Research ArticleInfectious diseases

VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans

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Science Translational Medicine  21 Jun 2017:
Vol. 9, Issue 395, eaam5434
DOI: 10.1126/scitranslmed.aam5434
  • Fig. 1. Experimental workflow for identification of heterotypic and homotypic RV human mAbs.

    Intestinal tissue from five bariatric surgery patients were isolated, B cells were enriched and live, TLP-binding IgA+ ASCs were single cell–sorted. Barcode-based amplification of paired IGHV/IGLV genes from the same B cell was used to generate dendrograms of paired intestinal Ab sequences from each donor. Selected clonal and singleton Ab sequences were cloned and expressed as recombinant human mAbs. mAbs were characterized in terms of binding specificity to RV proteins and in vitro and in vivo neutralization capacities against distinct RV serotypes.

  • Fig. 2. Identification of TLP-binding intestinal ASCs in adult donors.

    (A) CDC-9 TLP-binding intestinal ASCs were identified on the basis of CD20lo/−CD27hiCD38hi surface expression. Contour plots from a representative donor are shown. At least 200,000 events were acquired per sample. (B) The frequency of IgA+ and IgA ASCs as a proportion of total intestinal ASCs (left) and of TLP-binding IgA+ and IgA ASCs as a proportion of total IgA+ and IgA ASCs (right), as determined by FACS in five donors. (C) Frequency of IgA+ ASCs as a proportion of total intestinal B cells (left) and DLP-binding IgA+ ASCs as a proportion of total IgA+ ASCs (right), as determined by ELISPOT. Symbols represent the frequencies of individual donors. Red lines and bars represent the median frequency.

  • Fig. 3. Dendrograms of sorted RV TLP-reactive intestinal IgA+ ASCs.

    HC and LC dendrograms of sorted TLP-binding intestinal IgA+ ASCs from five donors. Each peripheral node depicts sequenced VH and VL regions derived from a single cell. Red lines indicate clonal families. Ig V gene sequences that were selected for cloning and expression of recombinant mAbs are numbered. Stars denote Abs that bound to RV proteins. Circled stars denote neutralizing Abs. Squares denote Abs that did not bind to RV.

  • Fig. 4. Minimally neutralizing VP8* mAb interferes with the capacity of strong neutralizing VP5* or VP8* mAbs.

    VP8* mAb #9 (200 ng/ml) was preincubated with Wa for 1 hour before the addition of potent neutralizing mAb #41 (VP5*) (A) or #47 (VP8*) (B) for an additional hour. The mixtures were then added to MA104 cells, and the neutralization assays were run as described.

  • Fig. 5. Human mAbs mediate heterotypic and homotypic protection from RV-induced diarrheal disease in mice.

    Five-day-old 129sv suckling mice (six to eight per group) were inoculated with 106 plaque-forming units (PFU) of indicated RV strains preincubated for 1 hour with human anti-VP7 (A and B) and anti-VP4 (C) mAbs (5 μg/ml) or with media. The percentage of pups with diarrhea up to 4 days after challenge is shown.

  • Table 1. Summary of the protein specificity of RV-specific human recombinant mAbs. CF, clonal family; S, singleton; ND, VP5* or VP8* specificity not determined.
    mAb no.Subject IDViral protein
    specificity
    Minimum
    concentration
    for TLP binding
    (ng/ml)
    G serotype or
    P genotype
    binding activity
    In vitro
    neutralization
    activity
    Clonal family or
    singleton
    2001VP4VP5*5.00P[4] and P[8]YesCF
    30002VP5*5.00P[1], P[4], P[6], P[7], and P[8]YesCF
    33002VP5*0.50P[1], P[3], P[4], P[6], P[7], and P[8]YesCF
    41003VP5*0.01P[1], P[3], P[4], P[6], P[7], and P[8]YesCF
    4001VP8*0.50P[4] and P[8]NoCF
    6*004VP8*5.00P[4] and P[8]NoCF
    8004VP8*0.05P[4] and P[8]NoCF
    9004VP8*0.01P[4] and P[8]YesCF
    11004VP8*0.01P[4] and P[8]NoCF
    12005VP8*0.05P[4] and P[8]NoCF
    13005VP8*5.00P[8]NoCF
    14005VP8*0.50P[4] and P[8]NoCF
    15005VP8*0.50P[4] and P[8]NoS
    16005VP8*0.01P[4] and P[8]NoCF
    18005VP8*0.05P[4], P[6], and P[8]NoCF
    19005VP8*0.05P[4] and P[8]YesCF
    20005VP8*0.01P[4] and P[8]NoS
    21005VP8*0.50P[4] and P[8]NoS
    23005VP8*50.00P[4] and P[8]NoS
    29002VP8*0.05P[8]NoS
    31002VP8*0.50P[4] and P[8]NoCF
    35002VP8*0.50P[1], P[4], P[7], and P[8]NoS
    43005VP8*0.50P[8]NoS
    44002VP8*0.50P[4] and P[8]NoCF
    55001VP8*500.00P[4], P[6], and P[8]NoCF
    60002VP8*0.50P[1], P[3], P[6], and P[8]NoCF
    47002VP8*0.50P[4] and P[8]YesS
    62003ND5.00P[4] and P[8]NoCF
    22005VP75.00G1NoS
    270020.01G1, G5, and G9YesCF
    460020.50G1YesS
    570020.50G1, G2, G3, G4, and G5YesS
    10*004VP60.5G1, G2, G3, G4, G5, and G6NoCF

    *IgA. All other mAbs were expressed as IgG.

    †Reacts with TLPs and DLPs. All other mAbs react only with TLPs.

    • Table 2. Neutralization titers of RV-specific human mAbs against indicated RV strains and G and P types. —, no neutralizing activity.
      RV strain neutralized (G,[P])*
      mAb no.Subject
      ID
      RV protein
      specificity
      WaDS1RRVST3OSUNCDVWI61L26Neutralizing
      activity
      G1P[8]G2P[4]G3P[3]G4P[6]G5P[7]G6P[1]G9P[8]G12P[4]
      2001VP4VP5*4.94.9625.0Heterotypic
      30002VP5*312.51.29.878.1312.51.2Heterotypic
      33002VP5*4.839.139.1156.3156.3Heterotypic
      41003VP5*0.639.19.84.978.119.52.44.9Heterotypic
      9004VP8*78.1312.5Homotypic
      19005VP8*78.1Homotypic
      47002VP8*1.24.978.1Heterotypic
      27002VP70.3312.5156.3Homotypic
      460022.4Homotypic
      570022.44.91.219.5Heterotypic

      *Minimum neutralizing concentration (nanograms per milliliter).

      • Table 3. Neutralizing potency of different VP4 or VP7 mAbs against escape variants.
        Parental
        virus/variants
        Mutation siteVP4 Neutralizing
        mAbs*
        #41#33#47
        Wa0.64.91.2
        Wa41v-1-1Amino acid 392 (A→E)>625.0>625.01.2
        Wa41v-2-1Amino acid 123 (G→D)0.64.9156.3
        VP7 Neutralizing
        mAbs*
        #27#57#46
        DxRRV0.62.42.4
        DRV57v-30-1Amino acid 126 (D→N)0.6>625.02.4
        DRV27v31-1Amino acid 151 (D→H)19.50.678.1

        *Minimum neutralizing concentration (nanograms per milliliter).

        Supplementary Materials

        • www.sciencetranslationalmedicine.org/cgi/content/full/9/395/eaam5434/DC1

          Fig. S1. Identification of RV-specific B cells by flow cytometry using TLP-Cy5.

          Table S1. Fluorescently tagged Abs used for FACS staining.

          Table S2. Human and animal RVs used in the study.

          Table S3. Summary of the binding reactivities of RV-specific human recombinant mAbs.

          Table S4. Molecular characteristics of RV neutralizing human mAbs.

          Table S5. Primers used for amplifying RV (Wa) genes.

          Table S6. IGHV and IGLV nucleotide sequences of all Abs in the study.

          Table S7. Primary data for in vivo RV challenge experiments.

        • Supplementary Material for:

          VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans

          Nitya Nair, Ningguo Feng, Lisa K. Blum, Mrinmoy Sanyal, Siyuan Ding, Baoming Jiang, Adrish Sen, John M. Morton, Xiao-Song He, William H. Robinson, Harry B. Greenberg*

          *Corresponding author. Email: hbgreen{at}stanford.edu

          Published 21 June 2017, Sci. Transl. Med. 9, eaam5434 (2017)
          DOI: 10.1126/scitranslmed.aam5434

          This PDF file includes:

          • Fig. S1. Identification of RV-specific B cells by flow cytometry using TLP-Cy5.
          • Table S1. Fluorescently tagged Abs used for FACS staining.
          • Table S2. Human and animal RVs used in the study.
          • Table S3. Summary of the binding reactivities of RV-specific human recombinant mAbs.
          • Table S4. Molecular characteristics of RV neutralizing human mAbs.
          • Table S5. Primers used for amplifying RV (Wa) genes.
          • Table S6. IGHV and IGLV nucleotide sequences of all Abs in the study.
          • Table S7. Primary data for in vivo RV challenge experiments.

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