Research ArticleHEPATITIS

Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection

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Science Translational Medicine  27 Jun 2018:
Vol. 10, Issue 447, eaap9328
DOI: 10.1126/scitranslmed.aap9328
  • Fig. 1 hNTCP/BAC-NRG mice facilitate uptake of HBVcc and HDVcc.

    (A) hNTCP expression as compared to housekeeping gene HPRT1 in hNTCP-BAC C57BL/6 (n = 4), hNTCP/BAC-NRG (n = 12) with C57BL/6 (n = 2), and NRG (n = 2) mice using relative RT-qPCR and comparing ΔΔCt. Mice were challenged with infectious 5-ethynyl-2′-deoxycytidine (EdC)–labeled HBVcc. (B) Quantification of HBVcc-EdC entry into murine hepatocytes (percent cells positive for HBV DNA in an entire slide section) of hNTCP/BAC-NRG (n = 3) versus NRG (n = 3) wild-type (WT) animals. (C) Representative images of HBVcc-EdC entry in hNTCP/BAC-NRG (top) and NRG WT (bottom) hepatocytes. HBV DNA (red) and nuclei (blue) were observed (scale bars, 200 μm). (D) HBsAg quantification over 2 weeks for hNTCP/BAC-NRG (blue, n = 5) and NRG WT (red, n = 5) mice challenged with HBVcc. Quantitation of HDV RNA in serum (E), HDV genomic RNA in liver (F), HDV antigenomic in liver (G), in hNTCP/BAC-NRG (n = 4) versus WT NRG (n = 4) mice with or without expression of HBV envelope proteins. The red dashed lines separate the hNTCP-expressing (left) from the nonexpressing mice (right) in each of the panels. hNTCP/BAC/1.3× HBV HDD NRG mice were challenged with patient-derived HDV (HDVpat) virions (n = 6), HDVcc, or were noninfected. (H) Longitudinal HBsAg data, (I) longitudinal HDV RNA in the serum of animals, and (J) quantification of genomic HDV RNA in the liver. All data are represented as ±SEM. Statistical significance was as follows: ***P ≤ 0.001, ****P ≤ 0.0001, using an ordinary one-way analysis of variance (ANOVA) with a Bonferroni’s multiple comparisons test. AU, arbitrary units.

  • Fig. 2 HDV infects hNTCP/BAC/1.3× HBV HDD NRG mice through native entry mechanisms.

    (A) Schematic of time course for peptide inhibition assay. Normalized quantification of HDV RNA in hNTCP/BAC/1.3× HBV HDD NRG mice treated with mock (black, n = 5), preS1–fluorescein isothiocyanate peptide (blue, n = 5), or a noninhibitory control (Ctrl) peptide (red, n = 5) in serum (B) or liver (C). All data are represented as ±SEM. Statistical significance was as follows: *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, using an ordinary one-way ANOVA with a Bonferroni’s multiple comparisons test.

  • Fig. 3 Characterization of persistent HDV infection in hNTCP/BAC-NRG mice.

    hNTCP/BAC/1.3× HBV HDD NRG (blue, n = 8) or hNTCP/BAC/AdV-HBV Env (red, n = 5) mice challenged with HDV. HDV RNA in serum (A); HBsAg quantification (B); firefly luciferase quantification in mice over time (C). (D) In vivo imaging system image of hNTCP/BAC/AdV-HBV Env mouse (left) compared to NRG WT mice (right). (E) HDV genomic RNA (red) and DAPI (4′,6-diamidino-2-phenylindole) (blue) visualization by PLAYR technique in HDV-challenged hNTCP/BAC/AdV-HBV Env (left) versus NRG WT (right). Scale bars, 200 nM. (F) Quantification of HDV genomic RNA in hNTCP/BAC/AdV-HBV Env versus NRG WT mice (percent cells HDV RNA positive/slide imaged). (G) HDV RNA quantification by RT-qPCR in the liver of hNTCP/BAC/1.3× HBV (blue), hNTCP/BAC/AdV-HBV Env (red), and NRG WT mice (black). (H) Highlighter plot of Rbz domain in hNTCP/BAC/1.3× HBV HDD mice at days 3 and 56 after infection (red, transversion mutations; light blue, transition mutations). (I) Highlighter plot of HDVAg sequence for hNTCP/BAC/1.3× HBV-NRG animals (orange, nonsynonymous mutations). All data are represented as ±SEM. Statistical significance was as follows: *P < 0.05, using an ordinary one-way ANOVA with a Bonferroni’s multiple comparisons test. Adeno, adenosine. bp, base pair.

  • Fig. 4 Single and combination therapies with MyrB and LNF efficiently suppress HDV viremia in vivo.

    (A) Schematic of drug treatment experimental time course. (B) Longitudinal HBsAg ELISA data. (C) Longitudinal analysis of HDV RNA in serum of mock carrier control, LNF, MyrB, and dually treated groups. (D) HDV RNA in liver at the treatment endpoint (18 days after HDV infection, 14 days after drug treatment) (n = 6 for each treatment condition). (E) HDV RNA in liver of HDV-challenged hNTCP/BAC-NRG mice that had drug treatment stopped (n = 4 for each drug condition). All data are represented as ±SEM. Statistical significance was as follows: *P < 0.05, using an ordinary one-way ANOVA with a Bonferroni’s multiple comparisons test.

  • Fig. 5 Characterization of HDV acute infection in immunocompetent hNTCP-BAC C57BL/6 mice.

    (A) Schematic of hNTCP/BAC C57BL/6 and 1.3× HBV tg mice crossed to generate hNTCP/BAC/1.3× HBV tg C57BL/6 mice. (B) HBsAg quantification of hNTCP/BAC/1.3× HBV tg versus HBV tg mice over a month’s time. NEG indicates the threshold for the assay above which HBsAg levels are considered positive. Normalized HDV RNA in serum (C) and liver (D) of hNTCP/BAC/1.3× HBV tg, hNTCP/BAC tg, and HBV tg animals. (E) Highlighter plot analysis of HDVAg sequences in immunocompetent hNTCP/BAC/1.3× HBV tg C57BL/6 mice (day 14) versus hNTCP/BAC/1.3× HBV-NRG mice (day 56) (red, site of mutation). For each time point, hNTCP only (n = 4), hNTCP/BAC/1.3× HBV tg (n = 4), and HBV tg (n = 4) mice were euthanized. All data are represented as ±SEM.

  • Fig. 6 Characterization of histopathology in HDV-challenged hNTCP/BAC/1.3× HBV tg animals.

    (A) ALT concentrations for hNTCP/BAC/1.3× HBV tg, hNTCP-BAC tg, HBV tg, and WT animals. (B) Histopathological analysis of the liver from hNTCP/BAC/1.3× HBV tg and HBV tg animals. Scale bars, 400 μm. (C) Normalized fold change of ISGs MX1, IP-10, OASL2, and PKR (RNA-activated protein kinase R) in the livers of hNTCP/BAC/1.3× HBV tg animals compared to HBV tg. (D) Cytokine analysis in sera of HDV-challenged hNTCP/BAC/1.3× HBV and HBV tg C57BL/6 animals at days 0, 3, and 14 after infection. Cellular immune response in the spleens of HDV-challenged hNTCP/BAC/1.3× HBV tg, hNTCP tg, HBV tg, and WT mice: NK cells (E), NK T cells (F), and mucosal-associated invariant T cells (MAIT) (G). Frequencies of CD45+ lymphocytes are indicated. Each data point is the average of four different animals. All data are represented as ±SEM. Statistical significance was as follows: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, using an ordinary one-way ANOVA with a Bonferroni’s multiple comparisons test. GM-CSF, granulocyte-macrophage colony-stimulating factor.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/447/eaap9328/DC1

    Materials and Methods

    Fig. S1. Schematic of the modified BAC containing the SLC10A1 gene used for generating the hNTCP tg mice on the NRG and C57BL/6 backgrounds.

    Fig. S2. hNTCP expression in liver and other organs of hNTCP/BAC-NRG mice on the C57BL/6 and NRG backgrounds.

    Fig. S3. EdC labeling of the HBV genome does not affect viral fitness.

    Fig. S4. Quantification of HBV replication intermediates in HBV-infected hNTCP/BAC-NRG mice.

    Fig. S5. Delivery of 1.3× HBV plasmid leads to sustained HBsAg secretion over time.

    Fig. S6. HBsAg levels remain stable in HDV-infected hNTCP/BAC/1.3x HBV HDD-NRG mice over the experimental time course.

    Fig. S7. Evidence for RNA editing in HDV genomes isolated from livers of HDV-infected hNTCP/BAC/1.3× HBV-NRG mice.

    Fig. S8. Adenoviral delivery of HBV envelope proteins leads to expression of all three forms of HBsAg large (L), medium (M), and small (S).

    Fig. S9. Assessment of HDVAg in HDV-challenged hNTCP/BAC-NRG mice.

    Fig. S10. Longitudinal sequence analysis of hypervariable domain in hNTCP/BAC/1.3× HBV HDD NRG mice.

    Fig. S11. Longitudinal sequence analysis of Rbz domain in hNTCP/BAC/1.3× HBV HDD NRG mice.

    Fig. S12. Predicted effect of the A/G mutation at position 46 of HDV Rbz on RNA secondary structure.

    Fig. S13. Longitudinal sequence analysis of HDVAg domain in hNTCP/BAC/1.3× HBV HDD NRG mice.

    Fig. S14. Longitudinal sequence analysis of HDVAg domain in immunocompetent hNTCP/BAC/1.3× HBV C57BL/6 mice.

    Table S1. Primary data.

  • Supplementary Material for:

    Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection

    Benjamin Y. Winer, Elham Shirvani-Dastgerdi, Yaron Bram, Julie Sellau, Benjamin E. Low, Heath Johnson, Tiffany Huang, Gabriela Hrebikova, Brigitte Heller, Yael Sharon, Katja Giersch, Sherif Gerges, Kathleen Seneca, Mihai-Alexandru Pais, Angela S. Frankel, Luis Chiriboga, John Cullen, Ronald G. Nahass, Marc Lutgehetmann, Jared E. Toettcher, Michael V. Wiles, Robert E. Schwartz, Alexander Ploss*

    *Corresponding author. Email: aploss{at}princeton.edu

    Published 27 June 2018, Sci. Transl. Med. 10, eaap9328 (2018)
    DOI: 10.1126/scitranslmed.aap9328

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic of the modified BAC containing the SLC10A1 gene used for generating the hNTCP tg mice on the NRG and C57BL/6 backgrounds.
    • Fig. S2. hNTCP expression in liver and other organs of hNTCP-BAC mice on the C57BL/6 and NRG backgrounds.
    • Fig. S3. EdC labeling of the HBV genome does not affect viral fitness.
    • Fig. S4. Quantification of HBV replication intermediates in HBV-infected hNTCP-NRG mice.
    • Fig. S5. Delivery of 1.3× HBV plasmid leads to sustained HBsAg secretion over time.
    • Fig. S6. HBsAg levels remain stable in HDV-infected hNTCP/BAC/1.3× HBV HDD mice over the experimental time course.
    • Fig. S7. Evidence for RNA editing in HDV genomes isolated from livers of HDV-infected hNTCP/BAC/1.3× HBV-NRG mice.
    • Fig. S8. Adenoviral delivery of HBV envelope proteins leads to expression of all three forms of HBsAg large (L), medium (M), and small (S).
    • Fig. S9. Assessment of HDVAg in HDV-challenged hNTCP/BAC-NRG mice.
    • Fig. S10. Longitudinal sequence analysis of hypervariable domain in hNTCP/BAC/1.3× HBV HDD NRG mice.
    • Fig. S11. Longitudinal sequence analysis of Rbz domain in hNTCP/BAC/1.3× HBV HDD NRG mice.
    • Fig. S12. Predicted effect of the A/G mutation at position 46 of HDV Rbz on RNA secondary structure.
    • Fig. S13. Longitudinal sequence analysis of HDVAg domain in hNTCP/BAC/1.3× HBV HDD NRG mice.
    • Fig. S14. Longitudinal sequence analysis of HDVAg domain in immunocompetent hNTCP/BAC/1.3× HBV C57BL/6 mice.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. (Microsoft Excel format). Primary data.

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