2A). (B) SAB-300 and SAB-301 antibodies were evaluated for the quantity of neutralizing antibody (μg) that inhibited infection of MERS-CoV in Vero cells as indicated by FRNA50 versus positive control anti-spike rabbit Ig. *P < 0.001, significant difference between SAB-301 and anti-spike control (Dunnett’s test following ANOVA; see table S2B). (C) Serial dilutions of SAB-300 and SAB-301 and negative control Tc hIgG were evaluated for the ability to neutralize MERS-CoV by TCID50 assay (LOD 158 TCID50/ml or 0.04% of media alone control) (see table S2C). Higher percentage means less inhibition of infection. *P ≤ 0.0003, significant difference between samples and media alone; **P = 0.0002, significant difference between SAB-301 and Tc hIgG control. Below LOD and no P value is calculable (Dunnett’s test following ANOVA; see table S2C). SD is given. For (A) to (C), all samples were independently tested in triplicate or more (n ≥ 3).

  • Fig. 3. ADE assay.

    RT-PCR analysis of MERS-CoV mRNA from nonpermissive Raji cells that were preincubated with serum containing SAB-300, SAB-301, negative control Tc hIgG or with mock control and then infected with MERS-CoV. At 48 hours after infection, RNA was isolated and assayed for amount of MERS-CoV–specific transcript by TaqMan RT-PCR. (A) Using primers specific for the E (UpE) gene, fold change and SD of MERS-CoV–specific genomic RNA are compared to those observed with mock samples (see table S3, A, B, and D). (B) MERS-CoV replicating RNA is analyzed with primers specific for the leader primer region (see table S3, A, C, and D). SD is given, and all samples were independently tested in triplicate (n = 3). No significant differences from mock control were observed (P > 0.05, all comparisons, Dunnett’s test following ANOVA).

  • Fig. 4. Inhibition of MERS-CoV replication in vivo.

    SAB-301 antibodies were injected into Ad5-hDPP4–transduced BALB/c mice (n = 3 per group per time point) intraperitoneally. (A and B) Mice received SAB-301 (A) 12 hours before or (B) 24 or 48 hours after intranasal infection with 1 × 105 PFU of MERS-CoV–EMC/2012 strain. Virus titers (log10 PFU/g tissue) in the lungs and SD were measured at days 1, 3, and 5 after infection in (A) and at day 5 after infection in (B). Mice receiving100 or 500 μg of SAB-301 12 hours before challenge had significantly lower lung titers (P < 0.0001) on days 1, 3, and 5 after infection compared to that observed with untreated controls or to control Tc hIgG (Dunnett’s test following ANOVA). Mice receiving SAB-301 antibodies 24 or 48 hours after MERS-CoV challenge had significantly lower lung viral titers at day 5 after infection compared to untreated controls or to control Tc hIgG (P ≤ 0.0001, Dunnett’s test following ANOVA). All samples were independently tested in duplicate or more (n ≥ 2).

  • Table 1. Human IgG subclass proportions of SAB-300, and SAB-301 antibodies.

    Human IVIG, SAB-300, and SAB-301 were analyzed for the IgG subclass proportions by ELISA. Percentage of each subclass is presented as compared to the positive control. Chimeric antibodies (containing human κ and bovine heavy chain) were less than 1%, and other bovine proteins were less than 100 ppm.

    AntibodyIgG1 (%)IgG2 (%)IgG3 (%)IgG4 (%)
    Human IVIG*67262.62.5
    SAB-300990.270.130.08
    SAB-301936.20.070.05

    *Human-derived IVIG served as the positive control and was run on the same IgG subclass ELISA plates with the other antibodies.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/8/326/326ra21/DC1

      Materials and Methods

      Tables S1A. Source data for Fig. 1B.

      Table S1B. P value of Dunnett’s test (V2, V3, V4, or V5 versus pre-V1D0) for Fig. 1B.

      Table S1C. Specific titer activity of purified antibodies for Fig. 1C.

      Table S1D. P value of Dunnett’s test (SAB-300 or SAB-301 versus negative control) for Fig. 1C.

      Table S2A. Source data for Fig. 2 (A and B).

      Table S2B. P value of Dunnett’s test for anti-spike antibody versus SAB-300 and SAB-301 for Fig. 2B.

      Table S2C. Source data and P value of Dunnett’s test for Fig. 2C.

      Table S3A. Source data for Fig. 3 (A and B).

      Table S3B. Source data for Fig. 3A.

      Table S3C. Source data for Fig. 3B.

      Table S3D. P value of Dunnett’s test for Fig. 3 (A and B).

      Table S4A. Source data for Fig. 4A.

      Table S4B. Source data for Fig. 4B.

    • Supplementary Material for:

      Human polyclonal immunoglobulin G from transchromosomic bovines inhibits MERS-CoV in vivo

      Thomas Luke,* Hua Wu, Jincun Zhao, Rudragouda Channappanavar, Christopher M. Coleman, Jin-An Jiao, Hiroaki Matsushita, Ye Liu, Elena N. Postnikova, Britini L. Ork, Gregory Glenn, David Flyer, Gabriel Defang, Kanakatte Raviprakash, Tadeusz Kochel, Jonathan Wang, Wensheng Nie, Gale Smith, Lisa E. Hensley, Gene G. Olinger, Jens H. Kuhn, Michael R. Holbrook, Reed F. Johnson, Stanley Perlman, Eddie Sullivan, Matthew B. Frieman

      *Corresponding author. E-mail: thomas.c.luke.ctr{at}mail.mil

      Published 17 February 2016, Sci. Transl. Med. 8, 326ra21 (2016)
      DOI: 10.1126/scitranslmed.aaf1061

      This PDF file includes:

      • Materials and Methods

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Tables S1A (Microsoft Excel format). Source data for Fig. 1B.
      • Table S1B (Microsoft Excel format). P value of Dunnett’s test (V2, V3, V4, or V5 versus pre-V1D0) for Fig. 1B.
      • Table S1C (Microsoft Excel format). Specific titer activity of purified antibodies for Fig. 1C.
      • Table S1D (Microsoft Excel format). P value of Dunnett’s test (SAB-300 or SAB-301 versus negative control) for Fig. 1C.
      • Table S2A (Microsoft Excel format). Source data for Fig. 2 (A and B).
      • Table S2B (Microsoft Excel format). P value of Dunnett’s test for anti-spike antibody versus SAB-300 and SAB-301 for Fig. 2B.
      • Table S2C (Microsoft Excel format). Source data and P value of Dunnett’s test for Fig. 2C.
      • Table S3A (Microsoft Excel format). Source data for Fig. 3 (A and B).
      • Table S3B (Microsoft Excel format). Source data for Fig. 3A.

      [Download Tables S1 to S4]

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