Research ArticleVASCULAR REMODELING

Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination

See allHide authors and affiliations

Science Translational Medicine  09 Sep 2015:
Vol. 7, Issue 304, pp. 304ra141
DOI: 10.1126/scitranslmed.aaa3787
  • Fig. 1. NS1 triggers vascular leak–mediated pathogenesis in vivo.

    (A) Ifnar−/− mice were infected intravenously with a lethal dose of DENV2 (107 PFU) alone (n = 7), a sublethal dose of DENV2 (106 PFU) with DENV2 NS1 [5 mg/kg (n = 6) or 10 mg/kg (n = 7)], a sublethal dose of DENV2 with OVA [5 mg/kg (n = 6) or 10 mg/kg (n = 7)] as a negative control, or DENV2 NS1 (10 mg/kg) alone (n = 7). A Kaplan-Meier survival curve is shown, with data derived from two independent experiments. Mice given NS1 (10 mg/kg) alone or OVA (5 or 10 mg/kg) + 106 PFU of DENV2 exhibited statistically significant survival using a nonparametric Mantel-Cox log-rank test (P = 0.022) compared to mice given NS1 (10 mg/kg) + 106 PFU of DENV2. (B to D) Evans blue dye was injected intravenously into mice 3 days after treatment with DENV2 NS1 (10 mg/kg) (n = 7), DENV2 NS1 (20 mg/kg) (n = 4), OVA (10 mg/kg) (n = 6), NS1 (10 mg/kg) + 106 PFU of DENV2 (n = 4), OVA + 106 PFU of DENV2 (n = 4), or 106 (n = 4) or 107 PFU of DENV2 alone (n = 7), as indicated. The dye was allowed to circulate for 1 h before mice were euthanized, tissues were harvested, and Evans blue was extracted in formamide and quantified in (B) lung, (C) liver, and (D) small intestine tissue by measuring absorbance at OD610. In all tissues, the mice given NS1 (20 mg/kg) or NS1 (10 mg/kg) alone or NS1 (10 mg/kg) + 106 PFU of DENV2 had significantly higher levels of Evans blue than the OVA controls. (E) Ifnar−/− mice were administered DENV2 NS1 (10 mg/kg) or OVA (10 mg/kg) alone, NS1 or OVA (10 mg/kg) plus 106 PFU of DENV2, or 106 or 107 PFU of DENV2 alone, as indicated (n = 4 per group). Plasma was collected 3 days after treatment, and the levels of NS1 were measured by NS1-specific capture enzyme-linked immunosorbent assay (ELISA). (B to E) Data were derived from two independent experiments and were analyzed by nonparametric Mann-Whitney analysis. *P < 0.05; **P < 0.01.

  • Fig. 2. NS1 causes increased production of inflammatory cytokines in vivo in the presence or absence of DENV2.

    Ifnar−/− mice were administered DENV2 NS1 (10 mg/kg) or OVA (10 mg/kg) alone, NS1 or OVA (10 mg/kg) plus 106 PFU of DENV2, or 106 or 107 PFU of DENV2 alone, as indicated (n = 4 per group). (A and B) Plasma was collected 3 days after treatment, and the levels of (A) IL-6 and (B) TNF-α were measured by ELISA. Mice given NS1 (10 mg/kg) + 106 PFU of DENV2 or NS1 (10 mg/kg) alone had significantly higher levels of both cytokines than controls receiving OVA. Data were derived from two independent experiments and were analyzed by nonparametric Mann-Whitney analysis. *P < 0.05.

  • Fig. 3. NS1 induces endothelial permeability in human pulmonary endothelial cells in vitro.

    Confluent monolayers of HPMECs grown on Transwell inserts were incubated for 48 h with NS1, and TEER (ohm) was measured at indicated time points. Relative TEER (ratio of resistance values between experimental and untreated cells) was plotted. Data were normalized to inserts containing medium only. (A) Increasing concentrations of DENV2 NS1 (0.5 to 5 μg/ml) or TNF-α (1 ng/ml) were added to HPMEC monolayers, and TEER was measured at the indicated time points. (B) DENV1, DENV2, DENV3, DENV4 NS1, WNV NS1, or DENV2 E protein (5 μg/ml) was added to HPMEC monolayers, and TEER was analyzed at indicated time points. OVA (20 μg/ml) was used as a treatment control. Results are representative of three independent experiments using duplicate Transwells. Error bars indicate SEM. NS1, DENV2 NS1 (5 μg/ml).

  • Fig. 4. NS1 vaccination protects against lethal DENV–induced vascular-leak syndrome.

    (A) Mice were immunized intraperitoneally with 20 μg of DENV2 NS1 (n = 12) or OVA (n = 8) combined with MPLA/AddaVax adjuvants on days 0, 14, and 42 or infected with a sublethal dose (105 PFU) of DENV2 PL046 (n = 12) at day 0; on day 56, half were challenged intravenously with a lethal dose of 107 PFU of DENV2 and half with lethal antibody-enhanced DENV2 infection. A Kaplan-Meier survival curve is shown. Mice immunized with NS1 were significantly protected compared to OVA controls from both ADE (P = 0.003) and high-dose (hi-dose) (P = 0.001) lethal challenge. (B) NS1 levels in serum were measured by NS1-specific capture ELISA in NS1-immunized (n = 6) or OVA-immunized (n = 6) mice and DENV2 PL046–immune (n = 3) mice 3 days after DENV2 lethal challenge. Circulating NS1 was significantly higher in OVA-immunized compared to NS1-immunized mice after challenge; *P < 0.05; **P < 0.01. (C and D) The viral RNA copy number was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in (C) serum and (D) bone marrow 3 days post-lethal challenge. OVA-immunized mice (n = 6) had higher levels of viral RNA in both tissues compared to NS1-immunized mice (n = 6); *P < 0.05; **P < 0.01. The limit of detection is indicated by a dashed line. GAPDH, glyceraldehyde phosphate dehydrogenase. GE, genome equivalent. (E) Mice were immunized with 20 μg of NS1 from DENV1 (n = 8), DENV2 (n = 8), DENV3 (n = 5), or DENV4 (n = 5) or OVA (n = 10) with MPLA/AddaVax adjuvants as above, or 105 PFU of DENV2 PL046 (n = 10) at day 0 and then challenged intravenously on day 56 with a lethal dose of DENV2. Mice immunized with the four DENV NS1 serotypes separately were significantly protected against lethal challenge compared to OVA-immunized groups (DENV1, P = 0.002; DENV2, P = 0.008; DENV3, P = 0.004; DENV4, P = 0.014). Data were derived from three independent experiments.

  • Fig. 5. NS1-immune serum and anti-NS1 mAbs block NS1-induced endothelial permeability in vitro.

    (A) Monolayers of HPMECs grown on Transwell inserts were incubated for 48 h with both NS1 (5 μg/ml) and anti-NS1 serum (1:10 dilution), NS1 alone, or anti-NS1 serum alone, and TEER (ohm) was measured at indicated time points. OVA (5 μg/ml) was used as a protein control. Anti-OVA and anti-DENV2 PL046 sera were also included as controls. Results are representative of two independent experiments with duplicate Transwells. (B) Monolayers of HPMECs grown on Transwell inserts were incubated for 48 h with both NS1 (5 μg/ml) and NS1-specific mAbs (10 μg/ml) or either NS1 or anti-NS1 mAbs alone, and TEER (ohm) was measured at indicated time points. A mAb specific to DENV E protein (3H5) was used as an isotype control. Results are representative of three experiments, each with duplicate Transwells. Error bars indicate SEM.

  • Fig. 6. NS1-immune serum and anti-NS1 mAb block NS1-induced lethality in vivo.

    (A) Pooled serum (300 μl) from NS1-immunized (n = 7), OVA-immunized (n = 7), or DENV2 PL046–immune (n = 4) mice was transferred intraperitoneally to naïve Ifnar−/− mice before intravenous injection of NS1 (10 mg/kg) + 106 PFU of DENV2. Anti-NS1 or anti-OVA serum was transferred to naïve mice before injection of OVA (10 mg/kg) + 106 PFU of DENV2 (n = 4 per group) as controls (ctrl). Mice were followed for survival for 14 days p.i. Mice administered NS1-immune serum followed by NS1 + 106 PFU of DENV2 were significantly protected (P = 0.014) against lethality compared to mice given OVA-immune serum (n = 7), using the log-rank Mantel-Cox test. Data were derived from two independent experiments. (B) Ifnar−/− mice were infected intravenously with 107 PFU of DENV2 alone (n = 3), 106 PFU of DENV2 alone (n = 3), 106 PFU of DENV2 with DENV2 NS1 (10 mg/kg), or 106 PFU of DENV2 with OVA (10 mg/kg), as indicated. Mice injected with DENV2 combined with DENV2 NS1 or OVA were concomitantly injected intraperitoneally with 200 μg of isotype control mAb or anti-NS1 mAb (1H7.4) (n = 4 for all groups). Kaplan-Meier survival curves are shown, where mice were monitored for morbidity and survival for 10 days p.i. Mice given anti-NS1 mAbs were significantly protected (P = 0.0069) against lethality compared to mice given the isotype control mAbs by log-rank Mantel-Cox test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/304/304ra141/DC1

    Fig. S1. Recombinant NS1 from DENV1, DENV2, DENV3, DENV4, and WNV is highly purified and oligomeric.

    Fig. S2. Morbidity and vascular leakage are induced by NS1.

    Fig. S3. NS1 increases endothelial cell permeability in two different endothelial cell lines, HUVEC and HPMEC.

    Fig. S4. Viremia after lethal DENV challenge is reduced by immunization with DENV2 NS1.

    Fig. S5. Immunization with DENV NS1 and DENV infection induces serotype cross-reactive anti-NS1 antibodies.

  • Supplementary Material for:

    Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination

    P. Robert Beatty, Henry Puerta-Guardo, Sarah S. Killingbeck, Dustin R. Glasner, Kaycie Hopkins, Eva Harris*

    *Corresponding author. E-mail: eharris{at}berkeley.edu

    Published 9 September 2015, Sci. Transl. Med. 7, 304ra141 (2015)
    DOI: 10.1126/scitranslmed.aaa3787

    This PDF file includes:

    • Fig. S1. Recombinant NS1 from DENV1, DENV2, DENV3, DENV4, and WNV is highly purified and oligomeric.
    • Fig. S2. Morbidity and vascular leakage are induced by NS1.
    • Fig. S3. NS1 increases endothelial cell permeability in two different endothelial cell lines, HUVEC and HPMEC.
    • Fig. S4. Viremia after lethal DENV challenge is reduced by immunization with DENV2 NS1.
    • Fig. S5. Immunization with DENV NS1 and DENV infection induces serotype cross-reactive anti-NS1 antibodies.

    [Download PDF]

Navigate This Article